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Antibiofilm and antimicrobial activities of papaya (Carica papaya L.) and stevia (Stevia rebaudiana Bertoni) leaf extracts against three biofilm-forming bacteria

Biofilm is a structural form of a microbial group that is protected by the Extracellular Polymeric Substance (EPS) matrix. The biofilm is considered as the main mediator of infection, and plays a major role in the occurrence of drug resistance. This study was aimed at determining the antimicrobial and antibiofilm activities of papaya (Carica papaya L.) and stevia (Stevia rebaudiana Bertoni) leaf extracts against three biofilm-forming bacteria. The antimicrobial assay showed that papaya leaf extract exhibits higher activity compared to stevia leaf extract in inhibiting the growth of the biofilm-forming bacteria. The optimum condition of papaya leaf extract to inhibit biofilm-forming bacterial growth occurred at 45% and 75% concentrations of the extract (pH 7). A 100% biofilm degradation by papaya leaf extract occurred at pH 6 and pH 9.


Introduction
A biofilm is a structural form of a microbial group that is protected by an extracellular matrix called Extracellular Polymeric Substance (EPS) (Prakash et al. 2003). Biofilms are currently considered as the primary mediator of infection by various species of pathogenic microorganisms, with about 80% of infections related to the biofilm formation (Archer et al. 2011). The biofilm handling can be done through physical, chemical, or biological treatments. In the present time, the handling of biofilms tends to be mostly carried out biologically, such as using various plant extracts because they have a low level of toxicity and are more environmentally friendly. Several natural materials from plants with potential as antibiofilm include the terpenoid, steroids, saponins, carotenoids, phenolics, furanones, alkaloids, peptides, tannins, and lactones (Viju et al. 2013).

Molecular identification of biofilm-forming bacteria DNA extraction, PCR amplification and sequencing
Bacterial DNA extraction was carried out directly from the 48-h colony on the Nutrient Agar (NA) medium. A total of 1-2 bacterial colonies grown on the NA medium was taken using a sterile ose, and then put into 50-100 µL nuclease free water and then homogenized. The bacterial suspension is considered as a DNA template. The DNA extraction was carried out using KAPA 3G Plant PCR Kit ® (KAPA Biosystems, USA) with the following mix solution composition per sample: 18.9 µL nuclease free water, 25 µL KAPA 3G Plant PCR Buffer (2x), 1.5 µL Primer 27F (5'-AGAGTTGATCMTGGCTCAG-3'), 1.5 µL Primer 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), 0.6 µL KAPA 3G Plant dNTPs, 0.5 µL KAPA enhancers, 0.5 µL MgCl 2 , and 1.5 µL DNA template. The PCR reaction was conducted using the following condition: initial denaturation at 95°C for 3 min, followed by 35 cycles consisting of denaturation at 95°C for 20 s, annealing at 50°C for 15 s, extension at 72°C for 30 s. A final extension at 72°C for 30 s was conducted after 35 cycles.

Phylogenetic analysis
A nucleotide sequence from the 27F and 1492R primer pair were edited and trimmed using the ChromasPro version 1.7.5 software (Technelysium Pty ltd, Australia). The ambiguous nucleotide base was checked to ensure that there is no readable base error. Homologous sequences were searched from the GenBank database using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were further aligned in the FASTA format using MUSCLE (MUltiple Sequence Comparison by Log-Expectation) (Edgar 2004) in MEGA (Molecular Evolutionary Genetics Analysis) version 6 (Tamura et al. 2013).
Bacterial identification was carried out using the Phylogenetic Species Concept (PSC), where species are defined as sequences that form independent clades. The phylogenetic tree was constructed using Neighbor-Joining (NJ) method in MEGA 6. The Maximum Composite Likelihood was used to estimate evolutionary distances between all pair of sequences simultaneously. Partial deletion was used to treat indels or missing data. The branch strength of the phylogenetic tree was tested by 1000 replications of bootstrap method.

Papaya (Carica papaya L.) and Stevia (Stevia rebaudiana Bertoni) leaves extraction
A total of 50 g of papaya leaf powder (˂ 80 mess) was extracted using 250 mL ethanol absolute, and then incubated in the rotary shaker at room temperature for 2 days at 120 rpm. The solution was further filtered and then concentrated in a rotary vacuum evaporator at a temperature of 40°C. The same procedure was used to extract stevia leaf powder.

Antimicrobial activity assay
Antimicrobial test was carried out using the method described by Ulyah et al. (2015) with a modification. Leaf extracts were prepared in several concentrations, namely 45%, 75% and 90%. The most optimum concentration in inhibiting biofilm-forming bacteria was carried out by agar diffusion method. The MHA (Mueller Hinton Agar) medium with different pH conditions (6, 7, and 9) were prepared. Three biofilm-forming bacterial isolates were prepared in 5 mL of Luria Bertani (LB) medium and incubated at 37°C for 24 h. After incubation, each bacterial suspension was swabbed on the surface of MHA medium and then incubated for 10 min. Three agar wells (3 mm in diam.) were prepared in the MHA medium. A total of 50 µL of each leaf extract concentration (45%, 75%, 90%) was inserted into the holes in the MHA medium. Chloramphenicol and sterile distilled water were used as positive control and negative control, respectively. Incubation was carried out at different temperatures (30°C and 37°C) and different times (24 h, 48 h, 72 h). The inhibition zone was measured as follows: Inhibition zone (mm) = clear zone diameter (mm) -diameter of the hole (mm) Inhibition effectiveness parameter was set as follows: -Diameter of inhibition zone > 20 mm: very strong inhibition activity -Diameter of inhibition zone > 10-20 mm: strong inhibition activity -Diameter of inhibition zone > 5-10 mm: moderate inhibition activity -Diameter of inhibition zone > 0-5 mm: weak inhibition activity Antibiofilm activity assay Biofilm inhibition assay was conducted using the microtiter assay method (Ulyah 2015) with a modification. First, the biofilm was formed on the microplate by growing biofilm forming bacteria for 24 h, then a total 50 µL of papaya leaf extract or stevia leaf extract was added into the microplate. This assay was conducted at pH (6, 7, 9)), temperatures (30, 37°C) and incubation times (24, 48, 72 h).
Microplate was rinsed with distilled water three times, then a 125 mL of 0.1% crystal violet was added, and the microplate was incubated at room temperature for 15 minutes. The microplate was rinsed again using distilled water three times, and then dried. A total 200 mL of ethanol was added into the microplate wells and was incubated at room temperature for 15 minutes. After the incubation, the absorbance was measured in the microplate reader at λ = 595 nm. The optical density (OD) was calculated as follows: % biofilm inhibition = 1 -(a corrected sample / a corrected blank) x 100% Notes: A corrected sample = fungal extract + plant leaf extract A corrected blank = DMSO 20% + plant leaf extract

Molecular identification of biofilm-forming bacteria
The BLAST results of the three bacterial isolates showed that the sequence from strain SA 1-3 had a close relationship with type sequences of the genus Bacillus (data not shown). The NJ tree showed that SA 1-3 sequence nested in the same clade with B. thuringiensis strain ATCC 10792, B. marcorestinctum strain LQQ, B. toyonensis strain BCT-7112, B. wiedmannii strain FSL W8-0169, B. mycoides strain NBRC 101228, B. weihenstephanensis strain DSM 11821, and B. cereus strain CCM 2010 with 92% bootstrap Figure 1. A phylogenetic tree generated from the NJ analysis showed a relationship of Bacillus sp. strain 1-3 with closely related sequences (1000× BS) support (BS) (Fig. 1). However, the SA 1-3 sequence was named as Bacillus sp. strain 1-3, due to formed an independent lineage within this clade.
The BLAST results showed that the nucleotide sequence of strain SA 1-5 showed a close relationship with type sequences of the genus Enterobacter (data not shown). The phylogenetic tree generated from the NJ analysis showed that the SA 1-5 sequence nested in the same clade with E. hormaechei subsp. steigerwaltii strain EN-5624, and E. cloacae strain ATCC 13047 with 67% BS (Fig. 2). Sequence of SA 1-5 was named as Enterobacter sp. strain 1-5 strains due to paraphyletic in this clade.

Figure 2. A phylogenetic tree generated from the NJ analysis showed a relationship of
Enterobacter sp. strain 1-5 with closely related sequences (1000× BS)
In addition, the papaya leaf extract at all concentrations showed a high antibiofilm activities (Table 4). The microtiter assay showed that the best antibiofilm activities from the papaya leaf extract against Bacillus sp. strain SA 1-3 and Enterobacter sp. strain SA 1-5 were found at 45% (37°C, pH 6) and 90% (37°C, pH 9), respectively (Table 3).

Discussion
Biofilm is the main mediator of an infection, with an estimated 80% of microbial infections related to the biofilm formation (Tarver 2009, Archer et al. 2011. This is because the biofilm can occur on surfaces of the organs of organisms (human, animal, and plant) and affect the function of the colonized organs. In general, biofilm can be treated using physical, chemical, and biological methods. The biological methods include application of microbial and plant metabolites (Hamieh et al. 2015, Sánchez et al. 2016, Bazargani & Rohloff 2016. Among the plant extracts, papaya and stevia leaves extracts were reported to have antimicrobial activities (Romasi et al. 2011, Gamboa & Chaves 2012, Miranda-Arámbula et al. 2017. Papaya leaf contains active substances such as carbainine alkaloids, carpaine, vitamin C and E, choline, proteolytic enzyme (papain), flavonoids, and tannins (Milind & Gurdita 2011). Meanwhile, stevia leaf contains active substances such as alkaloids, tannins, flavonoids, and phenol (Lamonthe et al. 2009).
The results of this study showed that both stevia and papaya leaves extracts exhibited antimicrobial and antibiofilm activities, but the papaya leaf extract showed higher activities than that of stevia leaf extract (Tables 1-4). This might be due to the activity of the papain enzyme from the papaya leaf extract. Papain is the proteolytic enzyme in the papaya leaves that has an antibiofilm and antimicrobial activities (Cynthya et al. 2014, Blanchette & Wenke 2018, Mohamed et al. 2018. Another important antimicrobial compound from papaya is the carpaine alkaloid (Julianti et al. 2014). Alkaloid compounds can inhibit bacterial cell wall synthesis through lysis activities of the bacterial cell wall. In addition, alkaloid compounds can interfere with the formation of peptidoglycan constituents in the bacterial cells, causing failure in the bacterial cell wall layer formation.
In the mechanism of antibiofilm formation by natural compounds such as papain, the biofilm degradation usually occurs in the Extracellular Polymeric Substance (EPS) layer. The EPS composition varies between microbial species and growth conditions (Borlee et al. 2008). This study showed that the bacterial biofilm responses to the stevia and papaya leaves extracts varies at different temperatures and pH conditions (Tables 3-4). Biofilm degradation ability of metabolites is related to their ability to penetrate into the EPS layer or the mucous layer that envelops the bacteria. Many antimicrobial compounds fail to penetrate biofilms because EPS, which acts as a barrier, protects bacterial cells inside. The formation of EPS is signaled by the secretion of Quorum Sensing (QS) (Tan et al. 2014). The QS is a mechanism used by the bacterial cells to determine the condition of their extracellular environment and takes a major role in the biofilm formation (Borlee et al. 2008). Therefore, disrupting the secretion of QS and preventing the formation of EPS is the main concern to prevent the biofilm formation (Ardani et al. 2010).