Nova Biotechnol Chim (2022) 21(2): e1337 DOI: 10.36547/nbc.1337 1 Nova Biotechnologica et Chimica Elucidation of antibacterial activity of Bacillus sp. and Alcaligenes sp. metabolites against multidrug-resistant bacteria Faheem Ullah1, Sadir Zaman1, Waheed Ullah1,, Shandana Ali3, Muhammed Qasim1, Niaz Muhammad1, Momina Mehmood1, Navid Ali1, Niamat Khan2 1Department of Microbiology, Kohat University of Science and Technology, Kohat 26000, Khyber Pakhtunkhwa, Pakistan 2Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat 26000, Khyber Pakhtunkhwa, Pakistan 3Department of Zoology, Kohat University of Science & Technology Kohat 26000, Khyber Pakhtunkhwa, Pakistan  Corresponding author: waheedwazir@gmail.com Article info Article history: Received: 22nd December 2021 Accepted: 1st July 2022 Keywords: Antibacterial activity Cholistan Desert Inhibitor Minimum inhibitory concentration Secondary metabolites Abstract Different resistance mechanisms are involved in exhibiting resistance to different groups of antibiotics. Researchers are searching for new therapeutic options to encounter the emerging trend of microbial resistance. Bacteria were isolated from the extreme environment of Cholistan Desert and were screened for characterization. Potential metabolites that showed broad-spectrum activity were partially purified using silica gel chromatography and determined their minimum inhibitory concentration. A collection of 50 bacterial isolates from soil samples was screened for metabolite production and among them isolate R19 of Bacillus sp. and isolate A8 of Alcaligenes sp. had high similarity with strong antimicrobial metabolite producers. The growth of A8 was stable at slight acidic pH while R19 was best at neutral pH. Similarly, the best growth of A8 was observed at 37 °C while R19 at 35 °C. Minimum inhibitory concentration of purified compounds of Bacillus sp. were determined at concentration range of (3.12 – 100 %) against multidrug-resistant (MDR) strains of Shigella, Enterobacter aerogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonia and produced 10 – 25 mm zone of inhibition. Metabolites of Alcaligenes sp. were sufficient to inhibit the growth of all selected MDR bacteria at concentrations 12.25 – 100 % and shows 10 – 20 mm zone of inhibition. Bacillus sp. and Alcaligenes sp. can be used as producers of potential antibacterial metabolites. Proper utilization of selected metabolites can be helpful in combating emerging drug resistant pathogenic bacteria. In addition, further proteomic analysis and structural insight should be considered to elaborate their active ingredients and its efficacy. Introduction Microbes are potential sources of different types of important active metabolites. In search of potential metabolites producer microbial strains, several studies have been conducted to explore different habitats for potential metabolites producer microbes. It can be deduced from different studies that microbes of diverse environments carrying strong antimicrobial potential according to Andayani et al. (2015), observed by others (Masand et al. 2018; Rajasabapathy et al. 2020). Microbial metabolites due to their increasing applications have allure the attention of scientists to mailto:waheedwazir@gmail.com Nova Biotechnol Chim (2022) 21(2): e1337 2 explore different untouched sites for potential metabolites producer microbes Kumar et al. (2018). Metabolites have various therapeutic applications in the treatment and eradication of different types of bacterial, fungal and parasitic diseases Demain (1999). In pre-antibiotic era, the treatment of infectious diseases was challenging due to unavailability of antibiotics Adedeji (2016). The discovery and commercialization of penicillin give new hope because morbidity and mortality associated with infectious diseases were reduced in infected population. Unfortunately, with passage of time resistant strains like emergence of methicillin Resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococci and multidrug resistant (MDR) strains of gram negative bacteria were reported worldwide particularly in developing world Hesse and Adhya (2019). World Health Organization estimates that annually millions of people lost their lives due to drug-resistant bacterial strains. According to Shrivastava et al. (2018) the World Health Organization enlist priority pathogens for which new and effective drugs need to be explored having bactericidal activity. Among these pathogens, carbapenem resistant strains are global threats Le Thanh Dong and Espinoza (2020). Carbapenems drugs are considered the last choice for treatment of MDR bacteria. Recently, few extreme drug resistant (XDR) strains of Salmonella sp. were isolated from infected patients in Pakistan, which revealed resistance to most of the available drugs including ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, ciprofloxacin, and ceftriaxone while fortunately sensitive to azithromycin and carbapenems Rasheed et al. (2019). In relation to the resistance, the induction of new effective drugs rate is low as compared to previously approved drugs that lose their efficacy Ribeiro da Cunha et al. (2019). To tackle antibiotic resistance issue, scientists are trying to explore new avenues for the isolation of potential metabolite producer microbes having strong inhibitory potential. Bacillus a gram positive spore forming bacteria, having potential to cope harsh environment has been frequently isolated from extreme habitats Nicholson et al. (2000). Their secondary metabolites exhibit strong antibacterial activity and many of their compounds have been commercialized to use as antibiotics such as bacteriocins Morikawa et al. (1992). Beside Bacillus, actinomycetes, fungi are also sources of antibiotics Singh et al. (2019). In the current scenario, researchers demonstrate key interest in bacterial characterization from the unexplored harsh environment like deserts and marine with the hope to explore strong bioactive metabolites producer strains according to Abdelkader et al. (2018); observed by Cita et al. (2017). Indeed, by 2008, more than 100 specialized metabolites had been isolated and identified from microorganisms isolated from such habitats. In addition, in the previous decade, 129 putative strains, producing 186 drug gable metabolites, were isolated from extreme strata Sayed et al. (2020). The current study was conducted for the exploration of antibacterial metabolites producer bacterial strains from the unexplored habitat of Cholistan Desert (Punjab, Pakistan) with harsh environmental conditions like high temperature, scarcity of nutrients and water depletion. Experimental Collection and isolation of bacteria from soil samples Samples from different locations of Cholistan Desert were collected in sterile bottles using standard procedure as previously described by Elbendary et al. (2018). All samples were brought to the Medical Laboratory, Department of Microbiology, KUST and were stored at -80 °C for further analysis. All samples were serially diluted up to 10-9 in sterilized distilled water. Aliquot of 100 µL was spread from last three dilution on tryptone soya agar (TSA) and nutrient agar (NA) and plates were incubated for 48 h at 37 °C. After incubation different colonies appeared on different culture media. Resistant bacterial strains profile MDR bacterial strains of P. aeruginosa, E. coli, E. aerogenes, K. pneumonia, S. aureus, and Shigella sp. were obtained from medical laboratory, Department of Microbiology, Kohat University of Science and Technology. To assess the Nova Biotechnol Chim (2022) 21(2): e1337 3 antibacterial potential of broth extract of metabolite producer strains, its inhibitory effects were screened against the resistant pathogens. All the selected bacterial strains revealed resistance toward different antibiotics like penicillin, cephalothin, tetracycline, amoxicillin, cefoperazone, ceftriaxone, and cefepime based on CLSI guidelines. Evaluation of Antibacterial activity of cell-free extract Isolated bacteria were grown in Luria Bertani broth (LB) for 5 to 7 days in shaking incubator at 170 rpm and 30 °C. After incubation, centrifugation was performed, and supernatants of each broth were filtered. The cell-free filtrates were assayed for antibacterial activity using well diffusion assay Oskay (2011). Growth optimization at different temperature and pH The isolates were incubated in nutrients broth at various temperatures like 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, and 75 °C. Growth conditions were optimized at different pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. The results were recorded using spectrophotometer at 600 nm OD. For proper growth, pH of the media was adjusted according to bacterial needs as previously described Khalil et al. (2009). Extraction of antibacterial metabolites Bacterial isolates from desert soil samples (n = 50) were screened for their antibacterial metabolites production. Among these isolates, A8 and R19 exhibit broad-spectrum activity against MDR bacteria and were selected for further analysis. An equal amount of ethyl acetate was added to the extracted filtrate and then metabolites were concentrated using rotary evaporator. Bacterial metabolites were purified using silica gel column chromatography, chloroform/methanol in the ratio of (100 : 0, 80 : 20, 60 : 40, 50 : 50, 40 : 60, 20 : 80, 0 : 100) as an eluent. Further each fraction were concentrated using rotary evaporator Rao et al. (2007). Minimum inhibitory concentration (MIC) determination of antibacterial metabolites Activity of each fraction of selected metabolites was examined on Mueller Hinton agar (MHA) against drug resistant strains by using agar well diffusion process. Fractions of best restraining were selected for MIC determination against resistant pathogens. Active fractions (2 mL) were considered 100 % and were serially diluted into two-fold to make 50 %, 25 %, 12.5 %, 6.25 %, and 3.125 %, and concentration respectively. Further 100 μL from each concentration was used for the MIC determination. Biochemical and molecular characterization of metabolites producer strains Strong metabolites producer strains were biochemically characterized using standard procedures of (Rahimnahal et al. 2017; Marathe et al. 2018). For molecular characterization, DNA from the selected active antimicrobial metabolites producers strains were extracted by standard phenol chloroform method (Neumann et al. 1992) using overnight culture. PCR reactions were carried out for the amplification of 16S rRNA gene using universal primers forward primer 5'- AGAGTTTCCTGGCTCAG-3' and reverse primer 5'-AAGGAGGTGATCCAGCC-3' (Lim et al. 2016). PCR conditions were set for amplification of 16S rRNA gene. The thermal cycling reactions were performed as: Initial denaturation at 94 °C for 5 min, then 25 cycles of 94 °C for 1 min, 50 °C for 40 s and 72 °C for 60 s and final extension was 72 °C for 5 min. The amplified products of PCR of the selected isolates were confirmed on 2 % agarose by adding 3.5 μL ethidium bromide as a staining dye and visualized by the digital gel doc system (Analytik Jena System, Jena, Germany). The products were subjected to sequencing and reactions were conducted at Macrogene (Seoul, South Korea) using the universal 16S rRNA forward primer. The resulted sequences were extracted using Bioedit software. NCBI BLAST analysis was performed for sequence alignment. Moreover, phylogenetic tree was constructed using MEGA 7 software. Nova Biotechnol Chim (2022) 21(2): e1337 3 Results Antibacterial activities of A8 and R19 metabolites Soil samples (n = 50) were screened for their antibacterial metabolites production and among these isolates A8 and R19 were expressed broad spectrum activity against resistant bacterial culture and were selected for further analysis. Morphologically isolate A8 stained gram-negative rod shape while R19 was identified as gram- positive rod shape and was also spore positive Fig. 1. Biochemically both isolates were catalase, oxidase, sugar fermenting, and motility recorded positive. a b Fig. 1. Morphological study of the selected isolates from Cholistan Desert. a – culture of bacteria on nutrient agar; b – pink colour Shows gram negative bacteria; c – the blue colour shows gram positive bacteria. Growth optimization of selected isolates on different temperature and pH Best growth of selected isolate A8 was observed at 37 °C and lowest growth were observed at 15 °C. Similarly isolate R19 have shown maximum growth at temperature 35 °C as shown in Fig. 2a. Similarly, pH also has a huge impact on the bacterial growth. A8 shows maximum growth at neutral pH while R19 showed maximum activity at pH 9.0. Both isolates growth was restrained at acidic pH as shown Fig. 2b. Gram negative rods Culture of bacteria Gram positive rods c 4 Nova Biotechnol Chim (2022) 21(2): e1337 3 0 0.2 0.4 0.6 0.8 1 1.2 1.4 O p ti c a l d e n s it y ( O D ) Temp = Temperature (°C) Effect on growth at different temperature A8 R19 0 0.5 1 1.5 pH 3.0 pH 4.0 pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0 pH 10.0 O p ti c a l d e n s it y ( O D ) pH Effect on growth at different pH A8 R19 Fig. 2. Effects of temperature (a) and pH (b) on growth of isolates (A8 and R19). MIC determination of isolate A8 collected through silica gel at different concentrations. The metabolites activities of strain A8, after silica gel purification, were observed for antibacterial activity against Shigella, S. aureus, E. aerogenes, P. aeruginosa, E. coli, and K. pneumonia MDR strains respectively at different concentration as shown in Table 1 and Fig. 3. Isolate A8 metabolites has the maximum zone of inhibition against MDR Shigella and S. aureus of about 25 mm, 24 mm, respectively. Similarly, it has strong inhibitory activity toward P. aeruginosa and 19 mm zone of inhibition while in case of E. aerogenes 20 mm zone of inhibition was recorded. While less inhibitory activity was observed against E. coli and K. pneumonia. a b 5 Nova Biotechnol Chim (2022) 21(2): e1337 3 Fig. 3. Antibacterial activity of A8 isolate against (a) Klebsiella pneumonia, (b) E. coli, (c) E. aerogenes, (d) S. aureus, (e) Shigella, (f) P. aeruginosa. Fig. 4. Antibacterial activity of R19 isolate against (a) K. pneumonia, (b) E. coli, (c) E. aerogenes, (d) S. aureus, (e) Shigella, (f) P. aeruginosa. Table 1. Inhibition zones of A8 metabolites against selected bacterial strains at different concentration. Tested bacterial strains 3.15 % [mm] 6.25 % [mm] 12.5 % [mm] 25 % [mm] 50 % [mm] 100 % [mm] Shigella -- -- 13 15 22 25 S. aureus -- -- 10 12 21 24 E. aerogenes -- -- -- 11 14 20 P. aeruginosa -- -- 10 15 17 19 E. coli -- -- -- 11 12 15 K. pneumonia -- -- -- -- 14 15 Antibacterial activity of R19 isolate The antibacterial activity of R19 metabolite was observed against Shigella, S. aureus, E. aerogenes, P. aeruginosa, E. coli, and K. pneumonia MDR strains at different concentration as shown in (Table 2, Fig. 4). R19 bacterial metabolites were more effective against all selected resistant bacterial strains as compared to A8. It has shown a strong inhibitory pattern from lower to higher concentration against Shigella and E. aerogenes. At lower concentration (3.12 %) it produced 10 mm zone of inhibition while at higher concentration (100 %) it generated 19 mm and 20 mm zone of inhibition against selected MDR strains respectively. Similarly, it has strong inhibitory action against MDR E. coli at concentration (50, 100 %) and manifest 19 mm and 23 mm zones of inhibition toward selected strains. Further their action against other tested isolates including K. pneumonia and P. aeruginosa revealed 16 – 18 mm zone of inhibition at concentration (100 %) as shown in Table 2. 6 Nova Biotechnol Chim (2022) 21(2): e1337 3 Table 2. Inhibition zones of R19 metabolites against selected bacterial strains at different concentration measured in diameter (mm). Test bacterial strains 3.12 % [mm] 6.25 % [mm] 12.5 % [mm] 25 % [mm] 50 % [mm] 100 % [mm] Shigella 10 13 15 16 18 19 S. aureus -- 8 9 12 16 20 E. aerogenes 10 11 12 13 14 20 P. aeruginosa -- 8 9 11 15 18 E. coli -- 5 6 10 19 23 K. pneumonia -- -- -- 10 15 16 Molecular characterization of A8 and R19 strains using 16S rRNA gene Further identification of both A8 and R19 metabolite producer strains were characterized through PCR amplification of 16S rRNA gene, size 1399 bp, as shown in Fig. 5. Amplified product of A8 and R19 were sequenced at Macrogen (Seoul, South Korea) using forward primer. Homology analysis of both isolates were conducted at NCBI BLAST and there were 97.7 % identity with Alcaligenes sp. for A8 isolate and 98.3 % identity with Bacillus sp. for R19. Based on sequencing results, phylogenetic trees were constructed using MEGA7 for the selected isolates (A8 and R19) as shown in Fig. 6 and 7. Fig. 5. Gel image of amplified product of 16S rRNA gene. Lane L shows ladder, lanes A8 and R19 bacterial isolates A8 and R19, respectively. Fig. 6. R19 phylogenetic tree using MEGA7. 7 Nova Biotechnol Chim (2022) 21(2): e1337 2 Fig. 7. A8 phylogenetic tree using MEGA. Discussions To combat extending bacterial resistance, exploration of new drugs is indispensable of a current scenario. Scientists are in search of new metabolites that have strong potential to address resistant strains. Soil harbour diverse microbial community which has potential to cherish strong antibacterial potential. Previously bacterial isolates particularly Bacillus sp. have been reported from different habitats having strong antibacterial activity. In recent study efforts were made to explore microbial community for potential of strong antibacterial compounds, from diverse environmental conditions. Deserts have diverse environmental conditions and have mixed microbial flora. Cholistan Desert of Pakistan has wide range of microbial community and have potential to cope with challenging environmental conditions Amin et al. (2020). In current study two isolates (A8 and R19) emerged as strong antibacterial metabolites producer strains that have been characterized based on ribotyping which revealed that A8 showed 97.74 % sequence similarity to Alcaligenes sp. and R19 unveil 98.33 % sequence similarity to Bacillus sp. Similar active producer strains have been screened from the unexplored unique habitat of mountain Himalaya which produced strong antibacterial metabolites according to Hussain et al. (2018). According to Das et al. (2018) study, streptomyces strains were isolated from protected forest land and most of these isolated strains carried broad spectrum antibiotic activity and were stable at 37 °C while some of the isolated strains were stable 42 °C. A8 and R19 isolate showed broad spectrum activity, against MDR clinical bacterial strains. Maximum growth of A8 and R19 isolates were observed on normal nutrient media. Other growth conditions like temperature and pH were also analysed, isolates that the best grown at temperature 37 ℃ and the lowest below 20 ℃ have been recorded. Our result are in line with the study conducted by Bharali et al. (2011) in which the Alcaligenes sp. isolates growth was documented at 42 °C. A8 showed maximum growth at neutral pH while R19 was most stable at slight alkaline pH. These results confirmed previously reported by Farag et al. (2019) in which metabolites of Bacillus manifest promising activity was observed at slight alkaline pH. In recent development, growth of MDR strains of Shigella and E. aerogenes were restricted at concentration of 3.12 % of the partially purified compound of Bacillus sp. Similarly, for S. aureus, P. aeruginosa and E. coli were determined 6.25 % while 25 % for K. pneumonia. The partially purified metabolites of isolated strain of Bacillus sp. exhibited promising activity against gram negative bacteria and it has also excellent inhibitory activity against S. pyogenes MTCC 442, B. cereus, E. faecalis, and S. epidermidis ATCC 12228. 8 Nova Biotechnol Chim (2020) 19(2): 124-137 Similarly, Ramachandran et al. (2014) isolated Bacillus sp. from soil samples and found a strong producer of metabolites that showed strong effective against broad-spectrum microbes. The metabolites of Alcaligenes sp. manifest strong activity at lowest concentration (12.25 %) against Shigella, S. aureus and P. aeruginosa while at concentration (25 %) growth of E. aerogenes and E. coli were inhibited. A thermophilic strain of Alcaligene sp. was isolated from petroleum contaminated soil having potency of antibacterial activity against Bacillus sp., K. pneumonia, E. coli, P. aeruginosa, and S. aureus observed by Bharali et al. (2011). Similarly, isolated strains of Alcaligenes sp. were reported from common effluent treatment plant which demonstrated antibacterial activity against MDR Enterobacter sp. and Serratia sp. being supported by the production of active a nucleoside antibiotic according to Kapley et al. (2016). Bacillus sp. are predominantly soil bacteria and can be found in different other habitats that their existence in soil in huge numbers as a result of their capacity to form resistance endospores and develop bioactive compounds which promote their resistance to variable environmental conditions reported by Onajobi et al. (2020). A study reported by Abd Sharad et al. (2016) of the Alcaligenes sp. isolated from marine habitat has strong antibacterial activity at 3 mg.mL- 1 against resistant bacteria. Conclusions Metabolites of Alcaligenes sp. (A8) and Bacillus sp. (R19) have shown the maximum zone of inhibition against MDR clinical bacterial strains. The active fractions of Alcaligenes sp. metabolites revealed strong inhibitory effects against all resistant pathogens. This study provides initial layout for researchers and drug developers to further characterize active ingredients. Furthermore, its growth was optimized on the best pH and temperature that will be helpful in extraction and stability of metabolites. Proper utilization of selected metabolites can be helpful in combating emerging drug resistance mess in pathogenic bacteria. In addition, further proteomic analysis and structural insight should be considered to elaborate their active ingredients and its efficacy. Acknowledgements This study was a part of MS research project. We extend our gratitude to Department of Microbiology, Kohat University of Science and Technology for providing lab facilities for conducting this research. Conflict of Interest The authors declare that they have no conflict of interest. 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