**2.** *Eucalyptus microcorys* **extracts**

#### **2.1 Bioactive** *Eucalyptus* **compounds**

Phytochemical screening showed that alkaloids, anthraquinones, flavonoïds and saponins are the major components of the aqueous extract of *E. microcorys*. Whereas anthocyanines, gallic tannins, polyphenols and triterpenes are found in negligible quantity [18].

#### **2.2 Effect of** *Eucalyptus microcorys* **leaves extracts on planktonic** *Escherichia coli*

A variation in the abundances of planktonic cells of *E. coli* was generally observed in the presence of the extract of *Eucalyptus microcorys* [18]. This variation is dependent not only on the concentration of the plant extract, but also on associated factors such as incubation temperature and lighting conditions. Thus, the incubation temperature affects the cultivability of *E. coli* cells with inhibition percentages varying from 3 to 100% for enteropathogenic *E. coli*, from 5 to 100% for commensal *E. coli*. *Eucalyptus microcorys* has a bactericidal property whose scope varies relatively according to the type of cell and the environmental conditions. Cultivable cells of *E. coli* happen to be relatively less abundant at temperatures 23°C and 37°C than 7°C, when grown in presence of *Eucalyptus microcorys* extract.

#### *Understanding of Cultivability of* Escherichia coli *in Aquatic Microcosm in the Presence... DOI: http://dx.doi.org/10.5772/intechopen.102861*

Planktonic cells of enteropathogenic *E. coli* have proven to be more resistant to bactericide properties of the *Eucalyptus microcorys* extract in psychrophilic conditions.

A gradual decrease in the abundance of cultivable enteropathogenic *E. coli* cells were observed during the period of exposure to light in the presence of the extract of *Eucalyptus microcorys* [20]. There is a progressive increase in the rate of cell inhibition in the cells tested in the presence of extract after exposure to light. Under dark conditions, the percentage of metabolically non-culturable enteropathogenic *E. coli* cells ranged from 17 to 99%. These inhibition rates increase under light conditions after each incubation period. Under an intensity of 1000 lx, the inhibition percentages fluctuated from 16 to 100% when considering all concentrations of *Eucalyptus microcorys* extract.

At 2000 lx, these inhibition rates fluctuated between 38 and 100%. At 3000 lx, peak inhibition rates of 100% were obtained after 12 hours of incubation at an extract concentration of 0.05%. Overall, the 3000 lx light intensity appears to result in the maximum inhibition of enteropathogenic *E. coli*.

The hourly inhibition rate of *E. coli* is very low in dark conditions and increases with increasing light intensity. In dark, cell inhibition rates are generally between 0.102 h−1 and 0.146 h−1. These inhibition rates increase considerably in the presence of light, with values sometimes reaching 0.662 h−1 at 3000 lx. The more the light intensity increases, the more the percentage of cell inhibition increases, whatever the concentration of the plant extract. For a light intensity of 3000 lx, the percentages of inhibition of *E. coli* cells are greater than 80%. The combined effect of light and herbal extract *E. microcorys* influence considerably the evolution of the percentage of cellular inhibition about each concentration of extract and each light condition.

The observation of commensal *E. coli* and enteropathogenic *E. coli* abundances is different when considering all the extract concentrations and incubation temperatures. Studies showed that the number of the colony forming units (CFU) of each of the cell strains in the presence of *Eucalyptus* extract, decreased in most cases for the increase of the concentration of aqueous extract and the incubation temperature. The cell inhibition percentage varied from one strain to another and with respect to the extract concentration and temperature incubation. The enteropathogenic *E. coli* strains seem to resist the effects of the extract concentrations 1% and 1.5% at 7°C, 23°C and 37°C, in contrary to the commensal strains with which the relatively higher percentages were observed in the same conditions.

Natural or acquired resistance to antibiotics would explain the observed resistance of enteropathogenic *E. coli* strains. Indeed, it is well known that bacteria can develop protective mechanisms such as changes in cell wall permeability and structure, production of inhibitory enzymes and alteration of antibacterial molecules [21]. This could explain the difference observed between CAIRs (Cell Apparent Inhibition Rates) of both bacteria strains. Indeed, whether we consider the enteropathogenic *E. coli* strain or the commensal strain, the rate of cell inhibition per hour for each incubation temperature increases as the concentration of the extract of *Eucalyptus microcorys* increases (**Figure 1**). Extracts of crushed and dried leaves of *Eucalyptus cloeziana*, *Eucalyptus microcorys*, *Eucalyptus saligna* and *Eucalyptus grandis* exhibit inhibitory activity against *E. coli* cells. Molecules present in *Eucalyptus* leaves that provide disinfectant properties are the monoterpenes, such as 1,8-cineole, alpha and beta-terpinene, 4-terpineol and tannins. 1,8-cineole has germicidal potential against *E. coli* cells.

## **2.3 Effect of** *Eucalyptus microcorys* **leaves extracts on adhered** *Escherichia coli*

The different percentages of adhered and detached enteropathogenic *E. coli* cells after contact with the *Eucalyptus microcorys* extract solution at the different

**Figure 1.**

*Hourly inhibitory rate of Enteropathogenic and commensal* E. coli *cells (determined as hourly values of cell apparent inhibition rates, CAIR) for each concentration of the* Eucalyptus microcorys *leaves extract (EM) at different incubation temperatures.*

concentrations chosen, were evaluated for the cells from each growth phase. They are presented in **Table 1**.

When the cells were from the lag phase, the percentages of cells remaining adhered after a stay in the extract solution fluctuated between 1.2 and 15%, 0.3 and 12.2%, and 0.2 and 6.4% after 1, 2 and 3 hours respectively when the concentration of the *Eucalyptus microcorys* extract was 1%. These percentages varied between 0.5 and 5.4%, between 0.3% and 3.1%, and between 0.2% and 4.7% after 1 h, 2 h and 3 h respectively in the 1.5% extract solution. In the 2% extract solution, these percentages fluctuated between 1% and 8.4%, between 0.1% and 4.9%, and between 0.9% and 4.4% after 1 h, 2 h and 3 h of residence respectively. At the same time, the percentages of detached cells varied from 1.7 to 11.0%, 0.5 to 9.2% and 2.0 to 4.5% respectively at extract concentrations of 1, 1.5 and 2% respectively (**Table 1**).

For cells from the exponential growth phase, the percentages of cells remaining adhered fluctuated between 0.4 and 10.2%, between 0.2 and 5.8%, and between 0.3 and 4.9% after 1 hour, 2 hours and 3 hours of contact with the 1% extract solution. They varied between 1.2 and 6.8%, between 0.6 and 5.4%, and between 1.0 and 5.2% after 1 hour, 2 hours and 3 hours of contact with the 1.5% extract solution. At the 2% extract concentration, these percentages fluctuated between 1.2 and 5.6%, between 0.3 and 6.5%, and between 1.0 and 1.8% after 1 hour, 2 hours and 3 hours of contact respectively. Under similar experimental conditions, the percentages of detached cells ranged from 1.6 to 11.4%, 0.9 to 68.8% and 1.0 to 12.7% at 1, 1.5 and 2% extract concentrations respectively (**Table 1**).

Solutions of *Eucalyptus microcorys* extract lead to detachment of the bacterial cells initially adhered to the polyethylene fragment. The importance of this cell detachment varies not only as a function of the concentration of the extract but also as a function of the residence time of the adhered cells in the extract solution. The bacterial adhesion to substrates involves two main steps: reversible adhesion and


*Understanding of Cultivability of* Escherichia coli *in Aquatic Microcosm in the Presence... DOI: http://dx.doi.org/10.5772/intechopen.102861*

> **Table 1.**

*Effect of 1%, 1.5% and 2%* Eucalyptus *leaves extracts on adhered EPEC cells from different growth phases and preincubation periods in 0.85% NaCl solution.*

irreversible adhesion [22]. The reversible adhesion is governed by physico-chemical interactions of type Van der Waals and Lewis acid–base [23]. The irreversible adhesion is slower than the previous one, the irreversibility of the membership using the bacterial metabolism step.

The detachment of enteropathogenic *E. coli* cells, initially fixed on the fragments of polyethylene, would be caused by the secondary metabolites present in the plant extract, which would cause the breakdown of the hydrogen bonds within the exopolysaccharide secreted by the enteropathogenic *E. coli* cells such as a protective matrix. In bacteria, the permeabilization of membranes by these compounds is associated with a loss of ions and degradation of the ATP potential, the aromatic molecules having the highest antibacterial activity being the phenols.

The polyphenols present in the extract of *Eucalyptus microcorys* would constitute stress factors and probably deprive the bacteria of their protective glycocalyx, thus causing a disorganization of the biofilm and the dislodgement of the bacteria from the surface of the polyethylene slides. However, studies showed that the rates of detached cells remain below 15%. This low rate would be linked to the exopolymer covering the bacteria which creates a concentration gradient so that the permeabilization of the protective layer is not complete. Thus, only bacterial cells from a certain distance from the support are affected and dislodged. Some bacteria carry specific genes in their plasmids, genes that code for virulence factors (type IV fimbriae, adhesins, toxins) and which play an important role in the cell adhesion process. They allow the interconnection of bacteria in micro-colonies, promoting their stabilization, which can lead to resistance to the effect of the detachment of the extract.

The change of strains from the adhered state to the planktonic state further exposes the bacterial cells to the antibacterial effect of the flavonoids and alkaloids contained in the plant extract. Alkaloids are hydrophobic cations with antibacterial properties and targeting cellular DNA. This inhibitory effect is modulated by the adherent cell-extract contact time, the long contact times acting on targets not reached by relatively short contact times. The percentages of inhibition of enteropathogenic *E. coli*, for all the four phases of cell growth, vary between 73.56% and 99.49%, the concentration of *E. microcorys* 2% being that which results in high levels of cell inhibition.

The presence of bacterial strains still living in the planktonic state in the extract could be explained by the phenomenon of resistance such as the phenomenon of microbial resistance to antibiotics. Bacteria can synthesize enzymes capable of destroying or modifying antibacterial molecules, the enzymatic reactions leading to this destruction or this modification, although varying with the bacterial strain. The resistance mechanism observed appears to be multifactorial. Indeed two mechanisms are generally advanced to explain the resistance of biofilms to antibacterials. It can be due either to a limitation of the diffusion of the antibacterial agents in the biofilm by the polysaccharide matrix which coats the bacteria, or to the particular physiological state (low growth rate) of the bacteria of the biofilm, consequence of the nutritional limitation that undergo bacteria within the biofilm. The hydrated polyanionic matrix that coats bacteria in biofilms, limits the diffusion of molecules from the surrounding medium and more particularly of charged molecules. The hydrated polyanionic matrix that coats bacteria in biofilms, limits the diffusion of molecules from the surrounding medium and more particularly of charged molecules.

The physiological state of the cell and the extract concentrations are the first factors influencing the adhesion process of *E. coli* through the detachment or maintenance of cells after the stay of the polyethylene fragment in the extract solution of *E. microcorys*. The action of disinfectant solutions on microorganisms could depend

*Understanding of Cultivability of* Escherichia coli *in Aquatic Microcosm in the Presence... DOI: http://dx.doi.org/10.5772/intechopen.102861*

on several factors, some of which are intrinsic to the organism and others are related to the environment. Knowledge of these factors should lead to better achievement of disinfection and sterilization. Resistance of the adhered cells of *E. coli* to the plant extract is observed after 9 hours of incubation when the cells have emerged from the exponential phase of growth. The lag phase corresponds to the adaptation of the inoculum to its new environment, while the decline phase is the period corresponding to the exhaustion of all nutritional resources. There is an accumulation of toxic metabolites. Under the action of endogenous proteolytic enzymes, cell lysis leads to a decrease in viable organisms.
