Volume 6, Issue 2, April Issue - 2018, Pages:342-351
|Authors: Reda Hassan Amasha|
|Abstract: The microbial diversity within cave ecosystems is largely unknown. This study aimed to studying the microbial communities of the three caves viz Mossy, Hotel and Reda caves which are located approximately 200 km of Riyadh region, between Riyadh and Al Kharj road, Saudi Arabia. These caves have an intricate cave system which developed in the calcareous sandstone and clay. Morphological interpretation revealed that Mossy cave was different from Hotel and Reda caves where it is tall and have a linear cave passage and narrow canyons. Various field studies revealed that many bacteria inhibited in caves and obtained their energy from degradation of inorganic substances, thus they directly interact with the surfaces where they live on. In present study, soil and wall dust samples were collected and cultured on Nutrient agar, starch nitrate agar and PDA plates. During study various Gram positive, Gram negative bacteria, actinomycetes and fungi were isolated from the cave soil and wall. The isolated bacteria were characterized and identified by using culture-dependent methods (morphological and physiological methods). Identifications of the most occurring bacterial genera were confirmed by using 16S rRNA gene amplicon sequencing which revealed the presence of 11 broad taxonomic species of bacteria. Among these, Proteobacteria were dominant in all caves and this was followed by Actinobacteria, Firmicutes and Bacteroidetes. Majority of the true bacterial isolates belong to the genera Alcaligenes, Bacillus, Brevibacterium, Klebsiella, Planomicrobium, Shigella, Pseudomonas and Staphylococcus. During study, percentage of resistance to some metallic cations, heavy metals and antibiotics were also determined. Result of study revealed that Bacillus cereus, Klebsiella pneumoniae and Pseudomonas earuginosa showed the highest resistant percentage to the tested heavy metals while Pseudomonas earuginosa, Klebsiella pneumoniae and Staphylococcus aureus were the highest resistant to the tested antibiotics. In conclusion, present study provide beneficial information about microbial diversity, taxonomic and their resistance to some heavy metals and antimicrobial agents in three uncharacterized caves, located in Alsoman region, north east of Riyadh.|
|Full Text: 1 Introduction All imaginable environment including unexplored caves must be examined for new microorganisms (Barton & Luiszer, 2005(. Saudi Arabia is a country with rarest wild caves with interesting and popular geologic and geomorphic features. Its caves have great exploration potential with unsurpassed beauty that provides unending curiosity to a caver. Jabal Al Qarah Cave is located between Dammam and Al-Hofuf, Saudi Arabia and its cool protected passages and considered a gathering place for visitation (Hotzl et al., 1978, Hussain et al., 2006). Harrat Khaybar lava caves, Dahl Rumahah, Kahf Al Shuwaymis, and Umm Jirsan Lava-Tube are some of the oldest caves which explored for their ecology and diversity in Saudi Arabia (Pint, 2009). Very few geological studies have been carried out about the caves located in Saudi Arabia (Forti et al., 2003; Al-Shanti et al., 2003). Microorganisms interact directly with the geology of our planet and their activities have provided the oil, oxygen and the essential nutrients such as carbon, nitrogen and phosphorus. Cave microbiology deals with microorganisms that are found in caves and have the ability to consume inorganic material for energy and survive under extreme conditions (Madigan et al., 2000, Shivaji et al., 2004). Extreme conditions included physical and chemical environmental limits, high temperatures, acid conditions, low light and growth under salt stress (Hathway, 2010). Generally, microorganisms from caves have been considered extreme microbes, because of their ability to grow at low nutrient accessibility, low or high temperature and moderately high humidity, under minimum light conditions of caves (Hathway, 2010). Attempts must be carried out to understand the difficulties associated with microbial activities in cave environments. The significant roles of cavers are the identification of new cave biota and conserving the microbial habitats of caves. Traditional methods such as growing microorganisms in Petri plates, Gram staining, type of respiration and mode of motion are generally used for bacterial identification but unfortunately if the number of bacterial species are higher than 5,000/gram, it is difficult to identify by above said technique (Amann et al., 1966). Thus, recently methods based on genetic sequences (16S rRNA gene) were used to identify and determine the diversity of the isolated bacteria (Al'Abri, 2011; Amasha, 2012, Almalki, 2012). Very little information is available about the diversity and importance of cave microbes, thus further researches on caves microbiology are needed. Moreover, the biodiversity of bacteria in Saudi Arabian caves are not received scientific attentions (Pint, 2006) and available published literature about the geology of the Saudi Arabian caves and their biological composition are in scarcity (Forti et al., 2003, Al-Shanti et al., 2003). There are several caves that have not been explored yet for their geomorphology and microbial diversity. This study aimed at studying and determining bacterial diversity of three caves viz. Mossy, Hotel and Reda caves near Riyadh, Saudi Arabia. 2 Material and methods 2.1 Study area: Three easily accessible caves in the vicinity of Riyadh named, Mossy Cave: 26°27’33.2?N, 47°14’03.3?E, Hotel cave: 26º 28' 14.2 47º 14' 23.8?E?, and Reda cave: 26°27’10.5?N, 47°15’1.9?E were visited with the help of a local cave explorer (Dr. Mahmoud Ahmed Shanti, Saudi geological Survey). All three caves are located in Somman area and the visitation was low because these were not opened for visitors. During the study period, the average temperature ranged from 11?C (January) to 43?C (July and August). Representative photos were taken for each cave and all samples were photo-documented at the collection area. Elevation of the cave (m) at the entrance, temperature, humidity and RH were determined during January 2017. Relative humidity is approximately the ratio of the actual to the saturation vapour pressure and is calculated from the following equation RH = (Actual vapor pressure) / (Saturation vapor pressure) × 100% 2.2 Sample collocation From each cave, ten soil samples were collected from surface soils and cave walls (microbial mats) from the entire selected cave by following protocol of Amasha (2012). All the collected samples were taken in sterile containers with a sterilized spoon and brought to the Laboratory of Microbiology, University of King Abdulaziz, Jeddah, Saudi Arabia for further analysis. All soil samples were spread on a clean paper sheet until dries. 2.3 Soil analysis Analysis of chromium (Cr), cadmium (Cd), cobalt (Co), copper (Cu) and iron (Fe) in each soil sample was carried out. All samples were homogenized; particle size, distribution and organic carbon (Org. C) content were determined using the loss-of-ignition method (Donkin 1991) and sieving method (Laker & Dupreez, 1982), respectively. The pH value was measured in soil extract (1:2 w/v) by the method of Sonneveld & Van Den Ende (1971). Soil sample digestion was carried by 10 N of mixture of ultrapure nitric acid (HNO3) and HCl. Metal detection was determined using Agilent 7500 Capillary Electrophoresis series Inductively Coupled Plasma Mass Spectrometer. Geo-accumulation index was calculated from the relation (Muller, 1969; Loska et al., 2004; Ji et al., 2008): I geo=log2 Cn/ 1.5 Bn (du Preez et al., 2016) Cn : measured concentration of n (element), Bn: measured geochemical background value. 2.4 Microorganism’s isolation and total microbial count One gram of each sample was suspended in 10 ml sterile distilled water and serial dilution carried out. From the suitable dilution, bacteria, actinomycetes and fungi were isolated on nutrient agar, starch nitrate agar (Pridham et al., 1957) and PDA, respectively. After 2 days of incubation at 37?C for bacteria and 7 days of incubation at 25?C for actinomycetes and fungi, colonies were counted, selected and streaked on new agar plates until pure colonies were obtained. Total bacterial, fugal and actinomycetes counts for different collected samples were determined using agar plate count methods. All pure colonies were preserved on agar slants of the same medium at 4?C until used. 2.5 Bacterial identification 2.5.1 Morphological and physiological characteristics Isolated bacteria were identified on the basis of various morphological characteristics such as Gram stain, cell shape, colony characteristics and growth on different nutrient media in addition to physiological characters like production of catalase, oxidase, gelatinase, chitinase and amylase (Plotnikova et al., 2010). For Gram negative bacteria, biochemical reactions were carried out using API20E system (Bio Mérieux S.A, France). 2.5.2 Molecular identification 16SrRNA sequencing was used to identify bacteria from caves sample. Genomic DNA was extracted from isolated and purified bacterial isolates and then PCR was used with the appropriate primers to produce large quantities of the 16SrRNA gene (Al'Abri, 2011). Phylogenetic grouping was performed using a multiplex PCR-based assay as described by Clermont et al. (2000). During molecular identification, the name and sequence of the used premiers have been given in Table 1. 16S rRNA gene was amplified using 10 p mol/μl of each forward 785F (5′- GGA TTA GAT ACC CTG GTA -3′) and reverse 907R (5′- CCG TCA ATT CMT TTR AGT TT-3'). DNA extraction, PCR and sequencing were carried out at Macrogen, Geumcheon-gu, Seoul 08511, Republic of Korea. 2.5.3 Metal tolerance and antibiotic resistance patterns All the obtained bacterial isolates were screened for resistance to heavy metals (1300 µg/ml), Cu, Cd and Cr using agar dilution method. After 24 hr of incubation, the plates were examined for bacterial growth (Washington & Sutter, 1980). Antibiotic resistance of the differed bacterial isolates were determined by standard disc diffusion method (Finegold & Martin, 1982) on Mueiler-Hinton agar (Lennette et al., 1974). Antibiotic paper discs (bioMerieux, Charbonnieres-les-Bains, France) containing Ampicillin (10 pg); Streptomycin (10 pg); Tetracycline (30 pg) and Neomycin (30 pg) were put on the surface of the inoculated agar and all plates were incubated at 30°C for 2 days. Zone of inhibitions were measured as described in the instructions (bioMerieux). 3 Results ad Discussion Caves are found in many types of rocks and are formed by different geological processes. Caves of Somman are formed by dissolving calcareous rocks and slow moving of the groundwater from the passages and cavities (Moore & Sullivan, 1978). In present study, three caves of the Somman were visited, characterized and microbial diversity of these three was determined. A part of the map of Saudi Arabia showing the three studied caves, north east of Riyadh city was represented in Figure 1. The caves under study named Mossy, Hotel and Reda caves. The three studied caves have interesting pattern, among these first two caves have horizontal and easy to walk while the third one viz, Mossy cave has deep pit, with 4 m long, and need ropes and a ladder to go inside (Figure 2). Similarly, Amasha (2012) studied, characterized and isolated various bacterial strains from Ghar Al Hibashi cave which is located 300 km southeast of Makkah, Saudi Arabia (21°10′ N, 42°10′ E). Environmental |
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