Antibacterial Activity and Tolerance towards Heavy Metals by Endolithic and Epilithic Bacteria Isolated from Rocks of Nathiagali, Lower Himalaya, Pakistan

Microorganisms are considered the most diverse, complex and important organisms in biosphere playing a significant role in various biological activities. Microorganisms are being isolated from almost all habitats around the biosphere (Muhammad et al., 2009; Jabeen et al., 2019). The rocks that are under oligotrophic condition are also inhabited by several species of extremophilic bacteria (Sajjad et al., 2015). The study of rock dwellers both endoliths and epiliths have been done in many parts of the world from different habitats (Horath et al., 2006; Norris and Castenholz, 2006; Walker and Pace, 2007; Walker et al., 2005; Chiellini et al., 2018; Yang et al., 2019). Endoliths were also reported from cliffs of the Niagara escarpment (Gerrath et al., 2000; Matthes-Sears et al., 1999) and from streams in the UK (Pentecost, 1992). These microbes were also reported from several harsh and unusual habitats like hot and arid desert environments (Friedmann, 1971; Bell et al., 1986; Bell, 1993) from travertine in Turkey (Pentecost et al., 1997) from Arctic and Antarctic locations (Ascaso and Wierzchos, 2002; Friedmann et al., 1993; Friedmann, 1982; Hughes and Lawley, 2003; Russell et al., 1998; Wierzchos and Ascaso, 2001) and also from marine littorals (Mason et al., 2007; Whitton and Potts, 1982). Rock-inhabiting microbes have also been isolated from other diverse habitats across the globe such as hydrothermal vents (Daughney et al., 2004), meteorite impact crater (Cockell et al., 2005, 2002), tsunami deposits (Cockell et al., 2007), deep subsurface (Amy et al., 1992; Pedersen, 1997) and also from the cultural heritage monuments (Scheerer et al., 2009). Several types of rocks have been studied for microbial diversity such as igneous rocks (both glassy and crystalline) (Herrera et al., 2009; Thorseth et al., 1992; Villar et al., 2006), sedimentary rocks such as sandstones, salts and limestones (Matthes et al., 2001; Weber et al., 1996; Wierzchos et al., 2006), and metamorphic rocks such as gneisses and granites (Cockell et al., 2002; De los Rios and Ascaso, 2005).

Extremophilic microorganisms produce molecules adapted to unusual living conditions and have been recognized as an important source of new biological moieties (Sánchez et al., 2009). Accordingly, these microorganisms are an important screening target for a variety of bioactive compounds such as secondary metabolites (Hackl et al., 2004). Antimicrobial compound production seems to be the general phenomenon for most bacteria. A commendable array of defense, i.e. broad-spectrum antibiotics, lytic agents such as lysozyme and metabolic by products such as organic acids are produced by bacteria. In addition, several other types of bacteriocins and protein exotoxins are also produced which are biologically active peptide moieties with the bactericidal mode of action (Riley and Wertz, 2002; Yeaman and Yount, 2003). Secondary metabolites are produced in the response of exhaustion of nutrients, biosynthesis or addition of an inducer and growth rate decrease (Demain, 1998).

Unwanted microorganism control is essential for life and diseases because microbes must be treated in humans, plants, and animals (Basavaraj et al., 2010; Gram et al., 2010). However, this leads to rapidly emerged and developed antibiotic-resistant microorganisms, mainly due to the misuse of antibiotics (Muhammad et al., 2009; Uzair et al., 2009). The problem of multidrug resistance is now recognized as a global health problem (Muhammad et al., 2009). The current solution for this problem involves the development of a more rational approach to antibiotic discovery and use of new antimicrobial agents (Bhavnani and Ballow, 2000). Most of the antimicrobial compounds originally isolated from the microbial source are being used in the past several decades (Pathania and Brown, 2008). The present study was aimed to investigate the antibacterial activity of endolithic and epilithic bacteria isolates against ATCC bacterial strains.

All the chemicals and reagents used in this research were of analytical grade and purchased from Sigma-Aldrich Chemical Co. and Merck.

Rock and soil samples were collected aseptically from Nathiagali (34°4’20”N, 73°23’55”E), District Abbottabad, KPK, Pakistan, (elevation 2600 meters), in pre-sterilized polyethylene zipper bags. On return to the laboratory, all the samples were stored at 4°C and preliminary isolation experiments were carried out immediately.

The outer surface of rocks was swabbed with sterile cotton-tipped swabs in Laminar flow hood. The swab was then immersed and shaken in 1 mL sterile water, the suspensions were then spread on nutrient agar plates for isolation of epilithic bacteria. The rocks were broken down with a hammer under aseptic conditions. Then the inner exposed surface was swabbed with sterile cotton-tipped swab under sterile conditions. The swab was then dipped and shaken in 1 ml sterile water and was then spread on nutrient agar plates for endolithic bacteria. The plates were incubated at 30°C for 3 days. For purification, visible colonies were selected based on morphological differences and cultured on nutrient agar plates separately.

All the isolates were identified through biochemical and molecular characterization. For molecular characterization, the bacterial DNA was extracted by CTAB method, and 16S rRNA gene was amplified by using both reverses and forward universal primers 27F’ (5’- AGAGTTTGATCCTGGCTCAG-3’) and 1494R’ (5’- CTACGGCTACCTTGTTACGA-3’) bacterial primers (47). 20 mL of PCR reaction mixture contains 1 mL DNA sample, 2 mL PCR buffer, 2 mL deoxynucleotide triphosphate (dNTP) mix, 2 mL each reverse and forward primers, ex Taq DNA polymerase of 0.5 mL and 10.5 mL distil water. Initially, the reaction mixture was incubated at 96°C for 4 min. Then 35 amplification cycles were run at 94°C for 40 sec, 55°C for 55 sec, and 72°C for 60 sec. Further, incubation of reaction was carried out for 7 min at 72°C. Both positive control (Escherichia coli genomic DNA) and negative control were run in parallel in the PCR. Sequencing products were resolved on an Applied Bio-Systems model 3100 automated DNA sequencing system (Applied BioSystems, USA) at the Macrogen, Inc., Seoul, Korea and the sequences were submitted to public gene bank NCBI and the accession number has been assigned (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic tree was constructed by the Maximum Likelihood method with the robustness of 1000 bootstrapping value in MEGA 6.0 (47).

ATCC bacterial cultures such as E. coli (10536), Staphylococcus aureus (6538) and Pseudomonas aeruginosa (15442) were used as test strains to check the antibacterial activity of endolithic and epilithic bacterial isolates.

A pure test microbial colony was transferred into test tubes having a normal saline solution and adjusting the turbidity comparing with 0.5 McFarland standard solution prepared by adding 0.5 ml of BaCl2 (1.17% w/v BaCl2.2H2O) into 99.5 ml of H2SO4 (1% w/v) with proper stirring. After adjusting, a sterile cotton swab was dipped into the normal saline suspension having test organisms and swabbed well over the entire surface of the plate containing Mueller Hinton agar (MHA) to ensure uniform distribution of the inoculum. Subsequently, a small portion of each bacterial isolate was point inoculated on plates that contain test microbial colony. These plates were incubated at 30oC for 24 h along with control having an antibiotic disc (ciprofloxacin and rifampicin). After 24 h of incubation, the diameter of the clear zones that showed inhibition of bacterial growth was measured in millimeter (mm). The experiment was done in triplicate and the mean value of zone inhibition was calculated with standard error.

Sensitivity test of isolates was performed to check their resistivity against antibiotics. Seven different types of antibiotic namely Amoxicillin + Clavulanic acid (AMC), Nalidixic Acid (NA), Gentamicin (CN), Piperacillin + Tazobactam (TZP), Cefepime (FEP), Imipenem (IMP), and Fusidic Acid (FD), of 30, 30, 10, 110, 30, 10 and 10 µg/disc, respectively were used for this purpose. A pure bacterial colony was transferred into test tubes having a normal saline solution and adjusted the turbidity comparing it with 0.5 McFarland standard solution. After adjustment, a sterile cotton swab was dipped into the normal saline suspension and swabbed well over the entire surface of the plates containing Mueller Hinton agar (MHA) to ensure uniform distribution of the inoculum. The antibiotic discs were placed on the plate very carefully.

To study the metal tolerance of all the strains, 24 h fresh cultures were streaked on nutrient agar plates supplemented with different concentrations (10-1000 ppm) of metal salts. Metal salts used included; ZnCl2, CrCl3, HgCl2, FeCl3 and CdCl2. Cultures were incubated for 24 h at 37°C and cell growth was observed.

Physical characters of the site were recorded during sampling. The pH of the site was 5.0 and the temperature was 20°C. The flora and fauna of the site were recorded. In flora, mostly shrubs and tall trees of pines were present around the sampling site. In fauna, insects, snakes, monkeys and various kinds of birds were seen there. Seventeen different endolithic and epilithic isolates were reported in the present study. Among 17 isolates, eight isolates were epilithic named as P17, P18, P19, P20, P21, P23, P24, and P26 while 9 were endolithic named as N12, N14, N21, N22, N26, N27, N28, N30, and N40. All these different strains were isolated on a morphological basis.

Morphological features such as size, shape, color, margins were checked and recorded (Table I). Gram’s staining results show that out of 11 endolithic bacteria two of them were Gram-positive rods while 9 were Gram-negative. While out of 10 epilithic bacteria, 2 were Gram-positive and 9 were Gram-negative.

The isolates were identified by BLAST search homology in NCBI. 16S rRNA gene sequences of the isolates were assigned the Accession numbers by the NCBI (Table II). Based on microscopic, morphological and biochemical tests the isolates were identified by comparing these characteristics with Bergey’s Manual of Determinative Bacteriology (9th Edition). Isolates N22, N21, P21, N14, N27 and P24 were identified as Alcaligenes spp., P17 and N40 were identified as Lysinibacillus spp., P20 was identified as Brevundimonas spp., P23 and N30 were identified Bordetella spp., P26 and P19 were identified as Pseudomonas spp., N12 as Pusillimonas spp.

-, no activity; +, weak activity; ++, moderate activity; +++, strong activity.

N26 as Fluviicola spp., N28 as Streptomyces spp. and isolate P18 identified as Parapusillimonas spp.

The phylogenetic tree constructed in MEGA 6.0 revealed that similar bacteria are grouped into the same group. The 1st cluster is of Proteobacteria group. Most of the study isolates belong to this group. This group contains bacteria similar to Advenella sp., Alcaligenes sp., Pseudomonas sp. and Bordetella sp. These all are gram-negative bacteria. The second cluster of gram-positive bacteria Actinobacteria. Isolate N28 is clustered into this group. The 3rd cluster is of gram-positive bacteria Firmicutes and isolate P17 belongs to this group (Fig. 1).

The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood (-2757.2947) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with superior log-likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 50 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 383 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Antimicrobial activity of all 17 isolates was checked against three ATCC strains; Pseudomonas aeruginosa (15442), Staphylococcus aureus (6538) and Escherichia coli (10536). The clear zone around the point inoculation of isolates showed inhibition of test microbes as with control (Table III). Alcaligenes spp. N27 and Lysinibacillus spp. P17 showed strong activity against all the three ATCC cultures while the strains Lysinibacillus spp. N40 and Brevundomonas spp. P20 showed no activity.

The sensitivity of all the isolates was checked against seven different broad and narrow spectrum antibiotics and the results were interpreted by measuring zones of inhibition according to Clinical and Laboratory Standards Institute (CLSI) protocol (Table IV). All the isolates were found resistant against Amoxicillin + Clavulanic acid and Cefepime except isolate Bordetella spp. P23, while Alcaligenes spp. N22 was resistant to Amoxicillin + Clavulanic acid and susceptible to Cefepime. Against Piperacillin + Tazobactam (TZP), Lysinibacillus spp. P17 and Alcaligenes spp. P24 were found resistant, whereas, Alcaligenes spp. N14, Pseudomonas spp. P26 and Pusillimonas spp. N12 were susceptible. It was observed that all strains were susceptible to Imipenem and Gentamicin. Majority of the strains were found susceptible to Nalidixic acid except for Lysinibacillus spp. P17, Streptomyces spp. N28, Lysinibacillus spp. N40 and Alcaligenes spp. P24. Against Fusidic acid, the isolates Parapusillimonas spp. P18, Pseudomonas spp P26, Alcaligenes spp. N27, Alcaligenes spp. N22, Lysinibacillus spp. N40, Pseudomonas spp. P19 and Bordetella spp. P23 were found resistant, whereas, the remaining were susceptible.

R, no zone produced or resistant; I, intermediate susceptibility; S, susceptible.

In the current study, five different metals including; Cd2+, Ar+2, Hg+2, Cr+3, and Fe+3 were examined for all isolates (Table V). The highest level of resistance was recorded by Alcaligenes spp. N14 as 920 ppm to iron, followed by resistance to arsenic by Alcaligenes spp. N21, cadmium again by Alcaligenes spp. N14, chromium by Bordetella spp. N30 and nickel by Streptomyces spp. N28. In the case of mercury, less resistivity was observed, yet Bordetella spp. P23 and Pusillimonas spp. N12 showed maximum resistivity i.e. up to 50 ppm. The level of resistance was recorded as; Fe > Ar > Cd > Cr > Ni > Hg.

There is a great deal of variation in the degree of antagonistic activity of different cultures (Pandey et al., 2002). The antimicrobial chemical compound efficiency has highly affected by its permeability, target site specificity and its effect on the host impact (Pathania and Brown, 2008). Antimicrobial compounds inhibition ability depends on the site of secretion of compounds, i.e. compounds are secreted outside the cell or accumulated in the cell (Nofiani, 2009). Increasing resistance pattern and the associated side effects of antibiotics have evolved the importance of some alternative source to be used as an antibacterial agent.

One of the options for the solution of this medical problem is the use of microorganisms from unusual habitats. These habitats are beyond the reach of anthropogenic activities, so that can be able to produce some unique types of secondary metabolites that may encounter the pathogenic microbes which is the main issue of the present period. Recently, one such study was conducted from the glacier environment and reported the antimicrobial activity of psychrophilic bacterial isolates (Rafiq et al., 2019). Bacteria may have certain genes responsible for encoding metabolites, but it does not express in normal circumstances (Demain, 1998). In this study, isolates showed an efficient antibacterial activity against other ATCC test bacterial species. This kind of environments holds nutrient deficient conditions and the microbes compete for nutritional requirements by producing anti-microbial agents against competing organisms. These antimicrobial compounds may demonstrate weak inhibition because some of them yield it in small amounts. Though, in high amount, these compounds may hold new and beneficial properties. Antimicrobial activity of isolates against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were checked and some isolates showed good activity against these ATCC bacterial cultures. Antibacterial activity of bacterial isolates from different extreme environments has already been done but the activity of endolithic and epilithic isolates is not reported yet. Endolithic and epilithic bacteria inhabit in the oligotrophic environment and possibly having the potential ability to produce antibiotics. Several studies focused on screening secondary metabolites produced by microorganisms that inhabit such extreme habitats as potential sources of useful compounds: antibiotics, immunosuppressant, and statins (Harvey, 2000), exopolysaccharides (Nicolaus et al., 2010), biosurfactants (Banat et al., 2010), extremozymes (Singh et al., 2011), radiation protective drugs (Singh and Gabani, 2011), antitumoral (Chang et al., 2011). Activity against these three strains was also checked by Singh et al. (2009), for the screening of antimicrobial activity of bacterial isolates from the soil of stressed ecological niches of eastern Uttar Pradesh, India.

Isolates also showed resistance to several broad and narrow spectrum antibiotics. This property may be adopted due to their survival in harsh conditions where they face unusual conditions by switching on certain genes to face harsh conditions and to encounter the effects of dominant competing microorganism. The sensitivity of isolates was checked against seven different antibiotics, and some isolates were found having resistivity against both broad and narrow spectrum antibiotics. This resistance against the known antibiotics reveals that these isolates can compete for other microorganisms for their nutritional requirements by producing certain secondary metabolites. Resistance against AMC, FD, and FEP was reported by endolithic and epilithic isolates. Similar study was reported by Parvathi et al. (2009), performing biochemical and molecular characterization of Bacillus pumilus isolated from coastal environment in Cochin, India, and Naphade et al. (2012) while performing isolation, characterization and identification of pesticide tolerating bacteria from garden soil in Kalyan, India. The antibiotic resistance of environmental isolates on the other hand also revealed the miss use of the antibiotics that may somehow be transferred from clinical to environmental microflora which is a big alarm for the current medical problems.

Isolates were also found to have resistance against certain metals. This property of these isolates is due to the fact that they are living in the harsh condition inside rocks which is abundant in metal concentration. Tolerance of isolates against six different heavy metals was checked and the results show that most isolates have great potential for metal tolerance. This capability of isolates could be exploited for certain applications such as for the bioremediation of heavy metals affected environments, extraction of metals from low-grade ores and their enzymes are metals tolerated. These isolates from such an unusual environment have several beneficial aspects, which could be exploited for the well-being of mankind.

Based on our study it was concluded that bacterial isolates from the unusual habitat of rocks not only showed resistance to metals and antimicrobial activity against ATCC isolates but also exhibited resistance towards several antibiotics. Moreover, purification and characterization of each bioactive chemical compound may be pharmaceutically more potential and significant. Further study is required to characterize and purify these antimicrobial compounds.

We are grateful to Quaid-i-Azam University, Islamabad for providing transport facility for the collection of samples and funds in order to accomplish research successfully on time.

The authors declare no conflict of interest.