30 Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria from Flowers in the University of the Philippines Diliman, Quezon City Gil M. Penuliar* Renz Joseph R. Artezuela Institute of Biology University of the Philippines Diliman ABSTRACT Fructophilic lactic acid bacteria (FLAB) is a recently discovered group of lactic acid bacteria that prefers fructose as carbohydrate source. The isolation of FLAB from fructose-rich niches like flowers, in particular, and the gut of insect pollinators suggests that it may be used as probiotics. The objective of this study was to determine if FLAB can be isolated from flowers at the Institute of Biology, University of the Philippines Diliman, and to screen them for antimicrobial activity against bacteria that are commonly associated with intestinal diseases. A total of 20 different isolates were obtained from 14 species of flowers. All isolates were identified as LAB, but only 17 out of 18 isolates were osmotolerant in 30% fructose, and only 8 out of 15 isolates had higher absorbance in Fructose Yeast Peptone Broth, which are characteristics of presumptive FLAB. Seven isolates exhibited inhibitory activity in at least three test bacteria in the primary screening and only four isolates had inhibitory activity in at least two test bacteria, particularly against Enterococcus faecalis and Campylobacter jejuni, in the secondary screening. DNA sequencing and phylogenetic analysis identified isolates MFPS 4.1 and MFRU 7.2 as Weissella spp. The in vitro antimicrobial activities of these isolates can be studied further for possible applications in food and medicine, and their low sequence similarities suggest that the isolates might be novel Weissella species. Keywords: FLAB, antimicrobial activity, Weissella spp. * Corresponding Author Science Diliman (July-December 2021) 33:2, 30-52 G. M. Penuliar and R.J. R. Artezuela 31 INTRODUCTION Fructophilic lactic acid bacteria (FLAB) is a unique group of lactic acid bacteria that preferentially utilizes fructose as their main carbohydrate source under anaerobic conditions. In addition, key characteristics of FLAB include a need for electron acceptors, such as pyruvate and oxygen, for effective glucose utilization, and poor carbohydrate fermentation capabilities (Endo et al. 2009). Studies have shown that FLAB can be isolated from fructose-rich environments such as flowers, fruits, fermented products made from fruits, and animal by-products such as honey (Endo and Okada 2008; Endo et al. 2011; Syed Yaacob et al. 2018). Some species of FLAB, like Lactobacillus apinorum, Lactobacillus kunkeei, and Fructobacillus fructosus, have been isolated from the guts of honeybees and are reported to exhibit antibacterial activity against bacteria that cause diseases in honeybees (Endo and Salminen 2013; Olofsson et al. 2014; Maeno et al. 2017). Development on possible probiotic application of FLAB has advanced in recent years with the discovery of kunkecin A, a bacteriocin produced by Apilactobacillus kunkeei FF30-6 isolated from honeybees, which had effectively inhibited Melissococcus plutonius, which causes European foulbrood in honeybee larvae (Zendo et al. 2020). It is possible that other members of FLAB isolated from other sources with observed antimicrobial activity can be used as probiotics for other animals. Probiotics are live microorganisms that confer health benefits when consumed in adequate amounts, such as restoration of normal gut microflora, prevention of colonization of pathogenic bacteria, prevention of viral infections, and promotion of oral health care by preventing or treating oral diseases (Reid 2002; Kang et al. 2019). The mechanism of action of probiotics includes the production of anti-adhering molecules to prevent the attachment of pathogenic bacteria to the epithelial lining of the gut; alteration of gut pH through the production of lactic acid and acetic acid; and modulation of the host’s immune response by enhancing the expression of genes involved in the integrity of the intestinal barrier (Bermudez-Brito et al. 2012; Collins et al. 2019). Currently, there are no published studies related to FLAB in the Philippines. Fructobacillus tropaeoli, the most recent species of the genus Fructobacillus, was isolated from the Nasturtium flower, Tropaeolum majus (Endo et al. 2011). Recent studies had also shown that other members of FLAB such as L. kunkeei and F. durionis were isolated from various fruit and flowers (Ruiz Rodriguez et al. 2019; Sakandar et al. 2019). It would be interesting to study the FLAB population present in the diverse flora found in the Philippines, especially the endemic floral species. Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria 32 The objectives of this study were (1) to isolate presumptive FLAB from flowers in the Institute of Biology, University of the Philippines Diliman, (2) to characterize and screen the isolates for in vitro antimicrobial activity, and (3) to identify the isolates through DNA sequencing. The study aims to contribute information on FLAB by detecting their presence in local flowers and by screening for isolates for antimicrobial activity against selected bacteria associated with intestinal diseases. MATERIALS AND METHODS Sample collection Fourteen flower species were aseptically collected from the Institute of Biology, University of the Philippines Diliman from 30 January 2019 to 8 February 2019. The samples were identified up to the genus and species level by the Jose Vera Santos Memorial Herbarium and were placed in sterile plastic bags before briefly storing in a refrigerator at 4°C prior to processing. Samples were dissected inside a biosafety cabinet, using sterile scalpel and forceps, to collect 1 g of floral nectaries (stamen and pistil), which were macerated inside a sterile 50 mL conical tube. Isolation and purification of isolates Enrichment, isolation, and purification methods were adapted and modified based on the study of Endo et al. (2009). Samples were enriched separately in 10 mL of Fructose Yeast Peptone Broth (FYPB) (HiMedia Laboratories, Mumbai, India), de Man, Rogosa and Sharpe Broth (MRSB) (HiMedia Laboratories, Mumbai, India), and Sobremisana, Artezuela and Penuliar Broth (SAPB), supplemented with 0.02% sodium azide and 0.01% nystatin as antifungal agent (ACME Laboratories, Dhaka, Bangladesh), and were incubated at room temperature (28°C) for 24 h. A loopful of enrichment culture was streaked on Fructose Yeast Peptone Agar (FYPA), de Man, Rogosa and Sharpe Agar (MRSA), MRSA supplemented with 1% fructose, and Sobremisana, Artezuela and Penuliar Agar (SAPA) plates, each supplemented with 0.5% CaCO 3 . The plates were incubated at room temperature (30°C) for 24– 48 h, and isolated colonies with distinct zones of clearing were purified on their respective isolation media using the 3-quadrant streak method. Purification plates were incubated at room temperature (30°C) for 24–48 h. Gram-staining Gram-staining was performed based on a method adapted from the American Society for Microbiology (Smith and Hussey 2005). Briefly, one loopful of a 24-h old bacterial culture was smeared onto a glass slide with one drop of distilled water G. M. Penuliar and R.J. R. Artezuela 33 and allowed to air dry. The smear was heat fixed by passing the glass slide over a flame three times. The slide was flooded with crystal violet for 30 s, rinsed with distilled water, then flooded with Gram’s iodine for 1 min. The slide was then rinsed with 95% ethanol drop-wise until the runoffs were clear. The slide was flooded with safranin for 30 s and was rinsed with distilled water. The slide was then blotted dry and viewed using a microscope with 100х magnification. Staphylococcus aureus and Escherichia coli were used as positive and negative controls, respectively. Catalase test Approximately 3 mL of 3% hydrogen peroxide was dispensed in each slant of 24-h old cultures. The presence of effervescence indicated that the isolate was catalase- positive. S. aureus and Streptococcus mutans were used as positive and negative controls, respectively. Polymerase chain reaction confirmation using LAB-specific primers The DNA of the isolates were extracted using the G-spinTM Genomic DNA Extraction Kit (iNtRON Biotechnology), following the protocol for Gram-positive bacteria. A preliminary PCR amplification of the isolates was performed to confirm their identity as LAB. Each 10 μL reaction mixture contained 5 μL GoTaq® Master Mix (Promega, Wisconsin, USA) (with 50 units/mL of Taq DNA polymerase in a proprietary reaction buffer [pH 8.5], 400 μM each of dATP, dGTP, dCTP and dTTP, and 3 mM of MgCl 2 ), 2.4 μL nuclease-free water, 1 μL DNA template, and 0.8 μL each of forward (L15f, 5’ GCTCAGGAYGAACGCYGG 3’) and reverse (L687r, 5’ CACCGCTACACATGRADTTC 3’) primers (Hou et al. 2018). PCR was performed in a MyCyclerTM Thermal Cycler System (Bio-Rad, California, USA), with the following cycling conditions: initial denaturation at 95°C for 2 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min. The PCR products were electrophoresed in a 1% agarose gel with 0.5 μg/mL ethidium bromide at 100 V for 24 min. The amplicons were visualized using a UV transilluminator. Fructose osmotolerance test The method used to determine the fructose tolerance of the isolates was adapted and modified from the work of Endo (Endo et al. 2009). Approximately 1.5 x 108 CFU/mL of 24-h old cultures were prepared in 0.9% saline, and 500 μL of each suspension was inoculated into scintillation vials containing 5 mL of FYPB with 30% (w/v) fructose. The vials were incubated at 37°C for 24 h under aerobic conditions on an orbital shaker (120 rpm) and observed for growth. Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria 34 Growth comparison in FYP broth and GYP broth Growth of the isolates was compared using FYPB and Glucose, Yeast and Peptone Broth (GYPB) under microaerophilic conditions. GYPB has the same components as FYPB except fructose is replaced with glucose. Approximately 1.5 x 108 CFU/mL of 24-h old cultures were prepared in 0.9% saline and 500 μL of each suspension was inoculated into separate scintillation vials containing 5 mL of FYPB and GYPB. The vials were incubated at 37°C for 24 h under microaerophilic conditions. An aliquot of 200 μL of each culture was transferred into a 96-well plate, and the absorbance of the cultures was measured by spectrophotometry (BMG Labtech, Offenburg, Germany) using 600 nm wavelength in triplicate and using sterile media as blank. Isolates with higher absorbance readings in FYPB compared to GYPB were considered FLAB. Agar plug diffusion assay The agar plug diffusion assay was used as primary screening method for antimicrobial activity against the following test bacteria: Acinetobacter baumannii MML ESAB1701, Campylobacter jejuni ATCC 33560, Enterococcus faecalis BIOTECH 10348, E. coli BIOTECH 1634, Klebsiella pneumoniae BIOTECH 1754, Proteus mirabilis MML ESPM1701, Pseudomonas aeruginosa BIOTECH 1335, and Salmonella Typhimurium BIOTECH 1756. Approximately 1.5 x 108 CFU/mL of 24-h old cultures of the isolates were prepared in 0.9% saline and were lawned on their respective agar medium. The plates were incubated at 37 °C for 24–48 h until the lawns were confluent. Similarly, about 1.5 x 108 CFU/mL of 24-h old cultures of the test bacteria were prepared in 0.9% saline and were lawned on Mueller-Hinton Agar (MHA) plates, except for C. jejuni, which was prepared using MHA with 5% lysed horse blood. Agar plugs 4.5 mm in diameter were made from the FLAB lawns and were aseptically transferred onto lawns of the test bacteria. The plates were incubated at 37°C, and 42°C for C. jejuni, for 24 h. The assay was performed in duplicates. The diameters of zone of inhibition (ZOI) were measured using a caliper and were classified as weak (diameter ≤ 3 mm), moderate (diameter ≤ 6 mm) or strong inhibition (diameter > 6 mm) (Pan et al. 2009). Isolates that exhibited moderate to strong inhibition were selected for secondary screening. Agar well diffusion assay The agar well diffusion assay was used to screen the cell free supernatant (CFS) of the isolates for antimicrobial activity. Approximately 1.5 x 108 CFU/mL of 24-h old cultures were prepared in 0.9% saline and 1 mL of each suspension was inoculated G. M. Penuliar and R.J. R. Artezuela 35 in a test tube containing 10 mL of their respective broth medium and incubated at 37°C for 24 h. An aliquot of 1 mL from each culture was transferred into separate 1.5 mL microcentrifuge tubes and centrifuged at 12,000 rpm for 1 min. The supernatants were collected and transferred into new 1.5 mL microcentrifuge tubes. About 1.5 x 108 CFU/mL of 24-h old test bacteria cultures were prepared in 0.9% saline and lawned on MHA plates using cotton applicators. Agar wells with a diameter of 6 mm were made on the lawns of test bacteria, and 50 μL of CFS was added into the wells. The CFS was allowed to dry and the plates were incubated at 37°C, and 42°C for C. jejuni, for 24 h. The assay was performed in duplicates. The ZOI of the CFS were measured using a caliper and were classified as previously mentioned. Identification of isolates by DNA sequence analysis The DNA of isolates were extracted as previously described, and uniplex PCR amplification of the 16S rRNA gene was performed for DNA sequencing. Each 30 μL reaction mixture contained 15 μL GoTaq® Master Mix (Promega, Wisconsin, USA) (with 50 units/mL of Taq DNA polymerase in a proprietary reaction buffer [pH 8.5], 400 μM each of dATP, dGTP, dCTP and dTTP, and 3 mM of MgCl 2 ), 7.2 μL nuclease-free water, 3 μL DNA template, and 2.4 μL each of forward (27F, 5’ AGAGTTTGATCCTGGCTCAG 3’) and reverse (1492R, 5’ TACGGYTACCTTGTTACGACTT 3’) primers (Lane 1991). PCR was performed in a MyCyclerTM Thermal Cycler System (Bio-Rad, California, USA) with the following cycling conditions: initial denaturation at 95°C for 2 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 74°C for 1 min, and final extension at 74°C for 5 min. Agarose gel electrophoresis was performed as previously described. Purified PCR products were sent to Macrogen (10F World Meridian Center, 60-24 Gasan-dong, Geumcheon-gu, Seoul 153-781, South Korea) for DNA sequencing. The consensus sequences were determined from the forward and reverse sequences using the Bioedit Sequence Alignment Editor 7.2.6.1 (Hall 1999), and were used to identify the isolates using the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI) Nucleotide database. The sequences were then deposited in GenBank. Phylogenetic analysis A phylogenetic tree was constructed to determine the relationship of the isolates to type strains of FLAB and LAB in GenBank. Full formats of 16S rRNA gene sequences of 43 type strains were downloaded from the nucleotide database of NCBI, including Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria 36 the sequence of Lactococcus lactis, which was used as outgroup. Multiple alignments of the sequences were performed using MEGA X and CLUSTAL_W algorithm (Kumar et al. 2018). The Kimura two-parameter model with gamma distribution was used in calculating the distance matrices for the aligned sequences (Kimura 1980). The Neighbor-Joining (NJ) method (Saitou and Nei 1987) was used to construct the phylogenetic tree, and a bootstrap of 1000 replicates was used to estimate the robustness of each branch (Felsenstein 1985). Statistical analysis Significant differences in the growth of the isolates in FYPB and GYPB were determined using student’s t-test (α=0.05) in Microsoft® Excel® for Microsoft 365 MSO. RESULTS Isolation of presumptive FLAB Flowers from 14 plant species were collected in the Institute of Biology, University of the Philippines Diliman. The plants were identified up to the genus and species level with the assistance of the staff from the Jose Vera Santos Memorial Herbarium at the Institute of Biology. The plant species were as follows: Acalypha indica, Begonia sp., Calotropis gigantea, Clerodendrum intermedium, Clerodendrum quadriloculare, Cosmos sulphureus, Hibiscus rosa-sinensis, Impatiens sp., Plumbago sp., Pseuderanthemum sp., Rosa sp., Ruellia sp., Tabernaemontana pandacaqui, and Thunbergia grandiflora. Enrichment and plating of the samples in four isolation media produced colonies of different morphologies ranging from raised and convex elevations, entire and undulate margins, and smooth and glistening appearance. Presumptive FLAB isolates were isolated in all samples except in T. grandiflora. Twenty-eight colonies with distinct morphologies and clearing zones were selected for purification and characterization (Table 1). G. M. Penuliar and R.J. R. Artezuela 37 Table 1: Number of distinct colonies observed in each flower species Specimen Media FYPA MRSA MRSA + 1% fructose SAPA Acalypha indica 0 1 1 0 Begonia sp. 0 1 1 1 Calotropis gigantea 0 0 0 1 Clerodendrum intermedium 0 0 1 1 Clerodendrum quadriloculare 1 1 1 0 Cosmos sulphureus 1 1 1 0 Hibiscus rosa-sinensis 0 1 1 1 Impatiens sp. 1 0 0 0 Plumbago sp. 1 0 0 1 Pseuderanthemum sp. 1 1 2 0 Rosa sp. 0 0 0 0 Ruellia sp. 1 1 1 0 Tabernaemontana pandacaqui 0 1 0 0 Thunbergia grandiflora 0 0 0 0 Subtotal 6 8 9 5 Total 28 All 28 isolates were Gram-positive, but only 20 isolates were screened for catalase activity due to loss of viability in eight isolates. Eighteen isolates were catalase- negative, while two isolates exhibited catalase activity. All 20 isolates were confirmed as LAB using LAB-specific primers (Table 2, Figure 1). Seventeen isolates were fructose osmotolerant, while eight isolates (FACQ 9, FAIM 21, MAPS 4, MABE 25, MAFPS 4.1, MFRU 7.2, MAFAI 8, and MFHR 16) exhibited higher absorbance in FYPB compared to GYPB and were selected for in vitro antimicrobial activity screening. Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria 38 Table 2: Characterization of the isolates based on flower origin, catalase test, amplification of LAB-specific primers, fructose osmotolerance and growth in FYPB and GYPB Isolate Flower source Catalase test PCR with LAB-specific primers Fructose osmotolerance Absorbance (600 nm) FYPB GYPB p-value FACS 2 C. sulphureus Negative Positive Negative - - - FACQ 9 C. quadriloculare Negative Positive Positive 0.649 0.505 0.074 FAIM 21 Impatiens sp. Negative Positive Positive 0.310 0.237 0.007 FAPL 23 Plumbago sp. Negative Positive - - - - MACS 1 C. sulphureus Negative Positive Positive 0.530 0.767 0.303 MAPS 4 Pseuderanthenum sp. Negative Positive Positive 0.553 0.446 0.059 MARU 7.1 Ruellia sp. Negative Positive Positive - - - MAAI 8 A. indica Negative Positive Positive - - - MACQ 10 C. quadriloculare Negative Positive Positive 0.548 0.856 0.166 MATP 17 T. pandacaqui Negative Positive Positive 0.138 0.169 0.048 MABE 25 Begonia sp. Negative Positive Positive 0.354 0.286 0.355 MFCS 1 C. sulphureus Negative Positive Positive 0.693 1.123 0.163 MFPS 4.1 Pseuderanthenum sp. Negative Positive Positive 0.734 0.517 0.043 MFPS 4.2 Pseuderanthenum sp. Negative Positive Positive 0.830 0.857 0.471 MFRU 7.2 Ruellia sp. Negative Positive Positive 0.838 0.483 0.001 MFAI 8 A. indica Negative Positive Positive 0.494 0.356 0.008 MFHR 16 H. rosa-sinensis Negative Positive Positive 0.523 0.404 0.011 MFBE 25 Begonia sp. Positive Positive Positive 0.034 0.113 0.051 SABE 30 Begonia sp. Negative Positive Positive 0.018 0.020 0.378 SAHR 35 H. rosa-sinensis Positive Positive - - - - Figure 1: Representative polymerase chain reaction (PCR) results using LAB-specific primers through agarose gel electrophoresis (AGE). Expected amplicon size was around 750 base pairs. G. M. Penuliar and R.J. R. Artezuela 39 In vitro antimicrobial activity of the isolates Seven isolates exhibited medium to strong in vitro antimicrobial activity in at least three test bacteria in the primary screening (Figure 2 and Figure 3). The largest ZOI was observed in isolate MFPS 4.1 against E. coli (9.7 mm) and Salmonella (8.925 mm), although a few colonies were seen in the inhibition zone. The largest complete ZOI was observed in isolate MAPS 4 against C. jejuni (8.3 mm), and in isolate MFRU 7.2 against E. faecalis (7.4 mm). Inhibition of all test bacteria was observed in isolates MFPS 4.1, MFRU 7.2, MFAI 8, and MFHR 16. Figure 2: Representative result of growth inhibition of isolate MFPS 4.1 against test bacterium E. faecalis. Measurements were done using a digital caliper and expressed in millimeter. Agar plug size was measured at 4.5 mm. Figure 3: Results of the primary screening of the isolates against four test bacteria. The grey dotted line represents the 3 mm diameter zone of inhibition (ZOI) threshold for isolates with moderate inhibition and the black dotted line represents the 6 mm diameter threshold for isolates with strong inhibition. Isolates that exhibited partial inhibition are marked with an asterisk (*). Isolates that did not exhibit inhibition are not included. Error bars represent standard errors. Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria 40 Only four isolates had inhibitory activity in at least two test bacteria in the secondary screening (Figure 4). The largest inhibition was observed against C. jejuni by CFS from isolates MFPS 4.1 (6.7 mm) and MAPS 4 (6.2 mm), although the CFS from isolate MFRU 7.2 also gave moderate to strong inhibition against C. jejuni (4.4 mm) and E. faecalis (3.5 mm). Figure 4: Results of the secondary screening of the isolates against test bacteria. The grey dotted line represents the 3 mm diameter zone of inhibition (ZOI) threshold for isolates with moderate inhibition and the black dotted line represents the 6 mm diameter threshold for isolates with strong inhibition. Isolates that exhibited partial inhibition are marked with asterisk (*). Isolates that did not exhibit inhibition are not included. Error bars represent standard errors. Identification of the isolates Only two isolates were selected for molecular identification based on their in vitro antimicrobial activity. DNA sequencing and BLAST identified isolates MFPS 4.1 and MFRU 7.2 as Weissella cibaria, although the sequence similarity with the top hit was less than 97%, 95.6% for MFPS 4.1 and 92.2% for MFRU 7.2. The two isolates formed a sub-cluster with W. cibaria and W. confusa with 95% and 100% bootstrap support (Figure 5). G. M. Penuliar and R.J. R. Artezuela 41 Fi gu re 5 : P hy lo ge ne ti c po si ti on o f th e is ol at es M FP S 4. 1 an d M FR U 7 .2 . T he n ei gh bo r- jo in in g tr ee b as ed o n th e 16 S rR N A ge ne s of t he i so la te s an d ty pe st ra in s of F LA B a nd L A B c on fi rm s th e id en ti fi ca ti on o f th e is ol at es a s m em be rs o f th e ge nu s W ei ss el la . Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria 42 DISCUSSION The Institute of Biology, University of the Philippines Diliman was chosen as the sampling site for the flower collection as it is deemed a suitable starting point because it is one of the few remaining green spaces in Metro Manila and also because it houses several native and endemic flowering plant species (Cabel 2019; Rodriguez 2019). Flowers possess nectaries that can produce nectars that are composed of varying concentration of sugars, most commonly a combination of sucrose, glucose and fructose, making flowers a fructose-rich niche (Chalcoff et al. 2006). Fructose-rich niches are probable sources of FLAB since fructose is the preferred carbohydrate source of FLAB over glucose in the absence of external electron acceptors (Endo and Okada 2008). Among the four different culture media used, both MRSA and MRSA with 1% fructose medium had more isolates observed than both FYPA and SAPA medium (Table 1). A possible explanation might be that formulated media like MRSA would be a better growth media than media created by mixing separate components like FYPA and SAPA. A similar study yielded the same results wherein MRSA with fructose medium was able to isolate both LAB and FLAB (Ruiz Rodriguez et al. 2019) while FYPA was able to isolate strictly FLAB species (Endo et al. 2009). Isolation of FLAB from fructose-rich niches requires enrichment with a medium that contains 1% - 2% (w/v) fructose or, alternatively, conventional medium for LAB isolation, such as MRSA, with fructose supplementation (Endo and Salminen 2013; Olofsson et al. 2014; Endo 2019). Optimum temperatures for isolation range from 30–35°C, depending on the sample used, to simulate the environment where the sample was taken, while the optimum pH for growth is pH 6.5, which was achieved by placing the isolation setup at room temperature (30°C) during the enrichment step (Endo and Okada 2008; Endo et al. 2011; Syed Yaacob et al. 2018; Olofsson et al. 2014). The addition of sodium azide to the enrichment medium inhibits the growth of Gram-negative bacteria, while the addition of nystatin inhibits the growth of yeasts (Snyder and Herman 1940; Barney et al. 2003). Two out of 14 flower species have the greatest number of distinct isolates. These are C. quadriloculare and Pseuderanthemun sp., which have four distinct isolates each. Both flower species have deep corolla tubes. A study conducted in temperate forests in South America stated that flowers with deep corolla tubes have low viscosity nectars due to low evaporation rates compared to more open flowers (Chalcoff et al. 2006). This might explain the number of distinct isolates obtained from the two flower species and agrees with the absence of isolates from the G. M. Penuliar and R.J. R. Artezuela 43 T. grandiflora, which are relatively open flowers with short corolla tubes, although further studies are needed to establish the correlation. All 28 isolates were Gram-positive and 18 out of 20 isolates tested were catalase- negative, which are typical characteristics of FLAB (Endo and Okada 2008; Endo et al. 2012). Similar results can be found in a study conducted by Endo et al. (2009) wherein isolates identified as Fructobacillus sp. were catalase-negative while other LAB isolated exhibited catalase-like activity. Members of FLAB are expected to have higher absorbance values when cultured in FYPB compared to GYPB under microaerophilic conditions, as observed in this study and particularly seen in eight isolates (FACQ 9, FAIM 21, MAPS 4, MABE 25, MAFPS 4.1, MFRU 7.2, MAFAI 8, and MFHR 16). However, two of the isolates (MAPS 4 and MABE 25) did not have significant difference when analyzed using t-test (p>0.05). Another study, however, reported that all their presumptive FLAB isolates were isolated using FYPB and FYPA, and had better growth in FYPB than GYPB (Endo et al. 2010). In contrast, the isolates in the current study were isolated using MRSA, MRSA with fructose, and FYPA. Members of FLAB are xerotolerant and can survive in an environment with low water activity (Endo and Okada 2008). Increasing the fructose content to 30% (w/v) in FYPB results in an environment with high osmotic pressure and low water activity, which is a condition conducive for FLAB selection (Endo 2012; Endo 2019). All eight isolates still proceeded to the next step based on the higher absorbance observed in FYPB compared to GYPB and to test more isolates for antimicrobial activity. Although not currently recognized as FLAB, our isolates that were identified as Weissella spp. were obtained using standard methods for isolating FLAB. Weissella is a genus of Gram-positive, catalase-negative, and non-spore forming LAB, with some species reported as osmotolerant. Weissella spp. have been isolated from fermented food, vegetation, and silage. Many of its species are heterofermentative LAB that utilize a wide variety of carbohydrate sources, including glucose and fructose, due to their numerous phosphotransferase systems (Fusco et al. 2015; Assamoi et al. 2016; Säde and Björkroth 2019). Antimicrobial activity of FLAB isolates Seven test bacteria that cause several gut related diseases, such as a variety of infections, diarrhea, campylobacteriosis, and salmonellosis, were chosen for the study. Several studies also included some of the test bacteria in determining the antimicrobial activity of LAB isolated from various sources particularly against E. coli and Salmonella sp. (Gao et al. 2019; Sakandar et al. 2019; Lakra et al. 2020). Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria 44 Numerous members of LAB have been used as probiotics in recent years, particularly W. cibaria, which had been studied for its possible growth inhibitory activity against different pathogens. Both isolates MFPS 4.1 and MFRU 7.2, identified as W. cibaria, exhibited strong antagonistic activity against C. jejuni, which causes campylobacteriosis. W. cibaria JW15 was shown to have inhibitory effects on the growth of C. jejuni in the study conducted by Yu et al. (2019). The same study also showed that W. cibaria JW15 inhibited the growth of E. coli and Salmonella Typhimurium, which was also observed on isolates MFPS 4.1 and MFRU 7.2, respectively. Based on the study, W. cibaria JW15 was shown to have an increased acetic acid production compared to other LAB, which explains its ability to inhibit the growth of several pathogens. Another study also observed that W. cibaria was able to inhibit the growth of E. coli (Lakra et al. 2020). In the study, it was concluded that acid production in the supernatant of W. cibaria inhibited the growth of E. coli, and loss of inhibitory activity was observed when the pH was neutralized. To date, studies on specific inhibition of W. cibaria against E. faecalis are scarce but other members of LAB, such as Lactococcus sp., were able to inhibit the growth of said test pathogen (González et al. 2007). Similar published studies on the antimicrobial activity of FLAB from the Philippines are also scarce, indicating that the current study might be the first one. Several studies also reported the production of other antimicrobial metabolites by Weissella sp. The bacteriocin Weissellicin 110 from W. cibaria inhibits growth of other Gram-positive bacteria, while weissellin A from Weissella paramesenteroides has been reported to inhibit food spoilage bacteria and Listeria (Srionnual et al. 2007; Papagianni and Papamichael 2011). Production of hydrogen peroxide by W. cibaria was found to inhibit the growth of Fusobacterium nucleatum (Kang et al. 2006). This was supported by another study that reported that W. cibaria CMU, an oral care probiotic, inhibits the growth of common cariogenic bacteria such as F. nucleatum and Porphyromonas gingivalis. The same strain had also effectively inhibited the biofilm formation of Streptococcus mutans (Jang et al. 2016). As mentioned in the previous studies, isolates MFPS 4.1 and MFRU 7.2, which were identified as W. cibaria, can have potential use in the food industry as food additive to prevent food spoilage and in the medical field as a probiotic. The growth inhibition exhibited by the isolates against the test bacteria in the primary screening were either reduced or absent in the secondary screening. Several studies have shown that the presence of stress in a microbial environment plays an important role in the production of antimicrobial substances. Cell-to- cell contact of the isolates with the test bacteria in the primary screening may G. M. Penuliar and R.J. R. Artezuela 45 be interpreted as stress in the form of competitive exclusion, thus the production of antimicrobial substance. However, the same stress was not present in the preparation of CFS since the isolates were grown in optimized culturing condition in broth media, therefore production of antimicrobial substance may have been reduced or limited. Assuming that the inhibitory substances produced by the isolates were bacteriocin or bacteriolysins, a possible explanation might be that the inhibitory substances produced were stress-induced. It has been reported that L. kunkeei produces bacteriolysin in the presence of microbial stressors, such as peptidoglycan, lipopolysaccharides, and lipoteichoic acid (Butler et al. 2013). This is in accordance with another study where the gene promoter for the production of bacteriocin, bactofencin A from L. salivarius, observed an increase in activity when cells were subjected to microbial stress such as exposure to bile salts, gastric fluids, and target microbiota (Guinane et al. 2015). Addition of sodium acetate in Weissella cibaria culture has been reported to produce an increased amount of H 2 O 2, which have inhibitory activity against F. nucleatum (Kang et al. 2006; bEndo et al. 2009). Another possible explanation for the lack of antimicrobial activity of the CFS is that the conditions, like temperature, pH, amount of carbohydrate source, and oxygen requirements, for the optimized production of either bacteriocin, bacteriolysin or organic acids were not met when the isolates were grown in broth cultures. CFS used in the assay were collected from cultures that were grown in optimized conditions for growth rather than for optimized bacteriocin, bacteriolysin or organic acid production. Several studies, however, used similar culture conditions for optimized bacterial growth and CFS preparation for antimicrobial assays (Pan et al. 2009; Endo and Salminen 2013; Syed Yaacob et al. 2018). A study on the production of bacteriocin-like inhibitory substance (BLIS) produced by Pediococcus acidilactici, for example, had different growth conditions when grown for the optimized BLIS production compared to conditions optimized for cell growth. In the study, 37°C was used as the optimal temperature and the culture was grown for 24 h for the growth of P. acidilactici, while 28.5°C was used as optimal temperature and the culture was grown for 28 h for optimized BLIS production (Md Sidek et al. 2018). Another study on Carnobacterium divergens reported different culturing conditions for optimized growth and for optimized bacteriocin production. In the study, optimized condition for growth of C. divergens were 15 °C, pH 6.5–8, and the amount of carbohydrate source was 2% (w/v) while the optimized condition for its bacteriocin activity were 15°C, pH 6.5, and the amount of carbohydrate source ranged from 0.2% - 2% (Brillet- viel et al. 2016). Isolation and Antimicrobial Activity of Fructophilic Lactic Acid Bacteria 46 Phylogenetic tree analysis The phylogenetic tree has 93% bootstrap support for the isolates clustering with the genus Weissella, and 95% and 100% bootstrap support for the isolates forming a sub-cluster with W. cibaria and W. confusa, respectively, which is consistent with the BLAST identification of the isolates based on their 16S rRNA gene sequences. The high support for the phylogenetic positions of the isolates confirms the identity of the isolates as belonging to genus Weissella. With regard to the species identification of the isolates, it has been reported that the recommended range of 16s rRNA gene sequence similarity for species differentiation is 98.9% - 99.0% (Stackerbrandt and Ebers 2006). It is therefore possible that isolates MFPS 4.1 and MFRU 7.2 are new species under the genus Weissella, or new species that are closely related to W. cibaria. However, this needs to be confirmed by further studies on the molecular and physiological level, which should include an extensive study of their cell morphology using scanning electron microscope (SEM), assessment of their carbohydrate fermentation capabilities and antimicrobial susceptibility, and additional phylogenetic analyses using multiple housekeeping genes to definitively determine the taxonomic positions of the isolates. CONCLUSIONS This work describes the first attempt to isolate FLAB from flowers in the Philippines. The results show that eight presumptive FLAB isolates were isolated and two Weissella spp. were identified from flowers from the University of the Philippines Diliman based on the antimicrobial activity. Isolates MFPS 4.1 and MFRU 7.2 displayed FLAB features such as fructose osmotolerance and preferential growth in FYPB, and exhibited in vitro antimicrobial activity against several enteric pathogens, particularly C. jejuni and E. faecalis, which can be studied further for possible applications in both the food industry and the medical field, such as as probiotics. Data Availability The sequences of the isolates described in the study have been deposited in GenBank with the following accession numbers: MW836947.1, MW836946.1, MW836945.1, MW836944.1, and OL629482. G. M. Penuliar and R.J. R. Artezuela 47 Conflicts of Interest The authors declare that there is no conflict of interest regarding the publication of this paper. ACKNOWLEDGEMENTS The authors would like to acknowledge Gilbert S. Sobremisana, Ma. Socorro Edden P. Subejano and Jann Eldy L. Daquioag. 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He has a Ph.D. in Medical Science and is the current Principal Investigator of the Medical Microbiology Laboratory.