**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 *E. coli* for coriander (*Coriandrum sativum* L.), peppermint (*Mentha piperita* L.), and juniper (*Juniperus communis* L.) [36].

#### *4.3.1. Materials and methods*

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,

**Figure 2.** High-power examination (1000× magnification). Gram-stained smear of endotracheal aspirate shows Gram-

**Figure 1.** High-power examination (1000× magnification). Gram-stained smear of endotracheal aspirate shows Gram-

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

provided in advance, could make the difference.

negative coccobacilli isolated or in diplo (*Acinetobacter* spp.).

**4.1. Prevention of respiratory contamination**

**4. Therapeutic issues**

negative capsulate rods.

56 Contemporary Topics of Pneumonia

We are interested in evaluating of the efficiency of some EOs against carbapenem-resistant *Acinetobacter baumannii* and *E. coli* ATCC 25922. *A. baumannii* is a ubiquitous nonfermenter species, found in soil, water, and clinical units, and *E. coli* is a constant presence of the normal microbiocenosis of humans and warm-blood animals. Of hundreds of natural products commercially available, without prescription, only 21 volatile extracts (**Table 1**) were tested undiluted by two methods—diffusimetric method and aromatogram—described elsewhere [36]. Diffusimetric sensibility testing demonstrates the antibacterial activity by charging 5 mm diameter sterile paper disk with 2 or 5 μl of EOs. The aromatogram method is illustrated in **Figure 3**. Each experiment was performed three times at intervals of 2–3 days. The results are presented as mean and standard deviation (SD).


*4.3.2. Results*

*4.3.2.1. Diffusimetric method*

deposed. After sealing the assembly, the volatile effect was tested.

**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.

**Figure 3.** Aromatogram. In the middle of a sterile paper disk, with diameter of a Petri dish lid, 10 μl of essential oil was

**No EO Species Family Producer Administration** 15 Sandalwood *Santal amyris* Rutaceae Herbavit Aromatherapy,

The Emerging Problems of Carbapenem-Resistant Gram-Negative Bacillary Pneumonia

16 Seeds of apricots *Prunus armeniaca* Rosaceae Herbavit Internal use 17 Incense *Boswellia serrata* Burseraceae Bionovativ Internal use

> *Chamommilla recutita, Lavandula angustifolia* extracts*, Propolis cera,*

19 Grapefruit *Citrus paradisi* Rutaceae Solaris Aromatherapy,

20 Orange *Citrus sinensis* Rutaceae Solaris Aromatherapy,

21 Rosewood *Aniba rosaeodora* Lauraceae Solaris Aromatherapy,

Eucalyptus oil

18 Inhalant *Pinus sylvestis, Salvia officinalis,* 

**Table 1.** The characteristics of the essential oils.

massage

59

massage

massage

massage

Tisofit Inhalations

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The Emerging Problems of Carbapenem-Resistant Gram-Negative Bacillary Pneumonia http://dx.doi.org/10.5772/intechopen.69630 59


**Table 1.** The characteristics of the essential oils.

commercially available, without prescription, only 21 volatile extracts (**Table 1**) were tested undiluted by two methods—diffusimetric method and aromatogram—described elsewhere [36]. Diffusimetric sensibility testing demonstrates the antibacterial activity by charging 5 mm diameter sterile paper disk with 2 or 5 μl of EOs. The aromatogram method is illustrated in **Figure 3**. Each experiment was performed three times at intervals of 2–3 days. The results are

**No EO Species Family Producer Administration** 1 Thyme *Thymus vulgaris* Lamiaceae Fares Internal use,

*cayophyllata*

*globulus*

*communis*

*angustifolia*

*officinalis*

*alternifolia*

6 Mint *Mentha piperita* Labiatae Fares Internal use,

7 Pine *Pinus silvestris* Pinaceae Fares Internal use,

10 Oregano *Origanum vulgare* Lamiaceae Steaua Divina Internal use,

11 Negril *Nigella sativa* Ranunculaceae Steaua Divina Internal use,

12 Lemon *Citrus limonum* Rutaceae Steaua Divina Internal use,

13 Fennel *Foeniculi fructus* Apiaceae Hofigal Internal use 14 Sage *Salvia officinalis* Lamiaceae Solaris Aromatherapy,

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

aromatherapy, massage

massage

Myrtaceae Fares Internal use,

Myrtaceae Fares Internal use,

Cupressaceae Fares Internal use,

Lamiaceae Fares Internal use,

Lamiaceae Fares Internal use,

Myrtaceae Fares Internal use,

presented as mean and standard deviation (SD).

2 Clove *Eugenia* 

58 Contemporary Topics of Pneumonia

3 Eucalyptus *Eucalyptus* 

4 Juniper *Juniperus* 

5 Lavander *Lavandula* 

8 Rosemary *Rosmarinus* 

9 Tea tree *Melaleuca* 

**Figure 3.** Aromatogram. In the middle of a sterile paper disk, with diameter of a Petri dish lid, 10 μl of essential oil was deposed. After sealing the assembly, the volatile effect was tested.

#### *4.3.2. Results*

#### *4.3.2.1. 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.

**Figure 4.** Diffusimetric method carbapenem-resistant *A. baumanii* tested by 2 μl EOs (see **Table 1** for the coresponding numbers).

*Thymus vulgaris*, *Eugenia cayophyllata*, *Origanum vulgare*, and *Aniba rosaeodora* oils not only are by far the most effective but also obviously inhibit *A. baumannii* more efficiently than *E. coli*. Alternatively, *Melaleuca alternifolia* is slightly more effective against *E. coli* when diffusimetric method was used. Diffusimetric method also permits observing other important details. Although only clear inhibition zone was measured, *E. cayophyllata* shows residual growing, which is not measured and demonstrates a very good inhibitory activity. Also, it demonstrates an agonist effect in combination with *T. vulgaris*. Contrary, *O. vulgare* and *Nigella sativa*

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61

**Figure 7.** Diffusimetric method *E. coli* ATCC 25922 tested by 5 μl EOs (see **Table 1** for the coresponding numbers).

Aromatogram method (**Figures 8** and **9**) reveals more interesting evidence of usefulness of volatile effects of at least seven essential oils: *T. vulgaris*, *E. caryophyllata*, *O. vulgare*, *A. rosaeodora*, *Lavandula angustifolia*, *Mentha piperita*, and *M. alternifolia.* Surprisingly, some EOs, like mint, proved to be more effective when volatile effect is tested. The susceptibility testing was repeated after several weeks, but the results are inconsistent, mostly for mint, which showed no inhibitory effect after several weeks after opening the bottle. For other EOs (thyme, clove, eucalyptus, oregano, and rosewood) the antibacterial effect was not changed in time. Anyway,

Synthetic results are listed in **Table 2**. At first glance, we noticed a remarkable antibacterial activity for *T. vulgaris*, *E. caryophyllata*, *O. vulgare*, and *A. rosaeodora*. Three other EOs could be considered as being efficient (*M. alternifolia*, *Lavandula angustifolia*, and *M. piperita*), but ten of them proved to have no antibacterial effect at any concentration tested. *Eucalyptus globulus* and *J. communis* could be considered for topical use, but not for their volatile properties. Four EOs (*E. globulus*, *Lavandula angustifolia*, *M. piperita*, and *Rosmarinus officinalis*) show multiple resistant colonies inside inhibition zone when *E. coli* was tested by aromatogram. Therefore, we consider them without antibacterial activity when volatile effect is

show an antagonistic effect.

using single-use vials may be an option.

*4.3.2.2. Aromatogram*

considered.

**Figure 5.** Diffusimetric method carbapenem-resistant *A. baumanii* tested by 5 μl EOs (see **Table 1** for the coresponding numbers).

**Figure 6.** Diffusimetric method *E. coli* ATCC 25922 tested by 2 μl EOs (see **Table 1** for the coresponding numbers).

The Emerging Problems of Carbapenem-Resistant Gram-Negative Bacillary Pneumonia http://dx.doi.org/10.5772/intechopen.69630 61

**Figure 7.** Diffusimetric method *E. coli* ATCC 25922 tested by 5 μl EOs (see **Table 1** for the coresponding numbers).

*Thymus vulgaris*, *Eugenia cayophyllata*, *Origanum vulgare*, and *Aniba rosaeodora* oils not only are by far the most effective but also obviously inhibit *A. baumannii* more efficiently than *E. coli*. Alternatively, *Melaleuca alternifolia* is slightly more effective against *E. coli* when diffusimetric method was used. Diffusimetric method also permits observing other important details. Although only clear inhibition zone was measured, *E. cayophyllata* shows residual growing, which is not measured and demonstrates a very good inhibitory activity. Also, it demonstrates an agonist effect in combination with *T. vulgaris*. Contrary, *O. vulgare* and *Nigella sativa* show an antagonistic effect.

#### *4.3.2.2. Aromatogram*

**Figure 4.** Diffusimetric method carbapenem-resistant *A. baumanii* tested by 2 μl EOs (see **Table 1** for the coresponding

**Figure 5.** Diffusimetric method carbapenem-resistant *A. baumanii* tested by 5 μl EOs (see **Table 1** for the coresponding

**Figure 6.** Diffusimetric method *E. coli* ATCC 25922 tested by 2 μl EOs (see **Table 1** for the coresponding numbers).

numbers).

60 Contemporary Topics of Pneumonia

numbers).

Aromatogram method (**Figures 8** and **9**) reveals more interesting evidence of usefulness of volatile effects of at least seven essential oils: *T. vulgaris*, *E. caryophyllata*, *O. vulgare*, *A. rosaeodora*, *Lavandula angustifolia*, *Mentha piperita*, and *M. alternifolia.* Surprisingly, some EOs, like mint, proved to be more effective when volatile effect is tested. The susceptibility testing was repeated after several weeks, but the results are inconsistent, mostly for mint, which showed no inhibitory effect after several weeks after opening the bottle. For other EOs (thyme, clove, eucalyptus, oregano, and rosewood) the antibacterial effect was not changed in time. Anyway, using single-use vials may be an option.

Synthetic results are listed in **Table 2**. At first glance, we noticed a remarkable antibacterial activity for *T. vulgaris*, *E. caryophyllata*, *O. vulgare*, and *A. rosaeodora*. Three other EOs could be considered as being efficient (*M. alternifolia*, *Lavandula angustifolia*, and *M. piperita*), but ten of them proved to have no antibacterial effect at any concentration tested. *Eucalyptus globulus* and *J. communis* could be considered for topical use, but not for their volatile properties. Four EOs (*E. globulus*, *Lavandula angustifolia*, *M. piperita*, and *Rosmarinus officinalis*) show multiple resistant colonies inside inhibition zone when *E. coli* was tested by aromatogram. Therefore, we consider them without antibacterial activity when volatile effect is considered.

**EOs Carbapenem-resistant** *A. baumannii E. coli* **ATCC 25922**

66.67 mm (5.77)

32.33 mm (2.52)

16.00 mm (3.61)

23.00 mm (1.73)

14.67 mm (2.52)

20.67 mm (6.51)

16.33 mm (±1.53)

24.67 mm (3.51)

70.00 mm (0.00)

6.33 mm (0.58)

11 Negril NI NI NI NI NI NI 12 Lemon NI NI NI NI NI NI

14 Sage NI NI NI NI NI NI 15 Sandalwood NI NI NI NI NI NI

 Incense NI NI NI NI NI NI Inhalant NI NI NI NI NI NI Grapefruit NI NI NI NI NI NI Orange NI NI NI NI NI NI

> 32.33 mm (11.59)

7 Pine NI NI NI NI NI NI

1 Thyme 38.33 mm

2 Clove 21.67 mm

3 Eucalyptus 9.67 mm

4 Juniper 16.33 mm

5 Lavander 10.33 mm

6 Mint 13.67 mm

8 Rosemary 12.33 mm

9 Tea tree 15.67 mm

10 Oregano 46.00 mm

13 Fennel 6.00 mm

apricots

21 Rosewood 16.00 mm

16 Seeds of

(10.41)

(6.51)

(2.52)

(2.52)

(0.58)

(2.31)

(±4.62)

(8.14)

(±2.00)

(0.00)

(1.00)

as the mean (SD—standard deviation) of three independent experiments.

NI—no inhibition; mrc—multiple resistant colonies.

**2 μl 5 μl 10 μl 2 μl 5 μl 10 μl Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD)**

NI 8.00 mm

NI 14.67 mm (4.62)

NI 10.33 mm

NI 6.00 mm

NI NI NI NI NI NI

80.00 mm (0.00)

**Table 2.** The inhibition of carbapenem-resistant *A. baumannii* and *E. coli* ATCC 25922 by 21 EOs. The results are reported

(0.00)

14.67 mm (0.58)

28.00 mm (3.61)

The Emerging Problems of Carbapenem-Resistant Gram-Negative Bacillary Pneumonia

19.00 mm (5.29)

11.67 mm (0.58)

7.00 mm (0.00)

(±3.51)

18.33 mm (7.64)

29.67 mm (0.58)

(1.73)

65.00 mm (8.66)

http://dx.doi.org/10.5772/intechopen.69630

29.33 mm (2.31)

16.00 mm (3.46)

27.33 mm (2.52)

20.33 mm (3.51)

15.00 mm (1.00)

18.67 mm (±1.53)

23.33 mm (2.89)

40.33 mm (1.53)

NI NI

30.33 mm (9.50)

**75.00 mm** (5.00)

63

**33.33 mm** (7.64)

NI mrc

NI

NI mcr

NI mcr

NI mcr

11.00 mm (6.56)

76.00 mm (1.73)

50.00 mm (2.00)

70.00 mm (5.00)

46.67 mm (2.89)

19.67 mm (5.51)

39.00 mm (6.56)

26.33 mm (4.16)

77.67 mm (2.52)

**Figure 8.** Aromatogram carbapenem-resistant *A. baumanii* tested by 10 μl EOs (see **Table 1** for the coresponding numbers).

**Figure 9.** *E. coli* ATCC 25922 tested by 10 μl EOs (see **Table 1** for the coresponding numbers).


**Table 2.** The inhibition of carbapenem-resistant *A. baumannii* and *E. coli* ATCC 25922 by 21 EOs. The results are reported as the mean (SD—standard deviation) of three independent experiments.

**Figure 9.** *E. coli* ATCC 25922 tested by 10 μl EOs (see **Table 1** for the coresponding numbers).

**Figure 8.** Aromatogram carbapenem-resistant *A. baumanii* tested by 10 μl EOs (see **Table 1** for the coresponding numbers).

62 Contemporary Topics of Pneumonia

#### **4.4. Discussion**

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 Gramnegative 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 [41], 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 [44].

of environmental origin was to assess the influence of medical activities in changing soil and

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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 [46]. 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.

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—*Homo sapiens* (NP\_057111) and *Caenorhabditis elegans* (NP\_497107). Even they show MBL-folding, their similarity with other class B carbapenemases are very low, being more close to one hypothetical protein from *Sulfolobus acidocaldarius* (WP\_01538554)—20.5% identity with NP\_057111;

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 *t*-test assuming unequal sample variance was performed. The limit of this approach is due to the differences in the sequences length. The Needle program calculates identity, similarity, and score according to number of residues. Anyway, observing the data from **Table 4**, we noticed that environmental serine beta-lactamases are more similar with their clinical enterobacterial counterparts than with nonfermenters carbapenemases. The most striking differences are observed for subgroups IMI-3 of uncultured bacteria (ALJ52278) isolated from river sediment in China and OXA-48-like of uncultured bacteria (AJF40233) isolated from river sediment in Portugal. For ALJ52278 (IMI-3) beta-lactamase, 73.03% identity mean (CI 95 ± 25.33) with Class A carbapenemases of clinical *Enterobacteria* was noticed. More, sequences pairwise comparison reveals 93.50% identity with subgroups IMI-2 (*Enterobacter asburidae* WP\_032491237), IMI-2 (*E. coli* AEU10762), and IMI-14 (*Enterobacter hormaechei*

water microbiota.

**5.2. Data collection and methods**

**5.3. Results and discussion**

23.2% with NP\_497107, respectively.
