**7. Natural anti-biofilm strategies**

### **7.1 Plant extracts**

Many extracts of plants and their derivatives were widely studied to eliminate the '*Propionibacterium acne'* biofilm [36]. It has been reported that out of 119 plant extracts, five showed strong antibiofilm activity i.e. *Rhodiola crenulata, Dolichos lablab*, *Malus pumila, Epimedium brevicornum,* and *Polygonum cuspidatum.* These scientists also suggested that extracts of *P. cuspidatum* and *E. brevicornum* and their active derivatives i.e. resveratrol and icartin show a potential antibiofilm activity even when used at lowest MIC. Bark extracts of *Melia dubia* were evaluated with 30 mg/mL concentration [37]. Furthermore, these extracts exhibit potential suppression of hydrophobicity, swarming motility, hemolysis, and biofilm production in *E. coli*. Other colleagues also reported similar results about *Capparis spinosa* (caper bush) extract, this extract shows inhibitory effect on the EPS production and biofilm production in *Serratia marcescens, Pseudomonas aeruginosa, Proteus mirabilis,* and *Escherichia coli* at 2 mg/mL concentration [38]. In addition, well-known biofilm formation of 3 microbes was dispersed. A medically important plant '*Lagerstroemia speciosa'* usually present in Southeast Asia, fruit extract from this plant is capable of inhibiting biofilm formation by '*P. aeruginosa'* PAO1 at 10 mg/mL concentration [39].

Other two plant extracts Dandasa (*Juglans regia* Tree Bark) and green tea (*Camellia sinensis*) show a potential antibiofilm activity individually. Recently, researchers observed that both Green tea and Dandasa exhibit potential antibiofilm activity of *Streptococcus mutans* at 12.5 and 6.2 mg/mL concentration, respectively, and on *E. coli* at 3.1 and 12.5 mg/mL concentration, respectively.

**93**

**7.2 'Honey'**

*Innovative Strategies for the Control of Biofilm Formation in Clinical Settings*

*Allium sativum* extract i.e. fresh garlic extract (FGE) has a potential inhibitory effect against biofilm formation, it has been observed that FGE decreased '*P. aeruginosa'* biofilm formation [40]. In-vitro screening of antibiofilm activity of '*Staphylococcus epidermidis'* of different 45 aqueous extracts from twenty-four Caatinga (Brazilian xeric shrubland) medicinal species was published. Extremely favorable extracts were taken from *Chamaecrista desvauxii* fruits, *Pityrocarpa moniliformis* leaves, *Bauhinia acuruana* fruits and *B. acuruana* branches, which show decreased the formation of biofilm even when they were tested at the lowest concentration. In addition, it was also suggested that *Senna macranthera* and *Commiphora leptophloes* fruit extracts decreased biofilms by 66.7% and 67.3% respectively. *Mycobacterium smegmatis* which plays a significant role in biofilm development was observed by using many quantitative and qualitative techniques. Other scientists examined different plants i.e. *Vaccinium oxycoccos*, *Hippophae rhamnoides, Azadirachta indica* and *Juglans regia* and spices to look for useful antibiofilm natural substitutes. When the efficiency of plant extracts as an antibiofilm agent was checked it showed that the extract of *Azadirachta indica* usually named as "Neem" was surprisingly helpful at removing and lowering *M. smegmatis* biofilms [41]. Another plant extract 'casbane diterpene' isolated from "*Croton nepetaefolius*" extract, is used to suppress the biofilm production of five Gram-negative bacterial species (*Klebsiella oxytoca, Klebsiella pneumoniae, Escherichia coli, Pseudomonas fluorescens* and *Pseudomonas aeruginosa*), two Gram-positive bacterial species (*S. epidermidis* and *S. aureus*), and three yeast species (*Candida glabrata, Candida tropicalis* and *Candida albicans*) [42]. Furthermore, another study demonstrated that *Candida* biofilm formation was remarkably decreased by *Boesenbergia pandurata* also known as "finger root oil" almost by 63–98% when MIC levels were used from 4 to 32 μL/mL [43]. Later studies showed that different plant extracts were isolated against *Enterohemorrhagic E. coli* (EHEC) O157:H7 biofilm. Furthermore, this study displayed that out of 498 plant extracts, almost 16 of them showed an inhibitory effect on biofilm formation of EHEC above 85% with no-growth of planktonic cells [44]. Certainly, these results specify that these different plant extracts show maximum inhibitory effect on biofilm formation of several microbes. Hence, it is suggested that further efforts are required to study the potential of these

A natural product extracted by 'honey' bee from floral nectar is called as 'honey' however, 'honey' is generally common and is usually used for its remarkable activity in wound-healing, anti-inflammatory, and antibacterial activity and used as an antioxidant. It has antimicrobial activities against 60 species of fungi and bacteria. 'Honey' was reported as a useful agent to control the biofilm formation. Furthermore, it was described that 'honey' is effective in the prevention of *Enterococcus spp.* biofilm production and can also use as a therapeutic agent against many *Enterococcal* infections that are biofilm-related. It can also decrease the biofilm production of EHEC O157:H7. Recent studies show that very low quantity of 'honey' can significantly decrease the formation of biofilm, the virulence of *E. coli* O157:H7 and Quorum sensing. So, a very low 'honey' concentration can decrease the formation of biofilm by preventing the virulence genes transfer in microbes and the expression of biofilmassociated curling QS, without inhibiting the cell growth. Due to its antimicrobial properties, high concentration of 'honey' can also prevent biofilm formation as well as adhesion of bacteria. Despite its antibacterial activity, it is also observed that 'honey' inhibits biofilm formation by antibacterial peptide which is bee defensin 1 that prevents microbial viability as well as biofilm formation indirectly [41].

*DOI: http://dx.doi.org/10.5772/intechopen.89310*

plant extracts as antibiofilm agents in detail.

*Innovative Strategies for the Control of Biofilm Formation in Clinical Settings DOI: http://dx.doi.org/10.5772/intechopen.89310*

*Allium sativum* extract i.e. fresh garlic extract (FGE) has a potential inhibitory effect against biofilm formation, it has been observed that FGE decreased '*P. aeruginosa'* biofilm formation [40]. In-vitro screening of antibiofilm activity of '*Staphylococcus epidermidis'* of different 45 aqueous extracts from twenty-four Caatinga (Brazilian xeric shrubland) medicinal species was published. Extremely favorable extracts were taken from *Chamaecrista desvauxii* fruits, *Pityrocarpa moniliformis* leaves, *Bauhinia acuruana* fruits and *B. acuruana* branches, which show decreased the formation of biofilm even when they were tested at the lowest concentration. In addition, it was also suggested that *Senna macranthera* and *Commiphora leptophloes* fruit extracts decreased biofilms by 66.7% and 67.3% respectively. *Mycobacterium smegmatis* which plays a significant role in biofilm development was observed by using many quantitative and qualitative techniques. Other scientists examined different plants i.e. *Vaccinium oxycoccos*, *Hippophae rhamnoides, Azadirachta indica* and *Juglans regia* and spices to look for useful antibiofilm natural substitutes. When the efficiency of plant extracts as an antibiofilm agent was checked it showed that the extract of *Azadirachta indica* usually named as "Neem" was surprisingly helpful at removing and lowering *M. smegmatis* biofilms [41].

Another plant extract 'casbane diterpene' isolated from "*Croton nepetaefolius*" extract, is used to suppress the biofilm production of five Gram-negative bacterial species (*Klebsiella oxytoca, Klebsiella pneumoniae, Escherichia coli, Pseudomonas fluorescens* and *Pseudomonas aeruginosa*), two Gram-positive bacterial species (*S. epidermidis* and *S. aureus*), and three yeast species (*Candida glabrata, Candida tropicalis* and *Candida albicans*) [42]. Furthermore, another study demonstrated that *Candida* biofilm formation was remarkably decreased by *Boesenbergia pandurata* also known as "finger root oil" almost by 63–98% when MIC levels were used from 4 to 32 μL/mL [43]. Later studies showed that different plant extracts were isolated against *Enterohemorrhagic E. coli* (EHEC) O157:H7 biofilm. Furthermore, this study displayed that out of 498 plant extracts, almost 16 of them showed an inhibitory effect on biofilm formation of EHEC above 85% with no-growth of planktonic cells [44]. Certainly, these results specify that these different plant extracts show maximum inhibitory effect on biofilm formation of several microbes. Hence, it is suggested that further efforts are required to study the potential of these plant extracts as antibiofilm agents in detail.

### **7.2 'Honey'**

*Bacterial Biofilms*

induced depolymerizes can easily disrupt biofilms. Now genetically engineering for phages have been introduced that explicit biofilm degrading enzymes during infection. The scientist has engineered a gene namely "dispersion" (dspB) into an *E. coli* specific T7 phage to yield an engineered enzymatic phage, which shows more

Despite the several benefits of phage use, there are some disadvantages also, for example, the release of a considerable amount of bacterial membrane-bound endotoxins, decreased number of phages encoding toxins, insufficient pharmacokinetic data and conversion of lytic phages to prophages is also a big concern. Some of the above-mentioned problems have been well determined through different processes like designing a recombinant phage from *Pseudomonas aeruginosa* filamentous phage to minimize the mortality rate in experimental animals and release of membrane-bound endotoxins to report the endotoxin release issue is major advances to overcome the above-mentioned concerns [35]. It has been observed that bacteriophages and antibiotics have a big potential to control biofilms such as phage PhilBB-PF 7A plays role in the removal of *Pseudomonas fluorescens* biomass and has shown almost 63–91% activity. Different studies show some of the strongest inhibitions, for example, the existence of biofilm EPS matrix hindering the control of biofilm via antibacterial agents and higher antibiotic resistance can be controlled through phage use. Furthermore, there are many limitations of phage use such as microbial resistance to phages, virulence genes that are phage-encoded can incorporate inside the host bacterial genome and the narrow host range. Phage efficacy can also be reduced by the immune system, and phage preparations that are improperly obtained can also contain endotoxin. To control these obstacles engineered phages or phage mixtures can be an effective alternative. Moreover, after proper selection and several studies

efficacy for the removal of biofilms as compared to non-cloned phages.

phages has become one of the most useful anti-biofilm agents.

and on *E. coli* at 3.1 and 12.5 mg/mL concentration, respectively.

Many extracts of plants and their derivatives were widely studied to eliminate the '*Propionibacterium acne'* biofilm [36]. It has been reported that out of 119 plant extracts, five showed strong antibiofilm activity i.e. *Rhodiola crenulata, Dolichos lablab*, *Malus pumila, Epimedium brevicornum,* and *Polygonum cuspidatum.* These scientists also suggested that extracts of *P. cuspidatum* and *E. brevicornum* and their active derivatives i.e. resveratrol and icartin show a potential antibiofilm activity even when used at lowest MIC. Bark extracts of *Melia dubia* were evaluated with 30 mg/mL concentration [37]. Furthermore, these extracts exhibit potential suppression of hydrophobicity, swarming motility, hemolysis, and biofilm production in *E. coli*. Other colleagues also reported similar results about *Capparis spinosa* (caper bush) extract, this extract shows inhibitory effect on the EPS production and biofilm production in *Serratia marcescens, Pseudomonas aeruginosa, Proteus mirabilis,* and *Escherichia coli* at 2 mg/mL concentration [38]. In addition, well-known biofilm formation of 3 microbes was dispersed. A medically important plant '*Lagerstroemia speciosa'* usually present in Southeast Asia, fruit extract from this plant is capable of inhibiting biofilm formation by '*P. aeruginosa'* PAO1 at 10 mg/mL concentration [39]. Other two plant extracts Dandasa (*Juglans regia* Tree Bark) and green tea (*Camellia sinensis*) show a potential antibiofilm activity individually. Recently, researchers observed that both Green tea and Dandasa exhibit potential antibiofilm activity of *Streptococcus mutans* at 12.5 and 6.2 mg/mL concentration, respectively,

**7. Natural anti-biofilm strategies**

**7.1 Plant extracts**

**92**

A natural product extracted by 'honey' bee from floral nectar is called as 'honey' however, 'honey' is generally common and is usually used for its remarkable activity in wound-healing, anti-inflammatory, and antibacterial activity and used as an antioxidant. It has antimicrobial activities against 60 species of fungi and bacteria. 'Honey' was reported as a useful agent to control the biofilm formation. Furthermore, it was described that 'honey' is effective in the prevention of *Enterococcus spp.* biofilm production and can also use as a therapeutic agent against many *Enterococcal* infections that are biofilm-related. It can also decrease the biofilm production of EHEC O157:H7. Recent studies show that very low quantity of 'honey' can significantly decrease the formation of biofilm, the virulence of *E. coli* O157:H7 and Quorum sensing. So, a very low 'honey' concentration can decrease the formation of biofilm by preventing the virulence genes transfer in microbes and the expression of biofilmassociated curling QS, without inhibiting the cell growth. Due to its antimicrobial properties, high concentration of 'honey' can also prevent biofilm formation as well as adhesion of bacteria. Despite its antibacterial activity, it is also observed that 'honey' inhibits biofilm formation by antibacterial peptide which is bee defensin 1 that prevents microbial viability as well as biofilm formation indirectly [41].

### **7.3 Essential oils**

Naturally plant-derived volatile substances are called as essential oils (EOs). Because of their antibacterial and preservative properties, these are effective and favorable natural products for the food industry. These essential oils are commonly used against a wide diversity of microorganisms since ancient time. These oils exhibit antimicrobial impact on the cell wall of microbes, leading to the destruction of microbes. Furthermore, it is suggested that these oils are very effective in inactivating many microbes without producing antimicrobial resistance [45]. Because of little mammalian toxicity, rapid degradation in the environment and availability of many essential oils make them beneficial antibiofilm agent [46].

*Cumin oil* scientifically named *Cuminum cyminum,* a derivative of an aromatic, therapeutic plant of "Apiaceae" family, has various medicinal properties and in the digestive system, it acts as an astringent. It has been widely used for acute gastric diseases as a carminative and eupeptic, and as an analgesic. It is also widely used to flavor foods, for example, added in food for fragrance. Cumin seeds have been used since ancient time. The efficiency of cumin seed against biofilm development on *Klebsiella pneumoniae* strains was observed, which showed that cumin seeds has decreased biofilm activity with improved ciprofloxacin efficiency [47].

*Cinnamon oil* is derived from the inner bark of the "*Cinnamomum zeylanicum'* as well as "*Cinnamomum cassia*" and is mostly used in the food industry due to its specific fragrance [48]. It is suggested that this oil is efficient against biofilm cultures *Lactobacillus plantarum, S. mutans*, and *S. epidermidis*. *Oregano* also is known as *Origanum vulgare* has inhibitory activity on biofilm production in case of *E. coli* and *Staphylococci.* A study revealed that Oregano essential oil exerts antimicrobial action on *E. coli, S. haemolyticus, S. sciuri, S. aureus,* and *S. lugdunensis* and could prevent biofilm formation. Moreover, it also able to detach active biofilm even at very low MIC. Inhibitory activity of *"Brazil nut oil*" named as *Bertholletia excelsa* (a vegetable oil) on commercially available dentifrice to prevent dental biofilm was also assessed. Scientists showed that by adding this vegetable oil to commercially available dentifrice, dental biofilm formation can be inhibited. Furthermore, this oil helps in preventing and controlling periodontal diseases [41].

The antimicrobial activity of "tea tree" essential oils scientifically named *Melaleuca alternifolia*, synergistically with ciprofloxacin was also evaluated against '*P. aeruginosa'* biofilms. The consequences showed that the combined effect of TTO with ciprofloxacin has decreased biofilm biomass significantly by more than 70% and lowered the number of cells at the lowest (1.25 μg/mL) ciprofloxacin concentration. The efficacy of essential oils from *cinnamon (Cinnamomum verum),* namely *thymol*, and *oregano* at sub-lethal concentrations on biofilm formation of 3 biofilmforming bacterial strains i.e. *Stenotrophomonas*, *Acinetobacter* and *Sphingomonas* were assessed. Researchers showed that at the MIC, two out of three strains revealed resistance on microbial biofilm formation. Furthermore, among the three tested oils, "*thyme oil*" was considered as more efficient and showed inhibitory effect even on sub-lethal concentrations of 0.001% (w/v) [41].

### **8. Conclusion**

Since biofilms are abundant in nature, the importance of biofilms in hospitals especially regarding their role in infections is often undervalued. Future studies should attempt to comprehend the biological forces controlling the colonization to develop innovative strategies for controlling biofilm biomass within a clinical context. Additionally, comprehensive research is required to recognize the potential

**95**

provided the original work is properly cited.

, Maria Rasool1,2, Naheed Akhter1

1 College of Allied Health Professionals, Directorate of Medical Sciences,

2 Department of Microbiology, Government College University Faisalabad,

, Muhammad Hidayat Rasool<sup>2</sup>

Government College University Faisalabad, Pakistan

\*Address all correspondence to: mohsin.mic@gmail.com

*Innovative Strategies for the Control of Biofilm Formation in Clinical Settings*

of various synthetic and natural quorum sensing inhibitors (QSIs) for their applicability for humans. As these QSIs do not encourage the antibiotic resistance, therefore they can surely be the future therapeutic agents for the management of

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Bilal Aslam2

and Mohsin Khurshid2

, Ali Hassan1

\*

,

*DOI: http://dx.doi.org/10.5772/intechopen.89310*

**Conflict of interest**

**Author details**

Aqsa Shahid1

Sadia Sana1

Pakistan

biofilm-based bacterial infections in clinical settings.

The authors declare no conflict of interest.

*Innovative Strategies for the Control of Biofilm Formation in Clinical Settings DOI: http://dx.doi.org/10.5772/intechopen.89310*

of various synthetic and natural quorum sensing inhibitors (QSIs) for their applicability for humans. As these QSIs do not encourage the antibiotic resistance, therefore they can surely be the future therapeutic agents for the management of biofilm-based bacterial infections in clinical settings.
