The introduction of antibiotics into medical practice was a revolutionary event, but the selective pressure on pathogens caused a backlash: the emergence of antibiotic-resistant forms. The World Health Organization (WHO) predicts that this situation may lead to a decline in the effectiveness of antibiotics and make infectious diseases caused by pathogens one of the leading death causes worldwide by 2050 (O’Neill, 2016). One way to solve the problem of antibiotic resistance is to find new effective antimicrobial compounds to expand the arsenal of available drugs. The principal producers of antibiotics are actinobacteria and fungi isolated from complex multicomponent biocenoses, mainly from soils (Bérdy, 2005). This is due to the fact that, in the course of evolution, competition among microorganisms has been forcing them to produce various antimicrobial substances as a means of defense. Animal gut microbiota is another multicomponent biocenosis that can be viewed as a source of antibiotic producers. At the same time, poorly studied habitats are also promising in terms of finding producers of new antibiotics (Donadio et al., 2007). Considering both circumstances, it can be expected that the intestines of invertebrate animals, including millipedes, are a promising ecological system for the isolation of antibiotic-producing bacteria (Kaltenpoth, 2009).

Bacterial communities of millipede guts participate in digestion and methane biosynthesis, as well as maintain the microbiome stability. However, not all bacteria excreted from the gut of Diplopoda millipedes belong to the gut microbiome; many of them are transient species consumed with food. Using scanning electron microscopy, it was shown that the intestinal wall and cuticle, as well as the rest of the peritrophic membrane, were colonized by various bacteria, including actinobacteria, for which the intestinal environment appeared favorable (Szabo et al., 1992; Polyanskaya et al., 1996). These results suggest that part of the bacteria excreted from the intestines represent the microbiome of millipedes (Byzov et al., 1993; Byzov, 2005; König and Varma, 2006).

The present study was undertaken to continue our work on antimicrobial properties of bacteria isolated from the gut of the tropical millipede Nedyopus dawydoffiae (Glukhova et al., 2018).

The goal of this work was to investigate the antibiotic activity of bacterial strains isolated from the gut of the tropical diplopods Nedyopus dawydoffiae and Orthomorpha sp. and their feeding substrates.

MATERIALS AND METHODS

Objects. The study concerned bacteria isolated from the gut of millipedes Nedyopus dawydoffiae and Orthomorpha sp. (class Diplopoda) and their food substrates: rotten wood and leaf litter, respectively (Fig. 1). Millipedes were collected manually in the Cát Tiên National Park located in an indigenous monsoon rainforest in the south of Vietnam; they were kept in laboratory soil microcosms on natural substrates, which also served them as food. Bacteria were isolated in pure cultures by inoculating gut contents and food substrates on Gause agar medium no. 1.

Fig. 1.
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Millipedes Nedyopus dawydoffiae (top) and Orthomorpha sp. (bottom).

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Test strains. Antibiotic activity of bacterial isolates was assessed using the following test strains of gram-positive bacteria: methicillin-resistant Staphylococcus aureus INA 00761 (MRSA), methicillin-sensitive Staphylococcus aureus FDA 209P (MSSA), Micrococcus luteus NCTC 8340, Bacillus subtilis ATCC 6633, B. pumilus NCTC 8241, B. mycoides 537, and vancomycin-resistant Leuconostoc mesenteroides VKPM В‑4177 (VRLM); gram-negative bacteria: Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 with multiple drug resistance (MDR); fungi: Aspergillus niger INA 00760 and Saccharomyces cerevisiae RIA 259.

Nutrient media. The strains under study were cultured on the Gause media nos. 1 and 2, oat-, and soya-based agar media. The composition of the media was as follows (% in tap water):

(1) complete modified Gause medium no. 2: glucose, 1.0; peptone, 0.5; tryptone, 0.3; NaCl, 0.5; agar, 2.0; рН 7.2–7.4;

(2) Gause medium no. 1: starch, 2.0; KNO3, 0.1; K2HPO4, 0.05; MgSO4, 0.05; NaCl, 0.05; FeSO4, 0.001; agar, 2.0; pH 7.2–7.4;

(3) oat medium: oat flour, 2.0; agar, 2.0; pH 7.2;

(4) soya medium: soybean flour, 2.0; glucose, 1.0; NaCl, 0.5; agar, 2.0; pH 7.2.

For submerged cultivation of actinobacteria, the following eight liquid growth media developed for actinobacterial antibiotic producers in the Gause Institute were used (% in tap water):

(1) STR: glucose, 1.0; peptone, 0.5, tryptone, 0.3; NaCl, 0.5; рН 7.2–7.4;

(2) A4: glucose, 1; soybean flour, 1; NaCl, 0.5; CaCO3, 0.25; pH 6.8;

(3) 6613: starch, 2; maize extract, 0.3; KNO3, 0.4; NaCl, 0.5; CaCO3, 0.5; pH 7.0–7.2;

(4) sucrose: sucrose, 2; soybean flour, 1; NaCl, 0.3; CaCO3, 0.3; pH 6.8–7.0;

(5) 2663: glycerol, 3; soybean flour, 1.5; NaCl, 0.3; CaCO3, 0.3; pH 7.0;

(6) 330: sucrose, 2.1; starch, 0.85; pea flour, 1.5; CaCO3, 0.5; NaCl, 0.5; NaNO3, 0.5; pH 7.0;

(7) 5539: glycerol, 2; soybean flour, 0.5; (NH4)2SO4, 0.15; NaCl, 0.3; CaCO3, 0.3; pH 6.8;

(8) Am: sucrose, 4; K2HPO4, 0.1; Na2SO4, 0.1; NaCl, 0.1; (NH4)2SO4, 0.2; dry yeast extract, 0.25, FeSO4·7H2O, 0.0001; MnCl2·4H2O, 0.0001; NaI, 0.00005; CaCO3, 0.2; pH 6.5–6.7.

Cultivation conditions. Test strain cultures were grown for 24 h at 28°C for fungi and L. mesenteroides and at 37°C for all other bacterial strains. Fermentation of actinobacterial cultures was carried out as submerged cultivation in 750-mL Erlenmeyer flasks containing 150 mL of medium on a rotary shaker at 220 rpm and 28°C in two stages. At the first stage, to obtain material for inoculation, a 1-cm2 fragment of oat agar with the bacteria under study was excised and transferred into STR medium; these cultures were grown for 4 days. At the second stage, 5-mL aliquots of the culture liquid were used to inoculate flasks with seven different media.

Species identification. Actinobacterial species were identified based on morphological traits and on analysis of 16S rRNA gene sequences. Morphological description included the structure of sporophores, pigmentation of aerial and substrate mycelium, and pigment secretion into the medium (Gause et al., 1983; Bergey, 1984). DNA was isolated from the biomass of 3-day-old cultures grown in liquid STR medium. Bacterial genomic DNA was isolated using a PowerSoil DNA Kit (MO BIO, United States). Fragments of 16S rRNA genes were amplified by PCR using a PCR Master Mix kit (Thermo Scientific, United States) with the universal bacterial primers 27f (aga gtt tga tcc tgg ctcag) and 1492r (tac ggy tac ctt gtt acg act t). The reaction was conducted in an Applied Biosystems 2720 Thermal Cycler (United States) according to the following protocol: (1) 94°C, 5 min; (2) 30 cycles of 1 min at 94°C, 1 min at 51°C, and 2 min at 72°C; (3) 7 min at 72°C. The PCR products were analyzed by electrophoresis in a 1% agarose gel (using Tris-borate buffer, TBE) at electric field strength of 7.6 V/cm. The products were purified by DNA precipitation under mild conditions using 0.125 M ammonium acetate in 70% ethanol. Nucleotide sequences were determined using the Sanger’s method with the universal bacterial primers 27f, 341f (cct acg gga ggc agc ag), 519r (gta tta ccg cgg ctg ctg), 785f (ggm tta gat acc tgg tag tcc), 907r (ccg tca att cct ttg agt tt), 1100r (ggg ttg cgc tcg ttg), 1114f (gca acg agc gca acc c), 1392r (acg ggc ggt gtg trc), and 1492r on an automated Applied Biosystems 3500 analyzer. Sequences were assembled using the Mega 7 software. The obtained sequences were compared to 16S rRNA gene sequences of bacterial type strains available in GenBank (blast.ncbi.nlm.nih.gov/Blast.cgi) and Ribosomal Database Project (RDP; http://rdp. cme.msu.edu/).

Assessment of antibiotic activity. To evaluate antimicrobial activity of the strains under study, 0.1-mL aliquots of the culture liquid were loaded into wells 9 mm in diameter made in Gause agar no. 2 inoculated with test microorganisms. Antibiotic activity of the culture liquid was assessed on days 4 and 7 of submerged cultivation for actinobacteria and on days 2 and 4 for bacteria of other taxonomic groups. The result was presented as the diameter (mm) of the zone where the growth of the test strain was suppressed. Statistical analysis of the data was performed with Excel 2016; antibiotic activity in the culture liquid was analyzed in five replicates.

RESULTS AND DISCUSSION

Altogether, we studied 72 bacterial strains presented in Table 1. The dominant group among the actinobacteria studied were streptomycetes. The obtained sequences of 16S rRNA genes were aligned with the sequences of type strains from the RDP database. Sequences that exhibited at least 98% identity were deposited into the GenBank database. Species identification of the isolated bacteria revealed great taxonomic diversity: the isolates represented 25 genera, and 16 of them belonged to the class Actinobacteria.

Table 1. Taxonomic affiliation of the isolated bacterial strains based on the analysis of 16S rRNA gene sequences
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Actinobacteria dominate in the gut bacterial communities of millipedes and represent the second most abundant group after γ-proteobacteria (König and Varma, 2006; Knapp et al., 2010). According to the previous publications, the most abundant genus of actinobacteria in the gut of Diplopoda is Streptomyces (Byzov et al., 1993). Actinobacteria of millipede guts participate in hydrolysis of the gut content; their abundance is the greatest in the posterior section (Byzov et al., 1993; Zvyagintsev et al., 1996; Polyanskaya et al., 1996). Subsequent studies revealed a great diversity of actinobacterial genera. For instance, König and Varma (2006) isolated members of the genera Micromonospora, Actinomadura, and Streptosporangium, as well as nocardioide-like forms. Data obtained in our work agree with these results.

Evaluation of antibiotic activity revealed a considerable share of active strains among the isolates listed in Table 1. The spectra of their antimicrobial activity against the collection test strains are presented in Table 2.

Table 2. Range of antimicrobial activity of the isolated bacterial strains
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As it follows from the data presented in the tables, all isolated streptomycetes exhibited antibiotic activity, as it could be expected from decades of the experience accumulated during the golden era of antibiotics. According to the MIBiG database (Minimum Information about a Biosynthetic Gene cluster), members of the genus Streptomyces have an average total genome length of 9 Mb; at the same time, this genus is a clear leader in the number of detected biosynthetic gene clusters (BGCs): to date, there are 637 of them (NCBI (Assembly), www.ncbi.nlm.nih.gov/assembly; MIBiG, http://mibig.secondarymetabolites.org). It is known that members of the genus Streptomyces produce 70–80% of known secondary metabolites, but after them, important producers of antibiotics are members of “rare” genera of actinobacteria (Bérdy, 2005; Ventura et al., 2007; Raja and Prabakarana, 2011). In this work, in addition to streptomycetes, we isolated members of rare actinobacterial genera: Actinoplanes, Amycolatopsis, Kitasatospora, Lechevalieria, Micromonospora, Nocardiopsis, and Saccharopolyspora, including some strains with antibiotic activity. Detection of antimicrobial activity in actinobacteria of rare genera, which are considerably less studied than streptomycetes, increases the probability of finding new antibiotics (Fig. 2).

Fig. 2.
figure 2

Distribution of the strains studied by bacterial groups: A, gut of Nedyopus dawydoffiae; B, food substrate of Nedyopus dawydoffiae (rotten wood); C, gut of Orthomorpha sp.; D, food substrate of Orthomorpha sp. (leaf litter).

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Since antibiotics are secondary metabolites, their biosynthesis is a trait that can exhibit intraspecies variation. For instance, Table 2 includes two strains of S. pratensis that differed in their antimicrobial activity against three strains of bacilli and the fungus Aspergillus niger: strain INA 01179 was active against Bacillus subtilis ATCC 6633, B. pumilus NCTC 8241, and B. mycoides 537, but not against the fungus; in contrast, strain INA 01182 was not active against these bacilli but exhibited antimycotic activity. Another example is two isolated strains of S. termitum: strain INA 01244 was active against the gram-positive bacterium Leuconostoc mesenteroides VKPM B-4177 and the gram-negative bacterium E. coli ATCC 25922, whereas strain INA 01245 did not possess such activity; in other tests, both strains exhibited identical activity (which indicates that members of this species synthesize more than one antibiotic). In a previous study, we found that S. antibioticus strains isolated from a nest of the black garden ant Lasius niger were heterogeneous in respect of production of actinomycin antibiotics (Efimenko et al., 2020). Heterogeneity by this trait is determined by the presence of competition in the biocenosis on the one hand and by available nutrient sources on the other hand. In the present work, we observed that two isolates of S. termitum obtained from the same population (leaf litter) and grown as submerged cultures in two media exhibited differences both in the level of antibiotic synthesis and in the spectra of antimicrobial activity (Fig. 3).

Fig. 3.
figure 3

Spectra of antibiotic activity of S. termitum strains grown on media 330 and 2663 after 4 and 7 days of cultivation.

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When assessing the antimicrobial spectra of the identified producers, we paid special attention to the test strains Staphylococcus aureus INA 00761 (MRSA), E. coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853, which are included in the WHO list of species of antibiotic resistance concern (O’Neill, 2016; Tacconelli, 2017; Efimenko, 2019). In this work, among the isolates representing rare genera of actinobacteria, there were strains producing antibiotics active against the above test strains, and their antimicrobial activity had not been described previously. For instance, Kitasatospora saccharophila was described as a new species in 2009 (Li et al., 2009). However, available publications do not mention any antibiotic production by this species. In our study, the strain Kitasatospora saccharophila INA 01226 produced antimicrobial compounds active against Leuconostoc mesenteroides VKPM B-4177 (VRLM), Micrococcus luteus NCTC 8340, and Aspergillus niger INA 00760. The species Nocardiopsis umidischolae was first isolated in 2001 from dust in a water-damaged school building, and the type strain Nocardiopsis umidischolae 66/93 (=DSM 44362 = NRRL B-24122) was described (Peltola et al., 2001). Available publications report no data on antibiotic production in this species. However, Nocardiopsis umidischolae strain INA 01230 isolated in our work from rotten wood, food substrate of Nedyopus dawydoffiae, exhibited activity against all test strains of gram-positive bacteria used, including methicillin-resistant Staphylococcus aureus INA 00761 (MRSA), and against the yeast Saccharomyces cerevisiae RIA 259. That is, the strains Kitasatospora saccharophila INA 01226 and Nocardiopsis umidischolae INA 01230 have for the first time been described to possess antimicrobial activity, in particular, against resistant forms of pathogenic microorganisms.

The genus Micromonospora is the second richest source of isolation of natural antibiotic producers after Streptomyces (Wagman and Weinstein, 1980). First reports on antibacterial activity of Micromonospora members appeared in 1942, and gentamicin, an aminoglycoside isolated from Micromonospora purpurea (the species was reclassified as Micromonospora echinospora Luedemann, Brodsky, 1964), was described in 1963, which subsequently inspired broad-scale screening of this rare genus actinobacteria for the presence of antibiotics (Wagman and Weinstein, 1980). In 2019, three novel isoflavonoid glycosides: daidzein-4'-(2-deoxy-α-l-fucopyranoside), daidzein-7-(2-deoxy-α-l-fucopyranoside), and daidzein-4',7-di-(2-deoxy-α-l-fucopyranoside), were isolated and identified from the fermentation broth of Micromonospora aurantiaca 110B. It was shown that all three compounds possessed cytotoxic activity, but did not exhibit activity against Candida albicans, MRSA, and E. coli (Wang et al., 2019). In 2020, the complete genome of M. aurantiaca sp. 01 was obtained; based on the 16S rRNA gene sequence, it had 99.28% identity with the type strain M. aurantiaca ATCC 27029T. Genome sequence analysis showed that strain M. aurantiaca sp. 01 possessed 77 clusters of genes involved in biosynthesis of secondary metabolites. It was predicted that they participate in production of polyketides, nonribosomal peptides, bacteriocins, and many other compounds. Among them, 42 genes clusters were annotated as associated with 37 known secondary metabolites, including antibiotics. Among antibiotics predicted based on genome analysis, targeted MS/MS analysis detected kanamycin, which was first obtained from a strain of M. aurantiaca. Furthermore, a study of organization of biosynthetic gene clusters revealed 21 clusters of antibiotic determinants, which is more than in other species of Micromonospora (Hu et al., 2020). In our study, the strain Micromonospora aurantiaca INA 01238 isolated from the gut of Orthomorpha sp. exhibited high levels of activity against all gram-positive test bacteria, including MRSA, as well as against Saccharomyces cerevisiae RIA 259. That is, antibiotic activity observed in this strain was different from the activity of antibiotics isolated previously from the same species, and we are planning further investigation of its the chemical nature.

The species Micromonospora tulbaghiae was first described in 2010; the type strain TVU1T (=DSM 45142T = NRRL B-24576T) was isolated from leaves of wild garlic (Tulbaghia violacea). This strain was shown to exhibit moderate activity against Mycobacterium aurum A+ and no activity against Enterococcus faecium (VanA), E. coli ATCC 25922, and Staphylococcus aureus ATCC 25923 (Kirby and Meyers, 2010). In our study, the strain Micromonospora tulbaghiae INA 01239 was found to be active against all gram-positive bacteria tested, including methicillin-resistant Staphylococcus aureus INA 00761 (MRSA), as well as against the yeast strain Saccharomyces cerevisiae RIA 259, which was different from the activity described previously.

In 2000, Fukami et al. described an antibiotic isolated from Actinoplanes capillaceus and active against B. subtilis, Sac. cerevisiae, and E. coli (Fukami et al., 2000). The strain Ac. capillaceus INA 01251 isolated in our work had a different spectrum of antimicrobial activity and was active against Leuconostoc mesenteroides VKPM B-4177 (VRLM) and Aspergillus niger INA 00760. Accordingly, the compound produced by strain INA 01251 can be considered promising and is worth further chemical investigation.

Previously, no information was available on antibiotic activity of two species belonging to the rare genera of actinobacteria, Amycolatopsis bullii and Saccharopolyspora dendranthemae, described in the recent decade (Zucchi et al., 2012; Zhang et al., 2013). In our work, we found that Amycolatopsis bullii INA 01250 was highly active against all gram-positive bacteria tested and against S. cerevisiae RIA 259, whereas Saccharopolyspora dendranthemae INA 01240 was active against Leuconostoc mesenteroides VKPM B-4177, which is naturally resistant to glycopeptide antibiotics of the vancomycin group, as well as against S. cerevisiae RIA 259.

Among prokaryotes of other taxonomic groups, the best-known producers are members of the class Bacilli (Bérdy, 2005). In our study, the most promising potential producers were Peribacillus simplex INA 01386 and Bacillus toyonensis INA 01397, which were active against MRSA. While Bacilli are a well-known source of antibiotic producers, descriptions of new antibiotic compounds of this origin keep appearing (Wang et al., 2020a, 2020b). Two strains of Stenotrophomonas rhizophila (INA 01299 and INA 01298) were isolated from leaf litter and rotten wood. These strains differed in their antibiotic activity: strain INA 01298 was active against MSSA, and strain INA 01299 was active against VRLM and mildly active against Aspergillus niger INA 00760. Previously, we showed that Stenotrophomonas rhizophila INA 01137 (endobiont of the mushroom-forming fungus Coprinellus micaceus) was active against St. aureus INA 0076 and E. coli ATCC 25922, as well as against Aspergillus niger INA 00760 (Efimenko et al., 2016). Previous publications indicate that Stenotrophomonas rhizophila can produce compounds active against a number of phytopathogenic fungi and the human pathogen Candida albicans (Wolf et al., 2002).

Bacteria isolated from the guts of millipedes and from their food substrates exhibited great diversity, both in terms of species affiliation and in spectra of antibiotic activity. The isolated strains were dominated by actinobacteria, which represented not only the genus Streptomyces, but also 14 other genera: Actinoplanes, Agrococcus, Amycolatopsis, Arthrobacter, Herbiconiux, Kitasatospora, Lechevalieria, Microbacterium, Micrococcus, Micromonospora, Mycobacterium, Nocardiopsis, Plantactinospora, and Saccharopolyspora. Among them, there were species that had not been previously described as antibiotic producers or that exhibited a different range of antimicrobial activity. For instance, it is for the first time that we report antimicrobial activity, in particular, against antibiotic-resistant forms of pathogenic microorganisms, for Amycolatopsis bullii, Kitasatospora saccharophila, Nocardiopsis umidischolae, and Saccharopolyspora dendranthemae. Two species of the genus Micromonospora (Micromonospora aurantiaca and M. tulbaghiae) were for the first time shown to exhibit activity against the methicillin-resistant strain Staphylococcus aureus INA 00761 (MRSA), and Actinoplanes capillaceus was found to be active against the vancomycin-resistant Leuconostoc mesenteroides VKPM B-4177 (VRLM). Activity against the resistant test strains St. aureus INA 00761 (MRSA) and L. mesenteroides VKPM В-4177 (VRLM) makes these isolates promising candidates for isolation of antibiotics that can overcome drug resistance.

The number of strains with antimicrobial activity isolated from millipede guts was as great as the number of active strains isolated from decaying plant debris and apparently was not affected by the gut environments. Presumably, this is related to the climate conditions in this habitat (Deshcherevskaya et al., 2013; Chernov et al., 2019). Previous studies showed that, judging by the numbers of ribosomal gene copies, tropical soil samples from largely intact monsoon forests of the Cát Tiên National Park are characterized by high levels of microbial abundance comparable to those of the richest soils of temperate latitudes, e.g., chernozems (Semenov et al., 2018; Chernov et al., 2019). The abundance of microorganisms promotes their intense competition in which biosynthesis of antibiotics represents an important evolutionary adaptation (Gause, 1934).

The great diversity of actinobacterial species isolated from the guts of tropical diplopods and their food substrates, as well as the observed high levels of their antibiotic activity, justify the need for further investigation of bacteria associated with this natural ecosystem. The isolated and selected actinobacteria will subsequently be subject to chemical studies as potential producers of new antibiotics.