Synergistic action of bacteriophage and metabolites of Pseudomonas fluorescens JB3B and Streptomyces thermocarboxydus 18PM against Enterotoxigenic Escherichia coli and Bacillus cereus and their biofilm

Abstract

Background

Foodborne disease and food spoilage are the prime public health issue and food security round the globe. Significant disease outbreaks mostly linked to the existence of pathogenic bacteria that extremely challenging due to the persistence of biofilm-forming. Proteins and bacterial metabolites have been shown to have good antibacterial activity and effectively removal bacterial biofilm. Recently, bacteriophage and their encoded lytic proteins such as lysin have attracted attention as potential alternative agent to control undesirable pathogens in human body infection, increasing food safety as advance preservations and medical treatment such as phage therapy. For these reasons, the efficacy of bacteriophage and their potential in combination with bacterial metabolites from Phyllosphere and Actinomycetes bacteria (Pseudomonas fluorescens JB3B and Streptomyces thermocarboxydus 18PM crude extracts) was the aim of this present study.

Results

In this study, bacteriophage BC-VP (1.28 ± 0.29 × 10¹¹ PFU/ml) and ETEC-phage-TG (8.9 ± 2.19 × 10⁸ PFU/ml) isolated from artificial lake water from previous study showed potential activity to control Bacillus cereus (BC) and Enterotoxigenic Escherichia coli (ETEC) population. The combination of BC-VP with metabolite (P. fluorescens JB3B and S. thermocarboxydus 18PM) which were known from previous study had antibiofilm activities were able to inhibit (86.1%; 83.3%) and destruct (41%; 45.5%) biofilm formation of B. cereus respectively. Likewise, the synergy of bacteriophage ETEC-phage-TG with the same crude extract also showed promising activity against biofilm of ETEC with percentage of inhibition (81.9%; 76.4%) and percentage of destruction (54.1%; 44.4%). Application in various food, combination of BC-VP and bacterial metabolite extract (P. fluorescens JB3B; S. thermocarboxydus 18PM) were able to reduce Bacillus cereus population in mashed potato (99.6%; 99.4%) at cold temperature (4 °C) and (68.9%; 56.6%) at room temperature (28 °C), boiled pasta (99.5%; 99.4%) and (84.7%; 75.7%), also soymilk (96.9%; 96.7%) and (42.4%; 39.4%) respectively. Likewise, combination of ETEC-phage-TG and bacterial metabolite (P. fluorescens JB3B; S. thermocarboxydus 18PM) potentially reduced ETEC population after two different temperatures (4 °C and 28 °C) incubation in bean sprouts (TFTC; TFTC) and (47.5%; 49.1%), chicken meat (TFTC; TFTC) and (58.1%; 54%), also minced beef (99.5%; 99.4%) and (41.1%; 28%). GC-MS determination performed, oxalic acid, phenol, phenylethyl alcohol, N-hexadecanoic acid, and pyrolol[1,2-a]pyrazine-1,4-dione, hexadro-3-92-methylpropyl was the most active compound in P. fluorescens JB3B. 2,4-Di-tert-butylphenol, phenyl acetic acid, N-Hexadecanoic acid, pyrolol[1,2-a]pyrazine-1,4-dione, hexadro-3-92-methylpropyl, and Bis(2-ethylhexyl) phthalate was most active compound in the S. thermocarboxydus 18PM isolates.

Conclusions

The combination of isolated bacteriophages and bacterial metabolite showed promising results to be used as biocontrol candidate to overcome biofilm formed by foodborne and food spoilage bacteria using their ability to produce antibiofilm compounds and lytic activity. In addition, this combination also potentially reduces the use or replace the drawbacks of common application such as antibiotic treatment.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-024-03557-1.

Background

Ensuring a safe and appropriate food supply is a cornerstone of human health and food security. However, foodborne disease and food spoilage are still not under control and become major threats of these issues worldwide. Foodborne disease is any illness that results from the consumption of contaminated foods [1]. Food spoilage is also broadly described as any change in appearance, flavor, smell, and nutritional value that would impact the food product and unacceptable for human consumption. As results, a significant portion of food is wasted annually on a global scale [2].

Pathogenic bacteria are the most common cause of foodborne-disease and food spoilage that exist in a variety of shapes, types, and abilities to survive such as sporulating and releasing the hazardous toxin. ETEC B. cereus (BC) are top common members of pathogenic bacteria that associated with food poisoning and food loss. ETEC is the major enteric pathogen that account for the tens of millions of diarrheal diseases each year in all ages. Also, ETEC is the key etiology for traveler’s diarrhea that affects travelers were diagnosed with gastrointestinal disturbance in some regions [3]. B. cereus related food poisoning and spoilage is not a notifiable disease in most countries. However, fatal incidences have been reported in several years and the outbreaks were most often attributed to farinaceous food as a human daily consumption such as rice dishes, pasta, noodle, and pastry. Inappropriate food handling and storage are most probably the cause of the outbreak and followed by massive food loss [4].

Most microorganisms including pathogenic bacteria also form biofilms as part of their survival mechanisms and have been recognized to be involved in many clinical and chronic infections. Biofilms are cluster of bacteria that are attached to a surface and embedded self-produced matrix called as extracellular polymeric substances (EPS). The biofilm matrix comprises of various substances like proteins, polysaccharide, and eDNA. In human body, the protection that offered by the matrix can employ several survival strategies to evade the host defense mechanism and accelerate collateral tissue damage [5]. Consequently, biofilm-related infections are rarely resolved by the immune respond and inconsistently to antimicrobial treatments such as antibiotics.

Food preservation is an adequate option to prolong the food shelf-life and elevate the food safety for consumers. Food preservation methods commonly can be classified into chemical preservation by using several chemical reaction and physical preservation such as heating, freezing, and radiating. However, the excessive usage of this method brings some disadvantages for human health and food quality such as significant loss of nutrients, numerous human side-effects, and develop bacterial resistance. Thus, the effective method is needed to supplant the previous method as an alternative biocontrol of food supplies [6].

Bacteriophage, a selective viral predator that solely infects and kills targeted bacteria due to their strong antimicrobial properties, is recently as the best option as a biocontrol agent. Natural abundance, high specificity, harmless in human, and self-replication are attractive ability that generally found in lytic phage. Naturally, lytic bacteriophage will act quickly to exterminate foodborne pathogenic and food spoilage bacteria existence in food surfaces. Also, the ability to survive in various condition will be promising factor that encourage researchers to use as an alternative biocontrol [7]. Additionally, bacteriophage also known can be worked synergistically with other components such as bacterial metabolite to increase the possibility rate of biofilm eradication with several mechanisms.

Microbial antibiofilm is one of bacterial metabolite that used to eradicate microbial biofilm formation. Anti-adhesive, anti-quorum sensing activity, and disaggregation of EPS matrix, are several mechanisms that effectively used to rid the survival tyranny of pathogenic biofilm formation on a surface [8]. Phyllosphere and Actinomycetes are a group of bacteria that are well-known to produce a wide range of secondary metabolites from their complex biochemical process such as antibiofilm. This statement is supported with recent study, it reported that Streptomyces sp. and Arthrobacter sp. crude extracts have antibiofilm activity against Streptococcus pneumoniae and Vibrio parahaemolyticus [9]. Another study showed that, phyllosphere bacteria are able to eradicate food spoilage bacteria such as Bacillus cereus and Bacillus subtilis that indicated crude extracts of phyllosphere performed bioactive compound like acidic and phenolic compound [10].

The objectives of this research are to screen the bacterial metabolites of Phyllosphere and Actinomycetes bacteria, investigate the efficacy of bacteriophage, and to further explore their potential in a combination.

Results

Bacteriophage titer determination

From this research, phage titer of both phages was determined by using double overlay agar assay (Table 1). The titer value of phage BC-VP was 1.28 ± 0.29 × 10¹¹ and for ETEC-phage-TG was 8.9 ± 2.19 × 10⁸.

Table 1.

BC-VP and ETEC-phage-TG bacteriophage titer enumeration

Phage	Host cell	Morphology of plaque	Diameter of plaque (mm)	Titer PFU/ml
BC - VP	Bacillus cereus	Circle and clear	± 1	1.28 ± 0.29 × 1011
ETEC -phage- TG	Enterotoxigenic E. coli	Circle and clear	± 1	8.9 ± 2.19 × 108

Data were shown in mean ± standard error value

Quantification of bacteriophage, antibiofilm, and their combination activity as biofilm treatment

Antibiofilm activity (Pseudomonas fluorescens JB3B and S. thermocarboxydus 18PM) and their combination with bacteriophage (BC-VP and ETEC-phage-TG) against biofilm formation of ETEC and BC was determined by 96 microplates well and quantified by using microplate reader to obtain the treated and untreated optical density value (OD 595 nm). P. fluorescens JB3B code stands for “Jambu Biji” that isolated from Psidium guajava leaves and S. thermocarboxydus 18PM code stands for “Pantai Mutiara” that isolated from marine sediment. Overall, all treatments (single and combination) are able to inhibit and destruct biofilm formation (range 40–90% ability). Detail result can be seen in Fig. 1.

Fig. 1.

Quantification results of bacteriophage (BC-VP), antibiofilm, and their combination activity to eradicate biofilm formation of Bacillus cereus (BC). (A) Percent destruction of B. cereus (BC) biofilm; (B) Percent inhibition of B. cereus (BC) biofilm

ETEC-phage-TG combined with antibiofilm also showed the same ability and similar percentage against biofilm formation of ETEC. The result can be seen in Fig. 2 below.

Fig. 2.

Quantification results of bacteriophage (ETEC-phage-TG), antibiofilm, and their combination activity to eradicate biofilm formation of Enterotoxigenic E. coli (ETEC). (A) Percent destruction of Enterotoxigenic E. coli (ETEC); (B) Percent inhibition of Enterotoxigenic E. coli (ETEC) biofilm

Application of bacteriophage and bacterial metabolite in various food

The potential ability of bacteriophage and bacterial metabolite combination against foodborne and food spoilage bacteria was proven by application in various food that commonly observed as a sufficient habitat of E. coli and B. cereus. The results in Table 2 showed that, combination of BC-VP and bacterial metabolite P. fluorescens JB3B or S. thermocarboxydus 18PM was able to reduce bacterial population in mashed potato, boiled pasta, and soymilk with two different temperatures incubation. In cold temperature (4 °C), percentage of reduction reach approximately up to 90% bacterial population. Likewise, in room temperature (28 °C), reduction ability was decreased slightly (up to 80%). The log reduction is used as additional information to determine the relative number of living bacteria that are eliminated by the treatment (1log reduction corresponds to inactivating 90% of bacteria target).

Table 2.

Application of bacteriophage and bacterial metabolite to control B. cereus

Samples	Incubation (°C)	Treatment (CFU/ml)		Reduction (%)		Log Reduction (log10)
		BC + Phage + JB3B	BC + Phage + 18PM
Mashed Potato	4	6.8 × 104*	11.4 × 104*	99.6	99.4	2.49	2.26
	28	6.6 × 106*	9.2 × 106*	68.9	56.6	0.5	0.36
Boiled Pasta	4	19.2 × 104*	22 × 104*	99.5	99.4	2.34	2.28
	28	6.4 × 106*	10.2 × 106*	84.7	75.7	0.81	0.61
Soymilk	4	40.2 × 104*	42.8 × 104*	96.9	96.7	1.51	1.48
	28	7.6 × 106*	8 × 106*	42.4	39.4	0.24	0.21

Control Positive: 21.2 × 10⁶ (Mashed Potato); 42 × 10⁶ (boiled pasta); 13.2 × 10⁶ (soymilk)

“*”: Significant difference between the control and the treatment with P ≤ 0.05

In addition, the similar result was obtained in the combination of ETEC-phage-TG with bacterial metabolite (Table 3). In cold temperature, percentage of population reduction were extremely high in bean sprouts, chicken meat, and beef (up to 90%). On the other hand, percentage of reduction was up to 60% in room temperature.

Table 3.

Application of phage and bacterial metabolite to control Enterotoxigenic E. Coli

Samples	Incubation (°C)	Treatment (CFU/ml)		Reduction (%)		Log Reduction (log10)
		ETEC + Phage + JB3B	ETEC + Phage + 18PM
Bean Sprouts	4	TFTC	TFTC	TFTC		TFTC*
	28	6.2 × 106*	6 × 106*	47.5	49.1	0.27	0.25
Chicken Meat	4	TFTC	TFTC	TFTC		TFTC
	28	6.2 × 106*	6.8 × 106*	58.1	54	0.37	0.33
Beef	4	9.8 × 104*	11.4 × 104*	99.5	99.4	2.33	2.27
	28	12.6 × 106*	15.4 × 106*	41.1	28	0.23	0.14

Control Positive: 11.8 × 10⁶ (Bean sprouts); 14.8 × 10⁶ (Chicken meat); 21.4 × 10⁶ (Beef)

TFTC: Too Few Too Count

“*”: Significant difference between the control and the treatment with P ≤ 0.05

Gas chromatography – mass spectrometry analysis of bacterial metabolite

GC-MS analysis showed that P. fluorescens JB3B and S. thermocarboxydus 18PM contained lots of constituents. A total of 116 and up to 150 compounds of S. thermocarboxydus 18PM and P. fluorescens JB3B were analyzed using GC-MS. However, all constituents must be chosen selectively according to its role related as antibiofilm agent. The following major compounds of P. fluorescens JB3B and S. thermocarboxydus 18PM bacteria were explained in Tables 4 and 5 respectively as representative data. Percent area of the target component is used to determine changes in concentration of a known sample mixture. In addition, the observation of bioactive compounds of P. fluorescens JB3B and S. thermocarboxydus 18PM (Tables 4 and 5) was screened from several factors such as the area, similarity index, and probability that have shown in a whole data result. Noteworthy was the observation of the compounds function that related of this research as biofilm destructive agents also must be considered.

Table 4.

Pseudomonas fluorescens JB3B GC-MS result

Name of Compound	Similarity Index (SI)	Area %	Molecular Formula	Biological Properties
Oxalic Acid	94	1.01	C2H2O4	Antibacterial, antifungal, anti-adhesive, destroy biofilm
Phenol	60	5.76	C6H6O	Antibacterial activites (damage membrane bacterial) and anti-quorum sensing
Phenylethyl Alcohol	46	8.11	C8H10O	Antimicrobial, anti-septic, and anti-quorum sensing
N-Hexadecanoic Acid	93	0.83	C16H32O2	Antimicrobial, antihypoxic activity and destroy biofilm
Pyrolol[1,2-a]pyrazine-1,4-dione, hexadro-3-92-methylpropyl)	71	0.99	C11H18N2O2	Algicidal cctivity and anti-quorum sensing

Table 5.

Streptomyces thermocarboxydus 18PM GC-MS results

Name of Compound	Similarity index	Area %	Molecular Formula	Biological Properties
2,4-Di-tert-butylphenol	97	1.99	C14H22O	Antibacterial, Antifungal and anticancer Activity
Phenyl acetic acid	60	1.99	C6H6O	Antibacterial activites (damage membrane bacterial and microbial enzyme inhibition)
N-Hexadecanoic Acid	99	7.39	C16H32O2	Antimicrobial, antihypoxic activity and destroy biofilm
Pyrolol[1,2-a]pyrazine-1,4-dione, hexadro-3-92-methylpropyl)	50	0.30	C11H18N2O2	Algicidal activity and anti-quorum sensing
Bis(2-ethylhexyl) phthalate	99	1.89	C24H38O4	Antimicrobial and cytotoxic activity

Oxalic acid (SI 94; 1.01% Area), phenol (SI 60; 5.75% Area), phenylethyl alcohol (SI 46; 8.11% Area), N- Hexadecanoic Acid (SI 93; 0.83% Area), and Pyrolol[1,2-a]pyrazine-1,4-dione, hexadro-3-92-methylpropyl) (SI 74; 0.99% Area) were the major compound in P. fluorescens JB3B.

2,4-Di-tert-butylphenol (SI 97; 1.99% Area), Phenyl acetic acid (SI 60; 1.99% Area), N-Hexadecanoic Acid (SI 99; 7.39% Area), Pyrolol[1,2-a] pyrazine-1,4-dione, hexadro-3-92-methylpropyl) (SI 50; 0.30% Area), Bis(2-ethylhexyl) phthalate (SI 99; 1.89% Area) were the major compound of S. thermocarboxydus 18PM.

Discussion

Double overlay agar results showed that titer of each bacteriophage was 1.28 ± 0.29 × 10¹¹ (BC-VP) and 8.9 ± 2.19 × 10⁸ (ETEC-phage-TG) which indicates high concentration bacteriophage (minimum titer 10⁶ PFU/ml is required). The high titer value is able to increase the reliability interaction between phage and their host in isolated media. The high number and clear plaques on agar plate also assumed that the phages are early categorized as lytic phages that has ability to eradicate pathogenic bacteria BC and ETEC effectively [11].

Biofilm treatment was successfully quantified by using microplate reader. All treatments can be assumed has ability to inhibit and destroy biofilm formation. In inhibition activity, either BC-VP or ETEC-phage-TG has high activity to inhibit biofilm formation of both pathogenic bacteria (up to 80%). In addition, antibiofilm extract of P. fluorescens JB3B and S. thermocarboxydus 18PM also effectively inhibit biofilm formation for up to 90% as single treatment. The similar results have obtained for combination treatment between phage and antibiofilm. This phenomenon can be explained while treatment and bacteria target were mixed at the same time in inhibition assay. Thus, either single and combination treatment are be able to reach bacteria target easily due to biofilm formation were not built perfectly. In destruction assay, the different results showed between single and combination treatment. The different result (single and combination) and this reduction ability was suggested due to some chemical compounds in crude extracts unable to build symbiotic mechanism with bacteriophage. In other words, several chemical compounds do affect phage adversely such as inhibit their motility to reach bacterial target [12]. On the other hand, not all bacterial metabolite were rapidly acting. The bacteriophage existence might be act as blockade of crude extract to work and potentially diluted away due to the difference of concentration.

Application in various food that commonly observed as a sufficient habitat of E. coli and B. cereus was used to prove bacteriophage and bacterial metabolite action. The result showed that (Table 2), combination of BC-VP and bacterial metabolite P. fluorescens JB3B or S. thermocarboxydus 18PM was able to reduce bacterial population in all food samples with two different temperatures incubation. Approximately up to 90% at 4 °C and up to 80% at 28 °C. In addition (Table 3), the similar result was obtained in the combination of ETEC-phage-TG and bacterial metabolite (P. fluorescens JB3B and S. thermocarboxydus 18PM). In cold temperature, percentage of reduction were high in all food samples (up to 90%). Meanwhile, in room temperature the reduction approximately up to 60%. From this result, it could be assumed that in cold temperature, bacterial population were unable to growth optimally and phage were kept in low temperature potentially had a stable titer concentration compared to room temperature condition. On the other hand, the capability of bacteriophage was possibly reduced against bacterial population due to the variation of food matrices. According to previous study, food matrices was evidence that might be able to trap and disturb bacteriophage activity. In other words, the complexity of food matrices would be easily inhibited bacteriophage movement due to Brownian motion was limited as the key feature of bacteriophage to move actively to reach bacterial target. Likewise, some of chemical compound in crude extract possibly work antagonistically with bacteriophage itself or compete with bacterial active compound that released by pathogenic bacterial in optimum temperature [13].

GC-MS analysis showed that P. fluorescens JB3B and S. thermocarboxydus 18PM contained lots of constituents. Oxalic acid was the first chemical compound that found in P. fluorescens JB3B extract. This constituent also known as ethane dioic acid, is an organic compound that has more acidic strength rather than acetic acid and it is effectively to eradicate harmful microbes such as Bacillus, Salmonella, and Vibrio genera [14]. The second chemical compound was phenol. This constituent was the most popular compound as an antimicrobe. The simpler natural phenol will incorporate as a complex phenolic compound such as polyphenol which were potentially found in phyllosphere microbiome to compete with other foreign bacterial to avoid biofilm formation. According to literature, Pyrolol[1,2-a] pyrazine-1,4-dione, hexadro-3-92-methylpropyl) demonstrated significant anti quorum sensing against P. aeruginosa by preventing biofilm formation, bacterial adherence, and biofilm development. N-hexadecanoic acid was the most abundant chemical compound that found in P. fluorescens JB3B and S. thermocarboxydus 18PM. According to literature [15]. N-hexadecanoic acid was the primary metabolite that produce during microbial degradation and has several biological activities. However, no literature has thus far published regarding antibiofilm activity of N-hexadecanoic acid and the rest of constituents such as Phenyl ethyl alcohol in P. fluorescens JB3B and 2,4-Di-tert-butylphenol, phenyl acetic acid, and Bis(2-ethylhexyl) phthalate in S. thermocarboxydus 18PM that had been found specifically.

Overall, the action mechanism of bacterial metabolite against biofilm formation is totally difference rather than other existing antibacterial and can be interrupted in several ways: by inhibiting surface adhesion (anti-adhesive), interfering quorum sensing activity, and disaggregating exopolysaccharide (EPS) matrix. Of note, EPS is the main culprit of biofilm formation due to the sticky protective nature that difficult to remove and 100 to 1000 times more resistant to existing antibacterial such as antibiotic. Briefly, adhesion is a prerequisite for bacteria to colonize and form biofilm on fresh produce. In addition, the bacteria are embedded precisely on a rough and hydrophobic surface. In this case, a hydrophilic bacterial metabolite such as oxalic acid can be applied as a chemical strategy to coat the surface as a physical barrier and reduce the adhesion force of bacteria [16]. Phenolic compound also known for their antimicrobial effects as a consequence of bacterial damage. Furthermore, phytochemical, such as phenol may compete with acyl homoserine lactone (AHL) and autoinducer peptide (AIP), a signaling molecule for binding to quorum sensing (QS) receptor. The microbial communication system is used by a wide variety of bacteria to build biofilm formation. However, the present of bacterial metabolite like phenol, being capable of interfering and dispersing established biofilm [17]. These mechanisms can be determined as difference major between bacterial metabolites as antibiofilm with other existing antibacterial.

Limitation

Application assay of this research was still limited. It needs to be assayed with more various types of food matrix to get more accurate results. In addition, other screening method is possible to apply as an option to collect other bacterial metabolites to be more informative results.

Conclusion

Combination of bacteriophage (BC-VP and ETEC-phage-TG) with bacterial metabolite (P. fluorescens JB3B and S. thermocarboxydus 18PM) was able to work synergistically to inhibit and destroy biofilm formation with range 40% until 90% ability. In addition, this ability was able and highly effective to work similarly on various food against BC and ETEC at two different temperatures (cold and room temperatures). For further research, it is required to do molecular characterization of bacteriophage BC-VP and ETEC-phage-TG to determine bacteriophage annotation, purify the bioactive compounds and characterize each compound in the crude extracts. Also, to test the crude extracts against several indicator bacteria to determine the wide mechanism of biofilm inhibition and destruction. Phage cocktail or difference concentration possibly applied to see potential efficacy on various food that might be higher than previous study also important for application in food safety.

Materials and methods

Bacterial cultivation

Food spoilage bacteria Bacillus cereus ATCC 10,876 (BC) and foodborne pathogenic bacteria Enterotoxigenic E. coli Nmr 1 (ETEC) stock were used in this study. Both isolates were sterilized in Luria Bertani (LB) agar (5 g tryptone; 2.5 g NaCl; 10 g bacteriological agar in 500 ml distilled water). Both bacteria were streaked onto new LB agar for refreshment and each plate were incubated at 37 °C overnight [18].

P. fluorescens bacteria JB3B [10, 19] and S. thermocarboxydus bacteria 18PM [20, 21] stock was cultivated in LB agar and Yeast Malt Extract (YME) agar (0.5 g yeast extract, 5 g malt extract in 500 ml distilled water) respectively. Both bacteria-producing antibiofilm were streaked onto new LB and YME agar for refreshment. Each plate was incubated at 28 °C for 48 h [22]. P. fluorescens JB3B and S. thermocarboxydus 18PM were obtained from previous study. P. fluorescens JB3B were isolated from Psidium guajava leaves [19]. S. thermocarboxydus 18PM were isolated from Pantai Mutiara region [20]. The previous study stated that a soil dilution technique using Strach Casein Agar (SCA) was performed for the isolation of Actinomycetes. Pre-treatment by heated at 50 °C for 1 h was done for all the sediment samples. Different solution of the marine, river, and paddy field sediment suspension were shaken under room temperature at 200 rpm for 1 min, then 100 µl of each suspension were spread onto SCA medium and incubated at 28 °C for 7 days. Then, isolates were grown onto YME agar for 7 days [20].

Bacteriophage enrichment and refreshment

BC and ETEC bacteria were cultivated in LB broth and incubated at 37 °C overnight. Optical density 600 = 0.132 (equal to10⁸ CFU/ml) were counted by using spectrophotometer. 150 µl of bacteria, 150 µl of bacteriophage lysate, 50 µl of 10mM CaCl_2, and 50 µl of 10mM MgSO₄ were mixed into LB broth and the mixture was incubated at 37 °C overnight at 120 rpm. After incubation, the mixture was centrifuged at 6000 x g for 10 min. Supernatant was collected by disposable syringe and filtered using 0.22 μm pore size into new conical tube. The volume of phage lysate was adjusted with 1/4 - strength sterilized Ringer Solution with ratio 1:1 (v/v). Phage lysate was stored in chiller at 4 °C [23].

Bacteriophage titer determination

In the previous study, both bacteriophages were successfully purified from lake water in South Tangerang, Indonesia, which were BC-VP phage isolated using B. cereus as a host and ETEC-phage-TG isolated using ETEC as a host [24, 25].

Phage lysate of BC-VP and ETEC-phage-TG was diluted with sterilized SM buffer pH 7.5 (5.85 g NaCl; 6.057 g Tris; 2.47 g; MgSO₄ in 1000 ml distilled water) by a series tenfold dilution. Double overlay agar assay was performed to determine bacteriophage titer. 100 µl of bacterial culture with OD600 = 0.132 nm (equal to10⁸ CFU/ml), 100 µl of phage lysate, 50 µl of 10mM CaCl_2, and 50 µl of 10mM MgSO₄ were mixed into 0.7% molten LB agar. Then, the mixture was poured gently onto 2% LB agar until it solidified. Petri dishes with different serial dilution were incubated at 37 °C overnight and the visible plaques (range 30–300) were counted following the general plating method for counting viable bacteria [22]. The phage titer was determined in plaques forming unit per mililiter (PFU/ml) [26].

Production of bacterial metabolite

P. fluorescens JB3B [27] and S. thermocarboxydus 18PM [28] were inoculated from previous studies which were known have antibiofilm activities were inoculated into 250 ml of LB broth and YME broth respectively. Both bacteria were incubated at 28 °C for 48 h by incubator shaker. The bacteria were transferred into 50 ml of sterile conical tube and centrifuged at 6000 x g for 15 min. The supernatant was harvested and mixed with ethyl acetate as a solvent at 1:1 (v/v) ratio, then re-incubated by incubator shaker at 28 °C overnight. After incubation period, the solvent layer was collected and evaporated by using rotary evaporator. Both extracts were dried by using a vacuum oven at 50 °C overnight or in a few days until solvent completely evaporated. DMSO 1% were added to reach 20 mg/ml final concentration of crude extract [29].

Biofilm treatment using bacteriophage

ETEC and BC were inoculated in LB broth at 37 °C overnight. Both bacteria (OD600 = 0.132 nm) were obtained to perform this assay. For inhibition assay, 100 µl of each bacterial culture was added into 96-well microtiter plate and combined with 100 µl of phage lysate directly (miMOI 0.001 for ETEC and 0.01 for BC). The mixture was incubated at 37 °C overnight. Meanwhile, for destruction assay, 100 µl of each bacterial culture was added into 96-well microtiter plate and incubated at 37 °C overnight. Then, 100 µl of phage lysate were mixed into each well of bacterial culture, and re-incubated at 37 °C overnight. After that, all liquid media were discarded and rinsed by distilled water for 2 times. Cells in 96 well microtiter plate were air dried before staining. 100 µl of 0.4% crystal violet solution were added into each well and let it stained for 30 min. The dye was removed and rinsed wells by distilled water for 5 times. Wells were air dried and diluted by using 200 µl of ethanol as a final step [29].

Biofilm treatment using bacterial metabolite

For antibiofilm treatment, 100 µl of each bacterial culture (OD600 = 0.132 nm) was added into 96-well microtiter plate and combined with 100 µl of crude extract of P. fluorescens JB3B and S. thermocarboxydus 18PM for each well. The mixture was incubated at 37 °C overnight (inhibition assay). Meanwhile, for destruction assay, 100 µl of each bacterial culture was added into 96 well microtiter plate and incubated at 37 °C overnight. Then, 100 µl of crude extracts were mixed into each well of bacterial culture, and re-incubated at 37 °C overnight. The staining method of antibiofilm was performed similarly refers to bacteriophage treatment [29].

Biofilm treatment using combination of bacteriophage and bacterial metabolite

For inhibition assay, 100 µl of each bacterial culture (OD600 = 0.132 nm) was added into 96 well microtiter plate and combined with 100 µl of crude extract for each well. Then, 100 µl of phage lysate (miMOI 0.001 for ETEC and 0.01 for BC) was added for each well at the same time. The mixture was incubated at 37 °C overnight. Meanwhile for destruction assay, 100 µl of each bacterial culture was added into 96-well microtiter plate and incubated at 37 °C overnight. Then, 100 µl of crude extract and 100 µl of phage lysate were added into each well, and reincubated at 37 °C for 24 h then continued for staining by crystal violet and eluting by ethanol.

After staining and eluting procedure, the optical density of 96 well microtiter plate was determined by using microplate reader at wavelength 595 nm, and percentage of inhibition and destruction assay was calculated by the following formula,

Application of phage and bacterial metabolite to control B. cereus

As much as 1 g of mashed potato, 1 g of boiled pasta, and 1 ml of soymilk (Choi and Kim 2020) were added into 4 different conical tubes and sterilized by using an autoclave. The first tube was used as positive control (BC only), the rest of the tube were used for single, combination treatment, and control negative respectively. Each treatment tube was doubled for incubation at two different temperatures (4 °C and 28 °C) overnight. The combination ratio of phage and crude extract was 1:1 (v/v) [30]. The ratio was selected to determine the effects of combination in antibiofilm assay as the minimum ratio.

Application of phage and bacterial metabolite to control Enterotoxigenic E. coli

Samples of 1 g of chicken meat (1 × 1 cm), 1 g of beef (1 × 1 cm) (Vikram et al. 2021), were sterilized by using an autoclave. Meanwhile, 1 g of mung bean sprouts were rinsed by distilled water and sterilized by ethanol 96% and UV light for 30 min. Each sample were added into 4 different conical tubes. The first tube was used as positive control (ETEC only), the rest of the tube were used for for single, combination treatment, and control negative. Each treatment tube was doubled for incubation at two different temperatures (4 °C and 28 °C) overnight. After incubation, each tube was diluted by 9 ml of SM buffer and vortexed for 2 min. 50 µl of mixture were spreaded onto LB agar and incubated at 37 °C. Then, the colony reduction was counted and compared with control [31]. The combination ratio of phage and crude extract was 1:1 (v/v) [30]. The ratio was selected to determine the effects of combination in antibiofilm assay as the minimum ratio.

Identification of bacterial metabolite using gas chromatography-mass spectrometry

The fraction of crude extract was identified by GC-MS method at BRIN Serpong. The crude extract of P. fluorescens JB3B and S. thermocarboxydus 18PM was dissolved by ethyl acetate in ratio 1:1 (w/v). 1 µl of crude extract was injected through the column type Agilent and Helium gas was used as carrier. The peak of chromatogram was obtained as a result and GC-MC Pyrolysis database was used as a reference [21]. GC-MS is considered to use due to the higher sensitivity, faster results, and more affordable method. Likewise, in the previous study, bacterial metabolites related to antibiofilm can be detected by GC-MS method.

Statistical analysis

Data are presented as the means and standard error. For statistical analysis, two-way ANOVA (Microsoft Excel office 16) with triplicate replication and level of difference defined at P ≤ 0.05 to indicate the significant differences from other samples in that column [32].

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

GC-MS

Gas Chromatography-Mass Spectrometry

miMOI

Minimum Multiplicity of Infection

Author contributions

D.R: Conducted the research and data analysis, prepared proposal writing and manuscript under advisory of D.E.W. and A.Y. D.E.W. and A.Y: Research design, data analysis and person in charge of all the research project. All authors read and approved the final manuscript.

Funding

This study was funded by Republic of Indonesia Ministry of Education and Culture (Kemendikbud). The funder has no contribution in design, collection, writing, and interpreting data in this study.

Data availability

The 16S rRNA gene sequences of the isolates have been submitted to the NCBI genebank database (GenBank Overview (nih.gov). Pseudomonas fluorescens strain UTB_111 JB3B OM763955 (Pseudomonas fluorescens strain UTB_111 16S ribosomal RNA gene, partial - Nucleotide - NCBI (nih.gov). Streptomyces thermocarboxydus strain NBRC 16323 18PM MW680906 (Streptomyces thermocarboxydus strain NBRC 16323 16S ribosomal RNA gene - Nucleotide - NCBI (nih.gov).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.