In the present study, a Lactobacillus plantarum FPL strain exhibiting fructophilic behavior has been isolated for the first time from honeydew. It is a probably syntrophic bacterium inhabiting the gastrointestinal tract of Coccus hesperidum L. and taking part in sugar metabolism. The promising growth characteristics and biochemical properties of Lb. plantarum FPL indicate that this may be a facultatively fructophilic species, whose properties are not associated with the loss of the alcohol/acetaldehyde dehydrogenase gene. The article attempts to classify the peculiar behavior of this strain by means of tests that are characteristic for FLAB as well as through a classic identification approach. In this study, we used a reference strain Lb. plantarum NRRL B-4496, which showed no fructophilic properties. With the FLAB group, the new strain shares the habit, such as a fructose-rich environment, the preference of this sugar for growth, and similar growth curves. However, it exceeds FLAB in terms of osmotolerance to high sugar content. The fructophilic Lb. plantarum FPL strain can proliferate and grow on a medium wherein the sugar concentration is 45 and 50% (w/v). Our findings indicate that honeydew can be a promising source of new fructophilic lactic acid bacteria. . . . . . Keywords Lactobacillusplantarum Coccus hesperidumL. Fructophiliclacticacidbacteria Honeydew Isolation Syntrophic bacteria Abbreviations FYP Fructose yeast peptone medium LAB Lactic acid bacteria GYP Glucose yeast peptone medium FLAB Fructophilic lactic acid bacteria HPLC High-performance liquid chromatography MALDI-TOF Matrix-assisted laser desorption/ ionization-time of flight mass spectrometry Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-018-1350-2) contains supplementary Lactic acid bacteria (LAB) are an example of organisms that material, which is available to authorized users. evolve depending on the environment in which they live (Douglas et al. 2015). Lactic acid bacteria are generally auxo- * Klaudia Gustaw email@example.com trophic for some compounds; they are quite demanding nutri- tionally and limited in their environmental tolerances Magdalena Michalak (Christiansen et al. 2008; Gomaa and Rushdy 2014). This firstname.lastname@example.org description contrasts with the biodiversity known today and Magdalena Polak-Berecka the ability to tolerate extraordinary habitats, given the progres- email@example.com sive knowledge of LAB genomes (Azcarate-Peril and Adam Waśko Klaenhammer 2010; Franz and Holzapfel 2011). The ongoing firstname.lastname@example.org reduction of the genome called Breductive evolution^ (van de Guchte et al. 2006) together with acquisition or overexpres- Faculty of Food Science and Biotechnology, Department of sion of genes (van de Guchte et al. 2006; Callanan et al. 2008; Biotechnology, Microbiology and Human Nutrition, University of Azcarate-Peril et al. 2009) may explain adaptation of LAB to Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland nutrient-rich and extreme environments. 460 Ann Microbiol (2018) 68:459–470 Beside their major nutritional characteristics, sugar-rich influence as potential probiotics (Endo and Salminen 2013; environments can inhibit or prevent bacterial growth and Vojvodic et al. 2013). Some FLAB have antibacterial activity cell division due to the presence of chaotropic solutes (e.g., against Paenibacillus larvae and Melissococcus plutonius phenols, ethyl acetate, ethanol, glycerol, fructose) and hy- causing foulbrood diseases (Forsgren et al. 2010;Rokop et drophobic stressors (such as hexane, ethyl octanoate, or al. 2015). These bacteria are capable of utilizing more com- octanol acetate) (Lievens et al. 2015). Fructophilic lactic plex carbohydrates than fructose and glucose, such as lignin. acid bacteria (FLAB) described recently by Endo and co- Degradation of lignin, which is a component of pollen, by workers were found to possess the ability to invade niches these bacteria helps to utilize this vital bee food (Alberoni et rich in high concentrations of sugar, especially fructose al. 2016). Therefore, it is believed that Fructobacillus bacteria (Endo and Okada 2008).Theycanbefoundinsuchenvi- can be syntrophic through the distribution/decomposition of ronments as flowers, nectar, fruits, and in regional foods more complex compounds and enhancement of their avail- like tempoyak (made mainly from fermented durian) or ability to other microbiome bacteria (Rokop et al. 2015). taberna (alcoholic beverage) (Endo et al. 2009). The first aim of this study was to isolate and identify Fructophilic LAB have also been discovered in the diges- fructophilic lactic acid bacteria from honeydew. The second tive tracts of pollinators such as bees, bumblebees, or in aim of our work was to characterize some biological proper- general in insects consuming significant amounts of fruc- ties of a newly isolated fructophilic Lb. plantarum FPL strain. tose, e.g., tropical fruit flies or ants from the genus Camponotus. Ants willingly feed on honeydew, which is a mixture of fructose-rich juices of plants damaged by Materials and methods aphids and the liquid excrement of these insects. Fructobacillus fructosus isolated from a flower in Japan Isolation of fructophilic lactic acid bacteria was described as FLAB for the first time (Endo and Okada 2008). Subsequent papers described instances of Honeydew produced by Coccus hesperidum L. was collected Fructobacillus from South Africa, Mexico, or the USA. in gardens in Lublin, Poland, Eastern Europe in August 2015. To the best of the authors’ knowledge, there have been Honeydew samples were placed in sterilized Eppendorf tubes no reports on Fructobacillus from Eastern Europe with saline. The samples were incubated for 1 h with shaking (Antunes et al. 2002;Endoetal. 2010, 2012;Endo 2012). on a heating ThermoMixer HLC (DITABIS AG, Pforzheim, The group of FLAB includes five species from the ge- Germany) at 30 °C and 1000 rpm. This solution was trans- nus Fructobacillus and two species from the genus ferred to a FYP (fructose yeast peptone) medium (Endo et al. Lactobacillus. The genus Fructobacillus prefers D- 2015) and MRS with fructose (2% (w/v)). The inoculated fructose to D-glucose as a main source of growth, due to medium was incubated at 30 °C for 24 h in aerobic conditions; the absence of the adhE gene encoding a bifunctional then, it was moved onto Petri plates on MRS with fructose and alcohol/acetaldehyde dehydrogenase. For glucose metabo- FYP. When colonies were visible, they were selected in terms lism, Fructobacillus species require fructose, oxygen, or of their morphological properties (shape, size, color). To ob- pyruvate as an external electron acceptor due to the short- tain pure cultures, the colonies were isolated by streaking on age of NAD+ (Endo et al. 2014). Under anaerobic condi- agar plates. tions where glucose is the only carbon source, the bacteria show no or very poor growth. This description applies to Identification of isolates using MALDI-TOF Bobligately^ fructophilic lactic acid bacteria, distinguished in the group of FLAB according to the two types of sugar Forty-nine isolates of bacteria were identified using the metabolism. Fructobacillus fructosus, F. ficulneus, F. MALDI-TOF Biotyper (Bruker Daltonics, Bremen, pseudoficulneus, F. durionis, F. tropaeoli,and Lb. kunkeei Germany). Lb. plantarum NRRL B-4496 (ARS Culture are classified as Bobligately^ fructophilic bacteria.Lb. Collection, Peoria, IL, USA) was used as a reference strain. florum represents the group of Bfacultatively^ fructophilic After a 24-h incubation, a single colony was transferred to an lactic acid bacteria. BFacultatively^ fructophilic bacteria Eppendorf tube with 150 μl of sterile deionized water. The can grow on glucose without an external electron acceptor samples were homogenized by repeated pipetting and and produce ethanol from glucose; however, the growth of vortexing. Four hundred fifty microliters of pure ethanol were FLAB on fructose is faster (Endo et al. 2012). Fructophilic added to the Eppendorf, and the content was mixed by lactic acid bacteria can also produce polyols such as glyc- vortexing for at least 1 min. After centrifugation for 2 min at erol, erythritol, or mannitol (Endo and Okada 2008;Endo 13000 rpm, the supernatant was removed; this step was re- and Dicks 2014; Tyler et al. 2016). peated twice. A 70% solution of formic acid was added in an As core members of the microbiome of honeybees and amount of 40 μl and vortexed, and the same volume of 99% other pollinators, FLAB are currently investigated for their acetonitrile was added as well. After vortexing for 1 min, the Ann Microbiol (2018) 68:459–470 461 samples were centrifuged (2 min, 13,000 rpm). One microliter pREV (59-TCG GGA TTA CCA AAC ATC AC-39). The of the supernatant was applied onto a metal plate in triplicate. composition of the reaction was 13 μl PCR Master Mix(2×) After drying at room temperature, the spots were covered with (Thermo Fisher Scientific, Bermen, Germany), 0.75 μl for 1 μlofmatrix(concentration of10 mgof HCCA- α-Cyano-4- each primer, and 10.5 μl nuclease-free water (Thermo Fisher hydroxycinnamic acid/ml) and left to dry. The plate was in- Scientific, Bermen, Germany); the reaction conditions were troduced to an UltrafleXtreme MALDI TOF mass spectrom- described previously (Torriani et al. 2001). The amplification eter (Bruker, Germany) with a 1000 Hz neodymium-doped products were separated on 1.5% agarose gel (Eurx, Gdańsk, yttrium aluminum garnet nitrogen laser (Nd-YAG). The sam- Poland) with 1 kb Ladder Perfect Plus (Eurx, Gdańsk, ples were analyzed automatically using a MALDI Bio-typer Poland). In order to clarify fructophilic properties, a PCR re- 3.0 software package (Bruker, Germany). The probability of action of the adhE gene was performed; the reaction condi- identification was expressed by a score in a scale from 0 to 3.0. tions were described previously (Maeno et al. 2016). A result above 2.0 denoted secure genus identification and probable species identification. Nine isolates were selected Biochemical characterization based on the high probability of identification for further ex- periments in this article. Carbohydrate fermentation was determined with a Hi-Carbo Kit (HiMedia, Mumbai, India). An inoculum with turbidity 16S rRNA gene sequencing and species-specific PCR 0.5 OD nm at 600 nm was added onto wells containing 35 sugars and incubated at 37 °C for 24 and 48 h. For carbohy- DNA extraction of nine strains was performed using Genomic drate utilization, a reference strain Lactobacillus plantarum Mini AX Bacteria Spin (A&A Biotechnology, Gdynia, NRRL B-4496 was additionally used. Gas production from Poland) according to the attached protocol. For amplification glucose was read with the Durham test, and catalase activity of the 16S rRNA gene, universal primers (27f) 5`- was determined by reaction with 3% (v/v)H O .API ZYM 2 2 AGAGTTTGATCCTGGCTCAG-3`, and (1495r) 5`- (bioMérieux SA, Marcy l′Etoile, France) was used for deter- CTACGGCTACCTTGTTACGA-3` were used (Genomed mination of enzyme production patterns. An inoculum with S.A., Warszawa, Poland). The PCR reaction was performed turbidity 0.8 OD nm at 600 nm was added onto 20 plates and in a total volume of 20 μl using a PCR Master Mix(2×) incubated for 4 h at 37 °C. (Thermo Fisher Scientific, Bermen, Germany) in a Labcycler (SensoQuest Göttingen, Germany). The amplifica- Biological activity tion reaction was characterized by the following steps in 30 repeat cycles: denaturation 95 °C for 1 min, annealing 48 °C Fructophilic properties of the isolates for 30 s, elongation 72 °C for 2 min, final extension 72 °C for 10 min, and cooling the samples to 4 °C. The amplification Fructophilic properties were determined using a Bioscreen C products were separated on 1.5% agarose gel (Eurx, Gdańsk, system (Labsystem, Helsinki, Finland). After a 24-h incuba- Poland). The nucleotide sequences were determined by the tion, bacterial cultures were centrifuged and removed from the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied medium. The bacterial cells were suspended in physiological Biosystems, USA), and the capillary sequencing system, saline, and the same optical density of 0.5 was set at 600 nm. −1 3730xl DNA Analyzer (Applied Biosystems, USA). The analyzed bacteria were grown in FYP with 10 g (L )D- −1 Sequences were assembled by a DNA Baser Assembler, sub- fructose, GYP with 10 g (L ) D-glucose, and GYP-P with 5 g −1 −1 sequently aligned with BLAST, and compared in the NCBI (L )D-glucose and5 g(L ) pyruvateas anexternalelectron −1 GenBank to find the closest relatives. A neighbor-joining tree acceptor, and MRS with 300 g (L ) D-fructose, with 300 g −1 −1 was made using MEGA 4 for the phylogenetic analysis based (L )D-glucose, with 400 g (L ) D-glucose, and with 500 g −1 on 16S rRNA sequences. Only one representative sequence (L ) D-glucose. Three hundred fifty microliters of the media was used to create the diagram, because there was no differ- were transferred onto honeycomb 100-well plates in triplicate, entiation after alignment. Sequences of the 16S rRNA gene and the wells were inoculated with 50 μl of the bacterial sus- used to construct the phylogenetic tree were approximately pension. The experiment was performed in aerobic and anaer- 1450 base pairs. obic conditions by measuring the OD every2hfor 48 h. 600nm Additionally, multiplex PCR, which detected the recAgene Anaerobic conditions were obtained by cutting off access to phylogenic marker, was used; this revealed distinction be- oxygen with a few drops of paraffin. Based on the growth tween Lb. plantarum, Lb. pentosus,and Lb. paraplantarum characteristics, nine strains were chosen for further examina- (Torriani et al. 2001). Multiplex was performed with four tion. Growth curve parameters (max specific growth rate, lag primers paraF (59-GTC ACA GGC ATT ACG AAA AC- time, doubling time, etc.) were determined using the 39), pentF (59-CAG TGG CGC GGT TGA TAT C-39), PYTHON script according to Hoeflinger et al. (2017). High planF (59-CCG TTT ATG CGG AAC ACC TA-39), and sugar tolerance was tested in FYP and MRS broth enriched 462 Ann Microbiol (2018) 68:459–470 with 20, 30, 40, and 45% (w/v) fructose and glucose or con- mundtii. According to the Brucker database, almost all isolates taining 5% of NaCl (w/v) by observing a significant amount of indicated Lb. plantarum; only three isolates showed other biomass in the probe. Production of lactic acid was checked species, but they can be considered as contamination. The by incubation on FYP-agar and MRS-agar containing 10 g results showed the dominance of Lb. plantarum in the honey- −1 (L ) CaCO and confirmed with HPLC with a UV-Vis detec- dew environment. Subsequently, spectra of the new FPL tor (Gilson Medical Electronics, Villiers-le-Bel, France). strains were aligned with the reference strain, and the shift of Production of sugars was determined after 3-day culture in some peaks indicates modification of proteins. The MALDI rotary shaker with aeration (150 rpm) (Minitron Incubator Biotyper analysis of the spectra shows that the surface of Shaker Infors AG, Bottmingen, Switzerland). The amount of certain proteins was modified, which may explain the adapta- glucose and fructose consumed after 3 days of incubation was tion to the fructose-rich environment. The results of the determined using an IR detector. A reference strain MALDI-TOF analysis revealed nine isolates preliminary Lactobacillus plantarum NRRL B-4496 was used as a control identified as Lb. plantarum, with the highest score of proba- in all tests. bility of identification. Antibiotic susceptibility test Species identification through 16S rRNA gene sequencing and multiplex PCR After 24 h incubation in 30 °C, the cells were centrifuged and removed from the culture medium with saline. The inoculum The identification of nine isolates of Lb. plantarum from hon- suspension in saline with McFarland density of 0.5 was care- eydew was performed by analyzing sequences of the 16S fully spread on Petri plates with 4-mm thick MRS agar. When rRNA gene. The DNA sequences obtained were aligned by the suspension was absorbed by the agar, rings with antibiotics BLAST with the nucleotide gene bank; it was revealed that all were distributed in triplicate. Erythromycin E15, kanamycin strains are > 99% similar to Lb. plantarum, Lb. K30, bacitracin B10, streptomycin S10, amoxycillin AML25, paraplantarum, and Lb. pentosus. In order to confirm the tetracycline TE30, trimethoprim WE, penicillin P10, species belonging of the isolates, a multiplex PCR was per- pirlimycin PIR2, chloramphenicol C30, and nalidixic acid formed. Reaction products of multiplex PCR for recAgene NA30 were purchased from Oxoid (Hampshire, England). with length of about 310 bp is specific to the Lb. plantarum species (Fig. 1). The phylogenetic tree constructed with the Statistical analysis neighbor-joining method shows strains that are the closest to Lb. plantarum FPL as well as the location of Fructobacillus The values from all measurements are mean ± standard devi- species. Lactobacillus kunkeei is the nearest phylogenetic ation. The data were analyzed using the Excel statistical pack- neighbor from the group of FLAB (Fig. 2). In the first reports age. Statistical significances were determined by Student’s t on FLAB, growth on various media was described; the 16S test and set at P =w0.01. rRNA gene was identified, and a few biochemical and fructophilic properties were characterized (Endo and Okada GeneBank accession number 2008). In this article, the studies proposed by Endo were con- ducted. We also used MALDI-TOF and multiplex PCR which GeneBank accession number for Lb. plantarum FPL 16S made it possible to identify strains from the honeydew. All rRNA gene: KY883188. Due to the sequence identity obtain- these methods facilitated quick and efficient selection of ed for the 16S rRNA gene, only one representative of this strains for further research. group was included. Biochemical properties Results and discussion The Lb. plantarum FPL strains utilize carbohydrates indicated in Table 1. The different strains are able to use also xylose, Species identification by MALDI-TOF galactose, raffinose, glycerol, and adonitol, which may indi- cate the dissimilarity of individual isolates. The reference The identification of 49 strains was performed by MALDI- strain Lb. plantarum NRRL B-4496 showed no differences TOF, and the spectra obtained were aligned with the Brucker in utilization of carbohydrates, except inulin, raffinose, and database. The spectra of the isolated strains had a high prob- mannitol, which were used only by the fructophilic Lb. ability of identification over two points, (experiments were plantarum FPL. carried out in triplicate). Analysis of 46 strains spectra indi- The results of API ZYM (Biomerieux) revealed production cated Lb. plantarum, three other spectra corresponded to of esterase (C4), esterase lipase (C8), lipase (C14), Staphylococcus haemolyticus, S. aureus, and Enterococcus leucinearylamidase, valinearylamidase, cystine arylamidase, Ann Microbiol (2018) 68:459–470 463 Fig. 1 PCR amplification products obtained from the multiplex assay. FPL4 298.5; line 7 Lb. plantarum FPL5 294 bp; line 8 Lb. plantarum Lane 1 contains a 1 kb Ladder Perfect Plus (Eurx, Gdańsk, Poland). FPL6 296 bp; line 9 Lb. plantarum FPL7 299 bp; line 10 Lb. plantarum Lane 2 contains the amplification product from Lb. plantarum FPL FPL8 308 bp; line 11 Lb. plantarum FPL9 310 bp. The length/number of with a length of 295.53 bp; lane 3 shows amplification products from base pairs was determined using Quantity One Software (Bio-Rad, Lb. plantarum FPL1 with a length of 295.56 bp; line 4 Lb. plantarum Illinois, USA) FPL2 295.6 bp; line 5 Lb. plantarum FPL3 298 bp; line 6 Lb. plantarum Fig. 2 Phylogenetic tree based on the sequence of 16S rRNA showing the relative positions of Lb. plantarum FPL 464 Ann Microbiol (2018) 68:459–470 Table 1 Carbon utilization profile of Lb. plantarum FPL and Lb. Mumbai, India), + positive (+++clearly visible change, ++ visible, + plantarum NRRL B-4496 determined with the Hi-Carbo Kit (HiMedia, poorly visible); − negative Carbon source Lb. Lb. Lb. Lb. Lb. Lb. Lb. Lb. Lb. Lb. plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum FPL FPL1 FPL2 FPL3 FPL4 FPL5 FPL6 FPL7 FPL8 NRRL Lactose + ++ ++ ++ ++ ++ + ++ ++ ++ Xylose + –––––––– + Maltose +++ ++ ++ +++ +++ +++ +++ ++ +++ ++ Fructose +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ Dextrose +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ Galactose – +++ – ++++ + Raffinose + – + –––––– – Trehalose ++ +++ ++ + + ++++++++ + Sucrose + ++ +++ ++ ++ ++ +++ ++ ++ ++ Mannose +++ +++ +++ +++ +++ +++ +++ ++ +++ +++ Inulin +++ +++ +++ ++ +++ +++ +++ +++ +++ – Salicin ++ +++ ++ ++ ++ +++ +++ ++ ++ ++ Sorbitol + ++++++++ + Mannitol + ++ ++++ ++++ – Cellobiose +++ +++ +++ +++ +++ +++ +++ +++ ++ +++ Melezitose ++ ++ +++ ++ ++++++++++ ++ α-methyl-D-mannoside + + + ++ ++ + ++ + + ++ Esculin +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ acid phosphatase, naphthol AS-BI-phosphohydrolase, β-ga- interactions between these bacteria through metabolic by- lactosidase, α-glucosidase, β-glucosidase, and N-acetyl-β- products. glucosaminidase. The strains had no activity of alkaline phos- phatase, trypsin, α-chymotrypsin, α-galactosidase, β-glucu- Antibiotic susceptibility test ronidase, α-mannosidase, and α-fucosidase. The study con- ducted by Siezen et al. (2010) tested carbohydrate utilization The antibiotic sensitivity slightly differs between the individ- by 185 strains of Lb. plantarum; all strains degraded trehalose, ual strains, as shown in Table 2. The strains are sensitive to all sucrose, melezitose (except one), and sorbitol similarly to the antibiotics used in this study except nalidixic acid. The Lb. plantarum FPL strains. There were differences in the case antibiotic-sensitivity test has shown that the Lb. plantarum of mannitol and inulin; this study has shown that only the Lb. strains are safe which is the first step to determining their plantarum FPL strains utilize these carbohydrates, which of- probiotic potential. The antibiotic-susceptibility test was car- ten occur in the plant environment. However, utilization of ried out earlier on FLAB that are inextricably linked to insects inulin by the Lb. plantarum species is not unique, as other and have probiotic potential for insects. Furthermore, FLAB strains have been reported to degrade grass fructan and inulin can produce and utilize substances supporting the growth of (Müller and Steller 1995;Siezen etal. 2010; Valan Arasu et al. the core gut microbiome of honey bees (Rokop et al. 2015). 2015). In contrast, the possibility of different carbohydrate metabolism by the Lb. plantarum FLP strains is significantly Fructophilic properties higher than that of the FLAB group, as the latter bacteria do not degrade salicin, sorbitol, cellobiose, and melezitose (Endo All tested carbon sources caused a considerable growth with et al. 2010, 2012; Lievens et al. 2015). All species in the the used bacteria (i.e., reached a final OD 660 > 0.9). Strains FLAB group can metabolize mannitol and fructose, as same isolated in this study, showed very similar growth curves; as fructophilic properties of the Lb. plantarum FPL strains therefore, only one representative strain is shown in Fig. 3. (Endo and Okada 2008). The strains do not exhibit acid phos- Growth parameters are shown in Table 3.However,distinctive phatase, trypsin, and chymotrypsin activity, which is present growth profiles were obtained within one species, where sugar in most Fructobacillus species. Lb. plantarum FPL has β- preferences are clearly visible depending on the strain. The galactosidase, α- glucosidase, and β- glucosidase activity, un- comparison of the growth curves of Lb. plantarum FPL and like the genus Fructobacillus, which may cause syntrophic Lb. plantarum NRRL-4496 shows different preferences for Ann Microbiol (2018) 68:459–470 465 Table 2 Antibiotic sensitivity of nine isolated Lb. plantarum FPL Antibiotic Concentration of antibiotic Average zone of inhibition A (mm) and Standard deviation SD(mm) FPL FPL1 FPL2 FPL3 FPL4 FPL5 FPL6 FPL7 FPL8 Total ASD A SD ASD ASD A SD ASD ASD ASD ASD A SD Eryhtomycin E15 15 μg 30.37 0.26 28.83 0.24 30.67 0.94 22.67 0.47 19.67 0.47 26.33 0.47 29.67 0.62 29.03 0.73 26.60 1.18 27.09 0.29 Kanamycin K30 30 μg 10.17 0.24 9.37 0.26 8.00 0.71 8.33 0.47 9.87 0.19 8.30 1.28 7.00 0.00 8.10 0.54 9.50 0.71 8.74 0.36 Bacitracin B10 10 units 21.87 0.66 22.83 1.43 11.67 0.47 16.00 1.41 17.50 0.41 13.17 3.06 20.67 1.25 19.83 0.62 19.20 1.57 18.08 0.78 Streptomycin S10 10 μg 12.83 0.62 14.93 0.33 8.50 0.41 10.33 0.47 10.50 0.71 12.17 0.85 9.90 1.58 11.17 1.43 9.50 1.87 11.09 0.53 Amoxycillin AML25 25 μg 37.60 0.22 36.20 0.59 27.50 1.22 30.33 3.77 31.67 1.25 29.33 0.47 30.83 0.85 34.00 0.82 31.67 2.49 32.13 1.07 Tetracycline TE30 30 μg 22.10 0.54 22.60 1.13 17.00 2.45 15.67 0.47 20.53 0.41 24.83 0.62 23.17 1.93 18.83 1.65 21.00 1.63 20.64 0.70 Trimethoprim WE 5 μg 25.13 0.81 25.00 0.00 24.50 1.08 25.00 0.00 22.97 0.82 21.33 1.43 19.00 0.71 20.27 0.21 20.50 0.41 22.63 0.46 Penicillin P10 10 units 24.17 0.24 26.63 0.45 24.00 0.82 20.00 0.00 22.83 0.62 20.67 3.30 19.00 0.82 25.47 0.34 19.00 2.16 22.42 1.01 Pirlimycin PIR2 2 μg 8.33 0.47 9.83 1.18 21.67 0.47 11.83 0.62 8.17 0.62 8.30 0.99 10.33 0.47 9.17 0.24 10.00 0.82 10.85 0.28 Chloramphenicol C30 30 μg 31.67 0.47 21.83 1.43 23.83 0.85 23.00 0.71 31.67 0.94 29.23 1.58 22.83 1.65 24.67 2.62 31.00 1.41 26.64 0.61 Nalidixic acid NA30 30 μg 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 466 Ann Microbiol (2018) 68:459–470 Fig. 3 Growth curves of Lb. plantarum FPL in a aerobic conditions, b anaerobic conditions and of Lb, plantarum NRRL B-4496 in c aerobic conditions, and d anaerobic conditions on various carbon sources (FYP, GYP, GYP-P) sugars as a growth substrate. The max specific growth anaerobic conditions, the growth of the reference NRRL-4496 rate of Lb. plantarum FPL on fructose was highest in strain, on the pyruvate medium, is slower compared to our the entire study. The origin of the Lb. plantarum FPL FPL strain. This can be explained by the fact that NRRL- strains had to determine their tendency towards fructose, 4496 strain does not exhibit fructophilic properties. which is a significant component of honeydew. Strains Furthermore, the latest articles report that Lb. plantarum spe- of Lb. plantarum FPL grew fast on FYP, both under cies can use pyruvate in various metabolic pathways (Zotta et anaerobic and aerobic conditions, which allowed us to al. 2017). state its fructophilicity, which is a niche-specific adap- Although strain NRRL B-4496 grows on fructose, it is not tation. Reference strain NRRL- 4496 does not exhibit able to grow on a medium with a high concentration of both such affinity for fructose. glucose and fructose. The reference strain did not develop Another significant difference between the strains is mechanisms that would allow survival in a sugar-rich envi- growth on the medium supplemented with pyruvate. ronment. The growth curves of FLAB already described Pyruvate as well as oxygen, citrate, and fructose can be used (Endo et al. 2009) are lower in comparison with Lb. by LAB as external electron acceptors (Zaunmüller et al. plantarum FPL. The experiments conducted in this article 2006), so Endo et al. used it in their experiments to show the show that MALDI-TOF and Bioscreen C facilitate rapid characteristic properties of fructophilic lactic acid bacteria screening of fructophilic lactic acid bacteria. (Endo et al. 2009, 2015). Our observations indicate that pyru- In addition, the newly described strain can grow on a me- vate, also stimulates the growth of Lb. plantarum FPL on dium with 50% (w/v) fructose; other FLAB, except F. glucose, especially with aerobic conditions. It is known that tropaeoli, tolerate 40% (w/v) fructose content. The growth Lb. plantarum grows better in aerobic conditions and what is curves in Fig. 4 show the adaptation to growth in high sugar more, it is able to use oxygen as a substrate (Zotta et al. 2012). concentrations. In the case of the Lb. plantarum FPL strain, Moreover, in the presence of oxygen, the expression of genes the fructophilicity are again visible. The strain grows best on a responsible for the consumption of sugars increases (Guidone medium with a concentration of 30% fructose, then 30% glu- et al. 2013; Zotta et al. 2013). Successively both in aerobic and cose. A slow growth rate can be seen on the medium with 40% Ann Microbiol (2018) 68:459–470 467 glucose, and even delayed on medium with 50% glucose. This osmotolerance is high, since generally bacteria and yeast tol- erate up to 50% (w/v) of sugar (Álvarez-Pérez et al. 2012). The tolerance to high concentrations of sugar in the MRS and FYP media containing 20, 30, 40, 45, and 50% (w/v)of glucose or fructose was evidenced by the presence of signifi- cant amounts of biomass at the bottom of the tube. The bac- terial growth was visible as a biomass after 24 h of incubation on broth with the 20 and 30% concentrations of glucose or fructose. After 48 h, the growth was visible in the medium with the 40, 45, and 50% sugar concentration; in addition to the biomass at the bottom, there was evident turbidity. The difference between the growth on fructose and glucose was not significant. A transparent zone, which indicated produc- tion of acids, appeared around the colonies on MRS and FYP agar with CaCO3. In the supernatant, 3.4% of lactic acid was detected by HPLC. The tested bacteria produced gas from glucose and were catalase-negative. HPLC detected that the cultured Lb. plantarum FPL strain produced glycerol from fructose; no polyols were detected. In addition, HPLC con- firmed that the strain utilized both carbon sources but first fructose. Many FLAB, as well as Lb. plantarum FPL, were isolated with the use of FYP containing 30% fructose. It was also noted that more isolates were cultured on FYP than on MRS with fructose. This confirms that FYP is a specialized media in which a high concentration of fructose selects FLAB and si- multaneously inhibits growth of other bacteria (Le Marrec et al. 2007;Endoet al. 2009). The absence of fructose in com- mercial media explains why fructophilic bacteria were not identified earlier (Endo 2012). Lb. plantarum strains are wide- spread in various environments, probably thanks to one of the largest genomes among lactic acid bacteria. Generally Lactobacilli have a relatively large number of transport and regulatory genes as well as sugar transport and utilization genes (Álvarez-Pérez et al. 2012). In the genome of Lb. plantarum WCFS1, 30 sugar transport systems have been found, which explains why this species can grow on a variety of carbon sources. Among the genes of Lb. plantarum that are most expressed besides housekeeping genes, there are genes of the Embden–Meyerhoff–Parnas (EMP) pathway and many genes encoding enzymes involved in pentose and hexose uti- lization. The sequencing of the whole genome showed that potentially highly expressed (PHX) genes included numerous of phosphotransferase systems (PTSs), especially fructose and mannose PTS systems (Kleerebezem et al. 2003). This flexi- bility may explain the appearance of the fructophilic proper- ties of the Lb. plantarum FPL strain. Moreover, in this article, we have described strains that prefer fructose as a source of growth, with resistance to high sugar concentrations. If the extended Lb. plantarum genome, which has allowed adapta- tion to the fructose rich environment, is the cause of the fructophilic properties of the new FPL strains, this stands in Table 3 Growth parameters of Lactobacillus plantarum FPL and Lactobacillus plantarum NRRL B-4496 Medium Strain Growth conditions Lag time Max specific Doubling Max OD Max OD Min. OD Min. OD Delta OD R RMSE (hours) growth rate Time (hours) (median filtered data) (median filtered data) (median (root-mean- −1 (hours ) filtered data) square error) GYP NRRL Aerobic 5.2473 0.2059 3.3657 1.6777 1.6760 0.0127 0.0127 1.6633 0.9970 0.0299 GYP FPL Aerobic 3.6182 0.0962 7.2084 1.0590 1.0508 0.0177 0.0177 1.0332 0.9990 0.0125 GYP NRRL Anaerobic 3.9524 0.1951 3.5537 1.8303 1.8303 0.1063 0.1327 1.6977 0.9981 0.0235 GYP FPL Anaerobic 4.2491 0.1010 6.8595 1.0267 1.0002 0.0153 0.0153 0.9848 0.9990 0.0122 FYP NRRL Aerobic 0.5822 0.1344 5.1568 1.5563 1.5550 0.0227 0.0227 1.5323 0.9870 0.0531 FYP FPL Aerobic 5.2862 0.2143 3.2347 1.4330 1.4313 0.0273 0.0273 1.4040 0.9973 0.0228 FYP NRRL Anaerobic 1.9264 0.1497 4.6312 1.5933 1.5933 0.0553 0.0553 1.5380 0.9836 0.0593 FYP FPL Anaerobic 4.9179 0.2244 3.0892 1.4630 1.4617 − 0.0063 − 0.0063 1.4680 0.9983 0.0181 GYP-P NRRL Aerobic 2.5441 0.0305 22.7309 0.9880 0.9880 0.0160 0.0160 0.9720 0.9995 0.0075 GYP-P FPL Aerobic 5.0423 0.1594 4.3497 1.2773 1.2750 0.0157 0.0157 1.2593 0.9986 0.0184 GYP-P NRRL Anaerobic 4.7016 0.0302 22.9741 1.0637 1.0637 0.0937 0.0937 0.9700 0.9991 0.0099 GYP-P FPL Anaerobic 5.2819 0.1141 6.0773 1.2243 1.2207 0.0220 0.0220 1.1987 0.9991 0.0134 468 Ann Microbiol (2018) 68:459–470 Fig. 4 Growth curves of Lb. plantarum FPL, Lb. plantarum NRRL-4496, Lb. florum DSM on various medium with high sugar concentrations 30, 40, and 50% of glucose or fructose in aerobic conditions opposition to the origin of the fructophilic characteristics of sugars in the genome, but the question remains why this strain FLAB. Adaptation to fructose in the FLAB group is due to the prefers fructose instead of glucose as opposed to Lb. lack of the adhE gene; in the case of Lb. plantarum FPL, the plantarum NRRL. The presence of Lb. plantarum in honey- mechanism of the fructophilic behavior has a different basis dew must have an impact on the ecosystem of aphids, bees, or (Fig. 5). Certainly, in part, this is explained by the huge num- ants. It is also very possible that Lb. plantarum inhabit the ber of genes responsible for metabolism and transport of digestive tracts of aphids and honeydew-consuming insects. Yeasts living in nectar increase the number of visits of polli- nators (Herrera et al. 2013), while the presence of certain bacteria in the nectar (Erwinia tasmaniensis, Lactobacillus kunkeei, Asai astilbes) repel insects from flowers by changing the chemical composition of the nectar (Good et al. 2014). More samples of honeydew from Poland should be investigat- ed to confirm the colonization of this habitat by Lb. plantarum and its effect on insects. Data availability statement All data generated or analyzed during this study are included in this published article Conclusion Fig. 5 PCR amplification products obtained with adhE primers. Lane 1 contains a 1 kb Ladder Perfect Plus (Eurx, Gdańsk, Poland). Lane 2 The main goal of this work was to isolate fructophilic lactic contains the amplification product from Lb. florum DSM 22689; lane 3 acid bacteria in honeydew from Poland to understand the var- shows amplification products from Lb. plantarum FPL; line 4 Lb. plantarum NRRL B-4496 iability of species in honeydew originating from an area with a Ann Microbiol (2018) 68:459–470 469 Douglas GL, Azcarate-Peril MA, Klaenhammer TR (2015) Genomic temperate climate. Our work indicates for the first time that evolution of lactic acid Bacteria. In: Biotechnology of lactic acid honeydew from the temperate climate of Europe can be a bacteria. Wiley, Chichester, pp 32–54 promising source of new fructophilic lactic acid bacteria. To Endo A (2012) Fructophilic lactic acid bacteria inhabit fructose-rich the best of our knowledge, the selected Lb. plantarum FPL niches in nature. Microb Ecol Health Dis 23. https://doi.org/10. 3402/mehd.v23i0.18563 strain is the first strain described as Lactobacillus plantarum Endo A, Dicks LMT (2014) The genus Fructobacillus. Lact Acid Bact with fructophilic behavior. The presence of Lb. plantarum in Biodivers Taxon 381–389. https://doi.org/10.1002/9781118655252. honeydew must have an impact on the ecosystem of aphids, ch22 bees, or ants. It can be concluded that Lb. plantarum FPL is a Endo A, Okada S (2008) Reclassification of the genus Leuconostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., syntrophic bacterium inhabiting the gastrointestinal tract of Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. Coccus hesperidum L. and taking part in sugar metabolism. nov. and Fructobacillus pseudoficulneus comb. nov. Int J Syst Evol Microbiol 58:2195–2205. https://doi.org/10.1099/ijs.0.65609-0 Compliance with ethical standards Endo A, Salminen S (2013) Honeybees and beehives are rich sources for fructophilic lactic acid bacteria. Syst Appl Microbiol 36:444–448. Conflict of interest The authors declare that they have no conflict of https://doi.org/10.1016/j.syapm.2013.06.002 interest. Endo A, Futagawa-Endo Y, Dicks LMT (2009) Isolation and characteri- zation of fructophilic lactic acid bacteria from fructose-rich niches. Syst Appl Microbiol 32:593–600. https://doi.org/10.1016/j.syapm. 2009.08.002 Open Access This article is distributed under the terms of the Creative Endo A, Futagawa-Endo Y, Sakamoto M, Kitahara M, Dicks LMT Commons Attribution 4.0 International License (http:// (2010) Lactobacillus florum sp. nov., a fructophilic species isolated creativecommons.org/licenses/by/4.0/), which permits unrestricted use, from flowers. Int J Syst Evol Microbiol 60:2478–2482. https://doi. distribution, and reproduction in any medium, provided you give org/10.1099/ijs.0.019067-0 appropriate credit to the original author(s) and the source, provide a link Endo A, Irisawa T, Futagawa-Endo Y, Takano K, Du Toit M, Okada S, to the Creative Commons license, and indicate if changes were made. Dicks LMT, du Toit M, Okada S, Dicks LMT (2012) Characterization and emended description of lactobacillus kunkeei as a fructophilic lactic acid bacterium. 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Annals of Microbiology – Springer Journals
Published: Jun 1, 2018
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