Microorganisms’ presence inside the cave systems are commonly oligotrophic or chemolithotrophic in nature and impose precise nutrients for their growth. Hence, it is very difficult to isolate some rare or specific bacteria from these sources (Portillo et al., 2008). In our study, the maximum number of bacteria was obtained from SCA media (n = 92; 39.1%) followed by ISP7 (n = 57; 24.2%), ISP5 (n = 26; 11.0%), LB (n = 24; 10.2%), TSA (n = 21; 8.9%) and TH2O media (n = 17; 7.2%). This finding was similarly reported by Adam et al. (2018) who state that 46 actinobacteria isolates collected from cave moon milk deposits were obtained from starch casein nitrate medium. Moreover, Tomova et al. (2013) reported that the greatest number of heterotrophic bacteria (46 isolates) collected from the Gallery with the drawings in Magura cave, Bulgaria was isolated from nutrient agar (NA) media. We have found that out of 235 isolates, 142 bacterial isolates were gram-positive (60.4%) and 93 of the gram-negative (39.5%). These findings were consistent with Bhullar et al. (2012) who state that out of 93 bacterial strains, 33% Gram-positive and 63% Gram-negative was isolated from Lechuguilla cave, New Mexico.
In this study, we have found significant antimicrobial activity in cave bacterial isolates against a gram-positive and gram-negative bacterial pathogen. In our study, out of 235 isolates, 136 (57.87%) strains showed significant antimicrobial potential at least five of the six tested pathogens whereas 48 (20.4%) isolates exhibited antimicrobial ability against all tested pathogens. This finding is similarly reported by Tomova et al. (2013) who suggested that 50% of the bacterial isolates obtained from Magura cave showed significant inhibitory activity against Pseudomonas aeruginosa and the yeast pathogen Rhodotorula mucilaginosa. Out of 136 isolates, 29 (21.3%) actinobacterial isolates and 19 (13.9%) bacterial isolates showed potential antimicrobial activities against all tested pathogens. Few researchers reported that mainly actinobacteria isolated from karstic caves showing significant antimicrobial activity against bacterial pathogens (Kim et al., 1998; Yucel and Yamac, 2010). Yucel and Yamac (2010) reported that 62% of actinobacteria isolated from the rock wall, speleothems surface and soil samples of karstic caves obtained from Turkey showed antimicrobial activity against four bacteria, two yeasts, and four filamentous fungi pathogens. Yasir et al. (2018) reported that out of 84 isolates, only 30 strains revealed antimicrobial activity against three pathogens i.e. Salmonella typhi, Staphylococcus aureus, and Candida albicans. Isolates BPSCV70 (Micrococcus luteus), BPSCV82 (Streptomyces sp.), BPSCV83 (Streptomyces sp.), BPSCV84 (Micromonospora sp.), BPSCV89 (Actinobacteria bacterium), BPSCV102 (Streptomyces sp.) and BPSCV120 (Actinomycetales bacterium) showed promising activity against Staphylococcus aureus (15 mm) and Bacillus subtilis (15 mm). This finding was similarly reported by Yasir et al. (2018) who suggested that only 15 isolates exhibited inhibitory activity against S. typhi whereas, 20 strains were highly significant against S. aureus. Moreover, only six isolates exhibited potent antibacterial activity against both tested bacterial pathogens. Further, genus Pseudomonas and Bacillus were also showing strong antimicrobial activity against tested pathogenic bacteria as suggested by Yasir et al. (2018). Adam et al. (2018) reported that three moonmilk Streptomyces strains (MMun141, MMun146 and MMun156) showed strong antimicrobial activities against all tested gram-positive bacterial pathogens (Bacillus subtilis, Staphylococcus aureus and Micrococcus luteus) whereas isolates Kocuria strain MMun160 and Amycolatopsis strain MMun171 showed active against gram-negative bacterial pathogens (Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii and Klebsiella pneumoniae). Cervimycins A-D produced from Streptomyces tendae strain HKI 0179, isolated from an ancient cave, the Grotta dei Cervi in Italy showed greatest antibacterial activity against gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and multi-drug-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) as reported by Herold et al. (2005). Higher activity of AMP assay against these microbes indicates that cave microbial community could be a potential source of future genomic resource discovery especially for wider applications in agricultural, domestic animal and human health (Logan 1988; Saglam et al., 2017; Bottcher et al., 2018; Edelmann et al., 2005).
Clarridge (2004) states that the 16S-rRNA gene sequence is the perfect technique to identify the isolates up to species level. In our study, we have found that all the isolates showed 98–100% identity with reference sequences in NCBI GenBank. All the isolates were divided into 13 families indicates that all isolates having a strong relationship with cave soil samples. In our study, the phylogenetic tree showed that actinobacteria were the dominant group followed by Proteobacteria and Firmicutes group. Similarly, Lee et al. (2012) reported that the phylogenetic tree of 16S-rRNA gene sequences obtained from 60 caves around the world. Among them, the most abundant groups belong to Proteobacteria followed by Actinobacteria, and Chloroflexi. The similar findings were reported by Yasir (2018) who state that the cultured isolates from both caves were divided into the phyla Proteobacteria, Actinobacteria, and Firmicutes. Among them, 13 genera were identified from the culturable study including Bacillus, Microbacterium, Pseudomonas and Staphylococcus.
Culture dependent is a commonly useful method to isolate microorganisms from any source, but they do not provide perfect identification of microorganisms. Hence, various molecular techniques have been developed to protect cultural heritage. For example, Pinar et al. (2014) has used two PCR based methods for the identification of the cultivable fraction of the halophilic microflora that inhabit the Catacombs of Palermo and used molecular approach to develop non-invasive and perfect sampling methods for DNA extraction for bacterial and fungal diversity analyses (Pinar et al., 2015 and 2016). The next-generation sequencing technologies were used to analyze bacterial diversity on bricks from barracks in the former Auschwitz IIeBirkenau Museum and to investigate the microbial population colonizing the medieval church of San Leonardo di Siponto (Italy). Interestingly, they have found three dominant bacterial phyla i.e. Proteobacteria Actinobacteria and Bacteroidetes (Gutarowska et al., 2015; Chimienti et al., 2016). In our study, total of 38 phyla (BHI80-139; Chlorobi; Elusimicrobia; Fibrobacteres; Gemmatimonadetes; GN02; MVP-21; OD1; FCPU426; ZB3; Synergistetes; Euryarchaeota; NKB19; TM6; GN04; Thermi; GAL15; WPS-2; Armatimonadetes; Chlamydiae; AD3; TM7; Crenarchaeota; Fusobacteria; WS2; WS3; Nitrospirae; Chloroflexi; Synergistetes; Acidobacteria; Cyanobacteria; Planctomycetes; Proteobacteria; Firmicutes; Verrucomicrobia; Bacteroidetes; Actinobacteria & unassigned other) was recorded in V3 and V4 region of cave sediment sample. Interestingly, we have recorded that the phylum Actinobacteria was the most dominant bacteria in both V3 and V4 regions. Previous researchers reported that the phylum Actinobacteria was found highly dominant in all the cave samples under V4 hypervariable region of 16S-rRNA (De-Mandal et al., 2016; Yasir, 2018). In the present study, we have identified under the phylum Actinobacteria were Saccharopolyspora, Nocardioidaceae, Streptomycetaceae, Pseudonocardia, Pseudonocardiaceae, Nocardiaceae, Streptomyces, Actinomycetales, Actinomycetospora, Frankiaceae, Actinosynnemataceae, Actinomadura, Mycobacterium, Amycolatopsis, Rhodococcus, Actinokineospora, Corynebacterium, Nocardioides, Nocardia, Micromonosporaceae, Micrococcaceae, Intrasporangiaceae, Acidimicrobiales, Kineosporiaceae, Kibdelosporangium, Sporichthyaceae, Cellulomonas, Propionibacteriaceae, Geodermatophilaceae, Bifidobacterium, Euzebya, Gaiellaceae, 0319-7L14, Solirubrobacterales and Rubrobacter. Similarly De-Mandal et al. (2017) reported that cave sediment samples collected from three different caves (Bukpuk [CBP V3], Lamsialpuk [CLP V3] and Reiekpuk [CRP V3]) of Mizoram and found that high abundance of dominant family (> 0.01%) under Actinobacteria were Nocardiaceae, Streptomycetaceae, Micrococcaceae, Frankiaceae, Gaiellaceae, Pseudonocardiaceae, Streptomycetaceae, EB1017, Mycobacteriaceae, Actinosynnemataceae, Corynebacteriaceae, Rubrobacteraceae, Nocardioidaceae, Micromonosporacea, Geoderma tophilaceae, Sporichthyaceae, Actinosynnemataceae, Nakamurellaceae, Pseudonocardiaceae, Cryptospora34ngiaceae, Kineosporiaceae, and Ruaniaceae. Moreover, few other genera under Actinobacteria were Mycobacterium, Corynebacterium, Rubrobacter, Actinoplanes, Saccharothrix, and Pseudonocardia. Various studies over the past decade suggested phylum Actinobacteria was highly present in all types of caves because of the favorable condition and environment to sustain inside the caves and are dynamically involved for the formation of crystals in cave walls and biomineralization process (Barton et al., 2001; Jurado et al., 2009). Laiz et al. (2009) also reported Actinobacteria under the genus Rubrobacter was isolated from biodeteriorated monuments that can induce crystal formation in caves and produce biofilm on the limestone. Yasir (2018) has isolated number of actinobacteria from two caves i.e. Koat Maqbari Ghaar (KMG) and Smasse-Rawo Ghaar (SG) of North-West region of Pakistan that showed significant antimicrobial activity against various bacterial pathogens.
Interestingly, the genera Streptomyces was recorded maximum under the family Streptomycetaceae that can synthesize various compounds including alcohols, sugars, amino acids, and aromatic compounds and poses abilities to produce clinically important antibiotics (Madigan and Martinko, 2005). Among the Proteobacteria, alpha-proteobacteria were highly dominant followed by gammaproteobacteria, beta proteobacteria, and delta proteobacteria. Previous studies state that few of the species under the Proteobacteria subphylum can subsist under very tremendous environment conditions by utilizing ABC (ATP-Binding Cassettes) and TRAP (Tripartite ATP-independent periplasmic transporters) mechanism (Kumbhare et al., 2015; De-Mandal et al., 2017). Proteobacteria isolated from Koat Maqbari Ghaar (KMG) and Smasse-Rawo Ghaar (SG) caves of Pakistan that showed alpha-proteobacteria (45.6% KMG and 34.1% SRG) and gamma-proteobacteria (35.2% KMG and 32.1% SRG) were dominant in both caves, followed by delta-proteobacteria (12.6% KMG and 16.9% SRG) and beta-proteobacteria (9.2% KMG and 16.7% SRG) as suggested by Yasir (2018). Moreover, the phylum Proteobacteria obtained from well-known Spanish Altamira cave, cosmopolitan was great abundance in dripping waters and cave walls (Portillo et al., 2008; Portillo et al., 2009). Schabereiter-Gurtner et al. (2002) reported that the phylum Proteobacteria was found half of the entire walls in the Tito Bustillo cave in Spain and this phylum can be used for chemolithotrophic energy production due to their versatile metabolic potential live on available ions in the rock contents. Further, the genera Rhodoplanes under the subphylum alphaproteobacteria were found in our study that possesses Photo and chemo-organ heterotrophic growth and produce hopanoids and carotenoids (Lakshmi et al., 2009; Lodha et al., 2015; Takaichi et al., 2012). Additionally, another genus Sphingomonas under the subphylum alphaproteobacteria was found in nutrient-limited subsurface environments that can metabolize a huge number of diverse aromatic compounds (Balkwil et al., 1997). Jin et al. (2012) reported that gram-negative heterotrophic bacteria Alteromonas under the subphylum gammaproteobacteria having abilities to degrade aromatic carbon rings investigated through an oil spill. Further genus Halomonas under the same subphylum having the capacity to resist extreme conditions and engross in sandstone formations as reported by Dong et al. (2014). Among the beta proteobacteria, the highly dominant genera were Achromobacter, Burkholderia, Neisseria and Ramlibacter were found in our study. Interestingly, the genus Burkholderia was diazotrophs bacteria and having abilities to degrade several xenobiotic compounds (Rusch et al., 2015). Moreover, the phylum Firmicutes was found dominant in our study. These findings were similarly reported by various researchers who state that the phylum Firmicutes obtained from various regions of caves was highly dominant and identified in more extreme ecosystems that are comparatively more resistant to nutrient stress (Chen et al., 2009; Ikner et al., 2007). In our study, we have obtained the Bacillus group from the Pukzing cave which can form endospores (Ikner et al., 2007). In addition, the archaea group under mesophilic Crenarchaeota and Euryarchaeota obtained from Pukzing cave were found in V4 hypervariable region of 16S rRNA. Similarly, both archaea group (mesophilic Crenarchaeota and Euryarchaeota) was detected in Koat Maqbari Ghaar (KMG) cave of Pakistan and found in the Lechuguilla Cave of United States (Ikner et al., 2007). Moreover, Legatzki et al. (2011) state that the presence of an archaeal community on calcite speleothems from Kartchner Caverns, Arizona, USA. Some important genera Methylobacterium, Rhizobium, Kocuria, Acinetobacter, Renibacterium, and Bacillus were found in our study. Similarly, these genera were reported from another cave and these genera having ability to utilize a carbon substrate as well as play a vital role in nitrogen fixation and calcification (Cuezva et al., 2012; Portillo et al., 2008; Schabereiter-Gurtner et al., 2002; Ikner et al., 2007; Northup et al., 2003). Numerous numbers of oligotrophic and facultative bacteria like Nitrospira, Sphingomonas, Bacillus cereus, Paenibacillus, Streptomyces sp., Brevibacillus, and Arthrobacter were obtained in our culture-independent study and were previously reported from various oligotrophic environments (Hayakawa et al., 2011). The phylogenetic analysis of community study indicated that the tree has divided into two major groups i.e. Bacteria and Archaea group. Among them, the bacteria group is the largest group and is divided into three another sub-groups such as FCB group; PVC group and Terrabacteria group. All the bacterial strains were clustered within these groups in our study. All the strains epitomize a novel potential isolate in caves biodiversity that indicates us isolated bacterial population properties to having significant discover a new micro-organism from the cave ecosystem (Jurado et al., 2009). Moreover, most of the isolated bacteria showed significant antimicrobial potential against bacterial pathogens. Interestingly, few researchers reported that isolated microbiota from cave ecosystem could be a potential source to discover new microorganisms and antimicrobial agents (Nakaew et al., 2009; Yasir, 2018) having wider applicability in health management of crop, animal and human.