**3. Results and discussion**

*Ecosystem and Biodiversity of Amazonia*

**2.4 Antibiotic susceptibility testing**

**2.5 Antimicrobial spectrum of the selected isolates**

activity (inhibit more than 5 indicator strains).

through vegan [35] and ggdendro [36] in R [30].

**2.6 Statistical and phylogenetic analyses**

included as the grouping variable.

Susceptibility to several antibiotics was determined by commercial discs of Amoxicillin (AMX: 25 μg), Ampicillin (AM: 10 μg) Gentamicin (CN: 10 μg), Kanamycin (K: 30 μg), Amoxicillin/Clavulanic Acid (AMC: 20/10 μg), Tetracycline (TE: 30 μg), and Cefuroxime (CXM: 30 μg). For the disk diffusion assay, we used concentrations recommended by the Scientific Committee on Animal Nutrition (discs provided by Merck) [12]. We used *Escherichia coli* ATCC 25922 as quality control. The microbiological breakpoints reported by the FEEDAP standards were used to categorize lactobacilli as susceptible or resistant [4]. *Lactobacillus plantarum* ATCC8014 and *L. fermentum* CNCM 1-2998 (Lacc) were used as a reference to the test. Results were qualified as R (resistance), I (intermediate), and S (susceptible).

The antimicrobial spectrum of the selected isolates was determined against Gram-positive and Gram-negative bacteria, as previously described by Garzon et al. [33]. The indicator bacteria were: *Salmonella enterica subsp. enterica* (Kauffmann and Edwards) Le Minor and Popoff ATCC 51741, *Salmonella enterica subsp. enterica* serovar Abaetetuba ATCC35640, *Escherichia coli* ATCC25922, *E. coli* ATCC10536, *Shigella sonnei* ATCC25931, *Streptococcus thermophilus* ATCC19258, *Enterobacter aerogenes* UTNEag1 (laboratory collection), S*almonella* UTNSm2 (laboratory collection), *Shigella* UTNShg1 (laboratory collection), and *E. coli* UTNEc1 (laboratory collection). LAB was grown in MRS broth at 34°C for 24–27 hours and the supernatants were collected by centrifugation at 13,000 x g for 20 minutes at 4°C. The crude extract (CE) was recovered and filtered with a 0.22 μm porosity syringe filter. The indicator strains (100 μl) were grown in broth medium (7 log CFU/ml) and mixed with 3.5 ml of soft MRS agar (0.75%). It was then overlaid on nutrient agar plates and incubated at 37°C for 2 hours. The CE of each strain (100 μl) was transferred onto the reaction wells (6 mm) on overlaid agar, incubated at 37°C and subsequently examined for the presence of an inhibition zone at 48 hours. To rule out the possible inhibitory activity of organic acids, the CE was heated at 80°C for 10 minutes, the pH adjusted at 6.0 and the activity was determined. *Lactobacillus plantarum* ATCC8014 and *L. fermentum* CNCM 1-2998 (Lacc) were used as reference strains. MRS broth was used as a negative control. The experiments were run in triplicate and the mean value of the inhibition zone was determined. A numeric scale from zero to ten was included in the statistical analysis and the results were also qualitatively defined as narrow (inhibit less than 5 indicator strains) or broad

The interpretation of the antibiogram results was assisted by the package AMR [34], which provided corresponding frequencies on the qualitative responses. The distances (Bray-Curtis) among samples were then projected in canonical space through a non-metric multidimensional scaling. Either putative genera of bacteria, assigned through the RDP Bayesian classifier algorithm [28], or the host plants were

The metabolic profiling resulted in a matrix that could be interpreted in binary form, and from which it was possible to determine a set of distances (binary Bray-Curtis) for classifying samples through a cluster analysis (unweighted pair group method with arithmetic averages, UPGMA). Ordination methods were carried out

**20**

#### **3.1 Wild fruits: A microenvironment of diverse lactic acid bacteria**

Out of 41 isolates, the most frequently observed genus was *Lactiplantibacillus* (31 isolates), followed by *Lactococcus* (3 isolates), *Weissella* (3 isolates), and *Enterococcus* (1 isolate). Three isolates showed large divergence and were not identified by the taxonomic assignment algorithm (i.e. UTN39, UTN41, and UTN88) (**Figure 2a**). The former three isolates may represent unreported lineages or species. The presence of *Lactococcus* in plants or fruit is rare: thus, we only found few isolates belonging to such genus.

Isolates showed a remarkable distance to the outgroup reference samples, and most were included within a clade formed by *Lactiplantibacillus,* where they showed relatively small distances (i.e. small branch lengths). However, within this inclusive group of *Lactiplantibacillus* there was an ingroup with strong support (Bayesian posterior probabilities = 1) and formed by *Weissella, Enterococcus,* and *Lactococcus*. This paraphyletic ingroup, within a more inclusive group formed mostly by *Lactiplantibacillus* should be eventually resolved by the inclusion of additional molecular markers (**Figure 2a**). The paraphyletic ingroup contains samples that belong to the plant species *Costus* sp., which occurs exclusively to this clade (**Figure 2b**).

#### **3.2 The metabolic profile reveals the divergent properties of selected LAB**

LAB strains may present specific metabolic traits as a result of their microenvironmental origin (i.e. different species of fruits) and possess a unique portfolio of enzymes that allow them to metabolize various compounds found in the host plant or fruit matrices. We present a metabolic profile together with other properties that were analyzed in the obtained isolates (**Figure 3**). Within *Lactiplantibacillus*, the isolates showed a variable capacity to ferment sugars and hydrolyze esculin. These features were strain-dependent. Among the available isolates, UTN39 and UTN76 were the only two samples that metabolized ARG, while UTN37 and UTN39 hydrolyzed urea. The latter is a relevant characteristic for selecting probiotic strains [2]. Of particular relevance were the locations of UTN76 and UTN39 on the previously presented phylogenetic hypotheses (**Figure 2**); these two isolates showed large genetic distances relative to the other samples, or unique positions in clades that diagnose them as different or remarkable lineages. The observed patterns in the metabolic profile are rather complex. UTN37, UTN39, and UTN76 show noteworthy properties; they, however, differ in their response to other substrates (**Figure 3**); thus, the corresponding dendrogram, which results from clustering the observed metabolic profile, hints to four main groups of isolates (**Figure 4**).

#### **Figure 2.**

*(a) Phylogenetic hypothesis of the isolated samples and corresponding genera of bacteria. (b) The hypothesis with the corresponding host genera of plants. Branch support values are posterior probabilities from the applied Bayesian inference.*

The ability to utilize the α-galacto-oligosaccharides-family (αGOS), d-melibiose [α-Gal-(1 → 6)-Glu], as well as the raffinose-family oligosaccharide (RFO) d-raffinose, seems to be a common feature among all the tested isolates. On the

**23**

property [39].

**Figure 3.**

and antibiotic profiles.

*spectrum (AS) is also presented for each isolate.*

*Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity…*

other hand, the metabolisms of the trisaccharide d-melezitose (α-Glu-(1 → 3)-β-Fru-(2 → 1)-α-Glu) and the disaccharides d-trehalose [α-Glu-(1 → 1)-α-Glu] and d-turanose (α-Glu-(1 → 3)-α-Fru) were strain-specific and restricted mainly to the *Lactoplantibacilaceae.* Although not classified, in a distinct lineage near the base of the tree, and with relatively large genetic distances, the isolates UTN41 and UTN88 did not metabolize most of the oligosaccharide substrates. The dissimilarity of individual isolates was supported by the different use of glycerol, trehalose, and sucrose. UTN40 was in the same lineage group as UTN37 and UTN38; however, it showed a distinct metabolic and antibiotic pattern, being the only one with resistance to AM10 and CN10. The isolate UTN76 showed a similar metabolic pattern to UTN88, both were the only isolates that did not metabolize p-n-p-β-D-cellobioside, p-n-p-phosphate, and proline. The *Lactococcus* species (UTN53, UTN86, and UTN87) were grouped in the same clade but were different in both the metabolic

*Annotated phylogeny and heatmap showing the metabolic profiles of the LAB isolates. The phylogeny is an alternative depiction of the one shown in Figure 2 and serves as a reference to the profiles. The antimicrobial* 

The results revealed the production of β-galactosidase, α-glucosidase, β-glucosidase, and p-n-p-α-β-galactoside in some isolates. Although all isolates were originated from plants, there were differences in the utilization of mannitol and fructose. Previous studies have indicated that strains of *L. plantarum*, which were isolated from plant environments, often were able to metabolize these carbohydrates, and which has been considered as a fructophilic

*DOI: http://dx.doi.org/10.5772/intechopen.94179*

*Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity… DOI: http://dx.doi.org/10.5772/intechopen.94179*

#### **Figure 3.**

*Ecosystem and Biodiversity of Amazonia*

**22**

**Figure 2.**

*Bayesian inference.*

The ability to utilize the α-galacto-oligosaccharides-family (αGOS), d-melibiose

*(a) Phylogenetic hypothesis of the isolated samples and corresponding genera of bacteria. (b) The hypothesis with the corresponding host genera of plants. Branch support values are posterior probabilities from the applied* 

[α-Gal-(1 → 6)-Glu], as well as the raffinose-family oligosaccharide (RFO) d-raffinose, seems to be a common feature among all the tested isolates. On the

*Annotated phylogeny and heatmap showing the metabolic profiles of the LAB isolates. The phylogeny is an alternative depiction of the one shown in Figure 2 and serves as a reference to the profiles. The antimicrobial spectrum (AS) is also presented for each isolate.*

other hand, the metabolisms of the trisaccharide d-melezitose (α-Glu-(1 → 3)-β-Fru-(2 → 1)-α-Glu) and the disaccharides d-trehalose [α-Glu-(1 → 1)-α-Glu] and d-turanose (α-Glu-(1 → 3)-α-Fru) were strain-specific and restricted mainly to the *Lactoplantibacilaceae.* Although not classified, in a distinct lineage near the base of the tree, and with relatively large genetic distances, the isolates UTN41 and UTN88 did not metabolize most of the oligosaccharide substrates. The dissimilarity of individual isolates was supported by the different use of glycerol, trehalose, and sucrose. UTN40 was in the same lineage group as UTN37 and UTN38; however, it showed a distinct metabolic and antibiotic pattern, being the only one with resistance to AM10 and CN10. The isolate UTN76 showed a similar metabolic pattern to UTN88, both were the only isolates that did not metabolize p-n-p-β-D-cellobioside, p-n-p-phosphate, and proline. The *Lactococcus* species (UTN53, UTN86, and UTN87) were grouped in the same clade but were different in both the metabolic and antibiotic profiles.

The results revealed the production of β-galactosidase, α-glucosidase, β-glucosidase, and p-n-p-α-β-galactoside in some isolates. Although all isolates were originated from plants, there were differences in the utilization of mannitol and fructose. Previous studies have indicated that strains of *L. plantarum*, which were isolated from plant environments, often were able to metabolize these carbohydrates, and which has been considered as a fructophilic property [39].

#### **Figure 4.**

*Dendrogram from a cluster analysis on the metabolic profile for the obtained isolates. Four groups are possible to define at the established distance (dotted vertical line). The assigned species and the host plant species are represented as color codes.*

## **3.3 The antimicrobial profile reveals that inhibitory activity is related to microenvironmental origin**

AMX25 was the only antibiotic for which all isolates showed susceptibility, while all isolates showed innate resistance to MET5. Previous studies have indicated that Lactobacilli have a high natural resistance to metronidazole, as well as antibiotics that inhibit the synthesis of proteins such as chloramphenicol, erythromycin, clindamycin, and tetracyclines [40]. UTN41 was susceptible to almost all antibiotics. Among the *Lactiplantibacillus* genus, the isolates UTN76 and UTN88 were resistant to AMC30, and UTN84 and UTN89 were resistant to TE30. However, this relevant pattern did not show consistency with the fruit host, as the plant species

**25**

**Figure 5.**

*Annotated phylogeny and heatmap showing antimicrobial profiles of the LAB isolates.*

*Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity…*

*Chrysophyllum oliviforme*, which was the origin of the resistant samples, was also the origin of other isolates in *Lactiplantibacillus*, but which did not coincide in their resistance pattern (**Figure 5**). Another relevant aspect was that of UTN68 (*Lactiplantibacillus*), which originated from *Theobroma grandifolium* and UTN40 (*Weissella*) from *Costus* sp., which were the only isolates showing resistance to

Although resistance to gentamicin and kanamycin is considered a health concern, the isolates that showed resistance to both antibiotics were control reference strains that were isolated from dairy and human intestine. In general, patterns in the antibiogram were broad and yet inconclusive, as the position of isolates and plant hosts in canonical space show that further evidence is necessary to establish definitive patterns of association (**Figure 6**). However, this is also evidence of large variability across samples, with no conclusive patterns in isolated species or their

Although antibiotic resistance is a criterium to be considered from a biosafety perspective, it has been shown that the probiotic strains of starter culture strains

*DOI: http://dx.doi.org/10.5772/intechopen.94179*

AM10 (**Figure 5**).

host plants.

*Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity… DOI: http://dx.doi.org/10.5772/intechopen.94179*

*Chrysophyllum oliviforme*, which was the origin of the resistant samples, was also the origin of other isolates in *Lactiplantibacillus*, but which did not coincide in their resistance pattern (**Figure 5**). Another relevant aspect was that of UTN68 (*Lactiplantibacillus*), which originated from *Theobroma grandifolium* and UTN40 (*Weissella*) from *Costus* sp., which were the only isolates showing resistance to AM10 (**Figure 5**).

Although resistance to gentamicin and kanamycin is considered a health concern, the isolates that showed resistance to both antibiotics were control reference strains that were isolated from dairy and human intestine. In general, patterns in the antibiogram were broad and yet inconclusive, as the position of isolates and plant hosts in canonical space show that further evidence is necessary to establish definitive patterns of association (**Figure 6**). However, this is also evidence of large variability across samples, with no conclusive patterns in isolated species or their host plants.

Although antibiotic resistance is a criterium to be considered from a biosafety perspective, it has been shown that the probiotic strains of starter culture strains

*Ecosystem and Biodiversity of Amazonia*

**24**

**Figure 4.**

*represented as color codes.*

**3.3 The antimicrobial profile reveals that inhibitory activity is related** 

AMX25 was the only antibiotic for which all isolates showed susceptibility, while all isolates showed innate resistance to MET5. Previous studies have indicated that Lactobacilli have a high natural resistance to metronidazole, as well as antibiotics that inhibit the synthesis of proteins such as chloramphenicol, erythromycin, clindamycin, and tetracyclines [40]. UTN41 was susceptible to almost all antibiotics. Among the *Lactiplantibacillus* genus, the isolates UTN76 and UTN88 were resistant to AMC30, and UTN84 and UTN89 were resistant to TE30. However, this relevant pattern did not show consistency with the fruit host, as the plant species

*Dendrogram from a cluster analysis on the metabolic profile for the obtained isolates. Four groups are possible to define at the established distance (dotted vertical line). The assigned species and the host plant species are* 

**to microenvironmental origin**

#### **Figure 6.**

*Non-metric multidimensional scaling (NMDS) that represents, in two dimensions, the distances among isolates and host plants according to the observed pattern of antibiotic resistance. a) Tile for the spatial relationships in NMDS space between antibiotics and the studied bacterial isolates. b) Tile for spatial relationships in NMDS space between antibiotics and the host plants of the studied bacterial isolates. Short distances among these objects in the canonical space represent strong associations of either resistance or susceptibility. The antimicrobial spectrum is shown here as a cofactor showing a directional trend. AMX25 was removed from this analysis as it was not informative (i.e. susceptibility as a zero-response constant). The colored areas facilitate the interpretation of the antibiotics position.* L. fermentum *and* L. plantarum *were used as a reference in the tests.*

are resistant to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracyclines, and chloramphenicol [4, 7]. Another study has reported that Lactobacilli isolated from fermented olives were resistant to cephalosporins, streptomycin, and kanamycin [41]. Overall, the observed pattern is for high variability or diversity in the response to antibiotics as there is considerable dispersion in the response within and among bacterial species or plant hosts (**Figure 6**). However, there were also isolates with unique resistance patterns, which will require a further inquiry into their molecular and physiological properties. Undoubtedly, a full safety assessment with a robust identification of the strains and an in vitro evaluation of the potential risks is needed; particularly if these are intended to be used as additives in food products.

It is known that some LAB strains produce a wide variety of anti-pathogenic compounds, like bacteriocins, ethanol, organic acids, diacetyl, acetaldehydes, hydrogen peroxide (H2O2), and peptides [42, 43]. When we analyzed the antimicrobial spectrum against ten Gram-negative and Gram-positive bacteria, including closely related species and pathogens such as *Salmonella enterica, Shigella sonnei, Escherichia coli, Enterobacter, Staphylococcus aureus,* we observed that the isolates showed high inhibitory potential, as none had values below six and were defined as broad-spectrum (**Figures 3** and **4**). The inhibitory effect of LAB strains may result from a combination of competition for metabolic substrates, growth suppression by organic acids, and bacteriocin secretion. Recently, we showed that some of the Lactobacilli strains inhibited *Salmonella enterica subsp. enterica* ATCC51741 and *E. coli* ATCC25922 at both the early and logarithmic stages of bacterial growth *in vitro* and *ex vitro* [44, 45, 46]. Also, we showed that one selected LAB strain from the

**27**

*Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity…*

culture formulations for dairy-based fermented food products [46].

*Lactococcus* genera harbored interesting functional properties to be used in starter

The Amazon rainforest is a sizeable reservoir of plants, animals, and bacterial diversity. For Ecuador, the Amazon region could be a significant source of new bioproducts, based on the transformation of biodiversity [47]. Subtropical wild fruits have a relevant ethnobotanical significance, as they are mostly consumed by indigenous people as food or natural medicine; however, the bacterial microbiota of those fruits has not been assessed. In this research, we investigated the lactic acid bacteria diversity associated with several wild fruits collected from the Amazon region of Ecuador. Their remarkable inhibitory potential towards Gram-negative bacteria might be related to their capacity to produce various antimicrobial substances, that when applied to food products might prevent the growth of undesirable microorganisms. A better understanding of the metabolic capacity of these microorganisms will further complement our knowledge about the development of a novel starter or preservative culture for fruit- and vegetable-based foods. The prospective comparative exploration of the genomes of LAB strains from various plant or fruit origins would be of particular interest to provide information on their adaptations to different food-matrices and to further explore biotechnological

Genotype-functional correlation studies contribute to the discovery of new biotechnological properties for several species. The results from the present study supported our hypothesis that LAB strains from wild fruits of the Amazon Region of Ecuador carry noteworthy characteristics that could be inherent to their ecological niches or environmental origin and that could be developed for biotechnological applications. Several strains were found capable of producing antimicrobials with high inhibitory potential against commensal and spoilage bacteria and are

This research was financed by the Technical University of the North, Centre of Research (CUICYT)-Grant no. 01388/2014 and Grant 0179/2016. The authors gratefully acknowledge the generous technical support of Ulcuango M, Torres J., and Benavidez A. GNT was supported in part by the Prometeo Project of the Secretary for Higher Education, Science, Technology and Innovation (SENESCYT,

The authors report no conflicts of interest. The authors are responsible for the

*DOI: http://dx.doi.org/10.5772/intechopen.94179*

**4. Conclusions**

applications.

promissory natural food preservatives.

**Acknowledgements**

**Conflict of interest**

content and writing of this article.

2014-2016).

*Lactococcus* genera harbored interesting functional properties to be used in starter culture formulations for dairy-based fermented food products [46].
