Identification and molecular characterization of potential probiotic subspecies of Bacillus tequilensis isolated from the gut of Masheer fish, using 16S rRNA gene sequencingAbstract
Over the past few decades, there has been increasing interest in using probiotics as an alternative to antibiotics. Researchers have conducted studies to investigate bacterial strains for their probiotic potential. Like other animals, fish also have several bacterial strains in their gut that possess probiotic properties, although this is limited to Bacillus species. Therefore, this study aimed to isolate and characterize probiotic Bacillus species from the gut of Masheer fish (Tor Puititora). Four pure bacterial isolates were selected as potential probiotic strains based on selection criteria, including survival rate in acid and bile salt. The isolates exhibited significant antimicrobial activity against pathogenic bacteria, including Escherichia coli (ATCC8739), Pseudomonas aeruginosa (ATCC9027), and Staphylococcus aureus (ATCC6538). MF2 and MF3 demonstrated clear zones, including antimicrobial activity against all three indicator pathogens. MF1 and MF4 exhibited antimicrobial activity against E. coli (ATCC8739) and P. aeruginosa (ATCC9027). Furthermore, the 16S rRNA gene sequences of all isolates exhibited a close association with Bacillus tequilensis (KCTC13622), with nucleotide similarity of 98.63%, 98.25%, 98.80%, and 98.35%, respectively. Our results demonstrate that these bacterial isolates show promise as an alternative to antibiotics in the fisheries food system. In this study, all isolates identified in the fish gut were subspecies of B. tequilensis.
Keywords:
Masheer fish; probiotics; 16S rRNA gene sequences; gut microbiota
Resumo
Nas últimas décadas, tem havido um interesse crescente no uso de probióticos como uma alternativa aos antibióticos. Pesquisadores conduziram estudos para investigar cepas bacterianas quanto ao seu potencial probiótico. Como outros animais, os peixes também têm várias cepas bacterianas em seus intestinos que possuem propriedades probióticas, embora isso seja limitado a espécies de Bacillus. Portanto, o objetivo deste estudo foi isolar e caracterizar espécies probióticas de Bacillus do intestino de peixe Masheer (Tor Puititora). Quatro isolados bacterianos puros foram selecionados como potenciais cepas probióticas com base em critérios de seleção, incluindo taxa de sobrevivência em ácido e sal biliar. Os isolados exibiram atividade antimicrobiana significativa contra bactérias patogênicas, incluindo Escherichia coli (ATCC8739), Pseudomonas aeruginosa (ATCC9027) e Staphylococcus aureus (ATCC6538). MF2 e MF3 demonstraram zonas claras, incluindo atividade antimicrobiana contra todos os três patógenos indicadores. MF1 e MF4 exibiram atividade antimicrobiana contra E. coli (ATCC8739) e P. aeruginosa (ATCC9027). Além disso, as sequências do gene 16S rRNA de todos os isolados exibiram uma associação próxima com Bacillus tequilensis (KCTC13622), com similaridade de nucleotídeos de 98,63%, 98,25%, 98,80% e 98,35%, respectivamente. Nossos resultados demonstram que esses isolados bacterianos mostram-se promissores como uma alternativa aos antibióticos no sistema alimentar da pesca. Neste estudo, todos os isolados identificados no intestino dos peixes eram subespécies de B. tequilensis.
Palavras-chave:
peixe purê; probióticos; sequências do gene 16S rRNA; microbiota intestinal
1. Introduction
Besides the medications, the demand for probiotics is increasing day by day. Probiotics can influence the gut microflora and their functions in treating gastrointestinal infections as well as cancer. Probiotics prevent, suppress and treat various diseases by modifying host immunity (Ejtahed et al., 2011). They modify the functions of both innate and specific immunity (Ren et al., 2009). Bacterial strains with probiotic aptitude synthesize various antibacterial substances such as organic acids, bacteriocins and hydrogen peroxide, which inhibit both gram-positive and gram-negative pathogenic bacteria. Secondly, these bacterial strains compete to adhere to the intestinal surface and block the adhesion site for pathogens. Thirdly, these strains degrade the toxin receptors studded on the mucosal wall and thus inhibit pathogenicity. Moreover, these strains consume the nutrients needed for the pathogen’s growth (Nayak, 2010).
The bacterial strains isolated from the guts of different fish are used as probiotics. Some of these strains include Lactococcus plantarum CLFP238, L. lactis CLFP101, L. fermentum CLFP242 (Balcázar et al., 2008), Pseudomonas PM11, Vibrio fluvialis PM17, Vibrio alginolyticus UTM102, Bacillus subtilis UTM126, Pseudomonas aestumarina SLV22, and Roseobacter gallaeciensis SLV03. These bacterial strains possess antipathogenic activities against fish pathogens. Additionally, these strains also help in the supply of essential nutrients, such as vitamins, enzymes, etc., to enhance nutrition in the host (Alavandi et al., 2004; Foster-Fishman and Behrens, 2007). The gut microbiota of animals is involved in various functions, including angiogenesis, digestion, and development of the mucosal system, and also acts as a protective barrier to diseases (Schiffrin and Blum, 2002). The current study aimed to isolate and identify the gut microbiota of the Masheer fish by using bacterial universal 16S rRNA gene primers and to investigate the isolated bacterial strains as probiotics against selected strains of pathogenic bacteria.
2. Methods
2.1. Sample collection and processing
This study was conducted partially at the Pakistan Agriculture Research Council, National Institute of Genomics and Advanced Biotechnology, National Agriculture Centre Islamabad and partly at Hazara University Mansehra, Pakistan, from 2022 to 2023. Masher fish were sampled from Tarbela Lake, Haripur. The fish were dissected, and the ventral surface of each fish was sterilized using 70% ethanol to remove the gut aseptically. The fish gut was incised longitudinally, opened, and thoroughly flushed using a sterilized, chilled 0.9% normal saline solution to remove all impurities. The fish’s gut was then homogenized using sterilized mortar and pestle and centrifuged for 5 minutes at 1000 rpm. The supernatant was serially diluted up to 10-6 times. A 100 µL solution was taken from the 10-3, 10-4, 10-5, and 10-6 dilutions and inoculated on pre-incubated De-Man Rogosa and Sharpe (MRS) agar (CM0361-Oxoid) culture media (pH 6.5) and allowed to incubate for 48 hours at 37 °C.
2.2. Isolation and identification
Culture plates were carefully examined after overnight Incubation for bacterial colonies. The growth of each sample was initially identified via colony morphology and gram staining. All suspected colonies were then further purified using a sub-culturing method to obtain pure bacterial strains. The isolated colonies were then further identified through different biochemical and other tests such as catalase, oxidase, motility test, urease, hemolysis activity, and probiotic properties tests (i.e., resistance to bile concentration, resistance to low pH, amylase, and amylolytic activity). A control strain of each isolate was screened while testing for bile concentration and pH. The tests were conducted in triplicate, and the mean was calculated. The identified samples were initially labelled as MF1, MF2, MF3, and MF4 (M = Masheer and F = Fish).
2.3. Antimicrobial activity assay
The antimicrobial activity of each sample was evaluated against known pathogenic bacterial strains (see Table 1) using the well-diffusion method. Nutrient agar (CM0003-Oxoid) plates were prepared and bored with the help of a sterilized borer aseptically. 1 mL of each marker pathogenic bacterial strain was inoculated on pre-incubated nutrient agar (CM0003-Oxoid), and each well was filled with 500 µL of broth containing a specific bacterial isolate and was labelled accordingly. The culture plates were allowed to incubate aerobically for 24 hours at 37 °C.
2.4. Susceptibility pattern
The susceptibility pattern of all samples was investigated using the Kirby-Bauer method on Mueller Hinton Agar (MHA) (CM0337-Oxoid) medium, which is suitable for sensitivity testing (Ali et al., 2020). Prior to the test, each isolate was inoculated in test tubes containing 5 mL tryptic soy broth (CM129-Oxoid) and allowed to incubate at 35 °C for 18 hours. After incubation, the growth was adjusted to a 0.5 McFarland index and inoculated onto a pre-incubated MHA (CM0337-Oxoid) medium. The susceptibility pattern of each sample was examined after overnight incubation by testing commercially available antibiotic discs (Oxoid) on MHA (CM0337-Oxoid) medium against bacterial isolates (mentioned in Table 2). The zone of inhibition (ZI) of each antibiotic against each sample was carefully examined, measured, and then compared with the standard chart.
Antibiotic susceptibility patterns of bacterial isolates were assessed for commercially available selected antibiotics.
2.5. DNA extraction and molecular characterization
The Miller protocol was used with minor modifications to extract DNA (Millar et al., 2000). The probiotic bacterial isolates were further investigated using bacterial universal 16S rRNA primers, including 27F (5ʹ-AGAGTTTGATCMTGGCTCAG-3ʹ) and 1492R (5ʹ-ACCTTGTTACGACTT-3ʹ). The PCR reaction was adjusted as follows: 2.5 µL PCR buffer, 13 µL deionized water, 0.5 µL Taq polymerase, 2.5 µL MgCl2, 1 µL each of forward and reverse primers, 2 µL template DNA, and 0.5 µL dNTPs were mixed according to the method followed by Zhang et al. (2004). The thermal profile was adjusted as follows: denaturation at 94 °C for 4-5 minutes, annealing at 55 °C for 20-40 seconds, DNA extension at 72 °C for 1 minute and final extension at the same temperature for 10 minutes.
2.6. Gel electrophoresis
Gel electrophoresis was performed using a 1% agarose gel in 0.5X Tris Borate EDTA (TBE) buffer to confirm the precise size of a specific amplified DNA fragment. 3-4 µL of each sample, mixed with 2 µL of loading buffer, were loaded into each well along with a 10kb ladder in a separate well. Ethidium bromide and bromophenol blue were added as loading and staining dyes, respectively, and were allowed to run on the agarose gel. The resulting DNA bands on the gel were visualized and recorded using the gel documentation system (UVIPro platinum, England).
2.7. Sequencing and phylogenetic analysis
The amplified PCR product of the 16S rRNA gene was purified using a commercially available kit (Invitrogen, USA). These samples were sent for sequencing to Macrogen, Korea. The primers mentioned above were also used for sequencing. The retrieved nucleotide sequences were further blasted in NCBI and Ez-Taxon server for confirmation and validation of their relatedness to the top-hit sequences available in these databases (https://www.ezbiocloud.net/identify). The 16S rRNA gene sequences obtained in this study are available in the NCBI database under the accession numbers MF1 MW133068, MF2 MW133774, MF3 MW136774, and MF4 MW137950. Clustal-W (version 1.6) was used for the sequencing alignment. The Neighbor-joining algorithm with bootstrap value was used to perform phylogenetic analysis using the MEGA-7 software package (Tamura et al., 2011).
3. Results
Based on colonial morphology, a total of 12 different bacterial colonies were isolated from the gut of Masheer fish. Furthermore, four different colonies were purified and selected based on their probiotic potential and labelled as MF1, MF2, MF3, and MF4. The isolates exhibited yellowish, rough, circular, and small colonies with shiny surfaces. They had a gram-positive purple colour and a rod-shaped morphology. All the samples were positive for catalase and oxidase, negative for urease, and were motile in nature, indicating that the isolates belonged to the Bacillus species.
3.1. Probiotic characterization
In the case of bile salt concentration, the isolated strains exhibited resistance when tested at a 0.3% bile salt concentration for 5 hours. MF3 exhibited the highest tolerance (7.91cfu/ml), while MF2 exhibited the second-highest survival rate (6.34 cfu/ml). MF1 and MF4 showed moderate tolerance, 4.72 and 4.51, respectively, to a 0.3% concentration of bile salt (Figure 1). None of the selected strains showed hemolytic activity around the colonies when inoculated on blood agar. All four strains exhibited amylolytic activity by producing a clear zone around the grown colonies when inoculated on starch-containing culture plates.
While studying pH, it was discovered that the bacterial isolates were found to tolerate lower pH levels ranging from 1.0 to 4.0. However, the cfu/ml of each of all Bacillus strains was limited at pH 1.0 and significantly increased at pH levels above 1.0. This proves that the isolates are able to survive at highly acidic pH levels (1.0). Furthermore, the isolates demonstrated the highest survival growth rate as the pH increased from 1.0 to 4.0 in terms of cfu/ml. In contrast to MF1 and MF2, MF3 and MF4 exhibited a high tolerance rate (3cfu/ml) across all tested pH ranges (Figure 2).
3.2. Antibacterial activity
Bacterial isolates were investigated for their antibacterial activity against selected pathogenic strains, including E. coli (ATCC8739), P. aeruginosa (ATCC9027), and S. aureus (ATCC6538). Among all isolates, MF3 exhibited the highest antibacterial activity against the selected pathogenic strains. At the same time, MF2 was noted to have the second-highest antibacterial activity and in contrast to MF2 and MF3, both MF1 and MF4 exhibited the lowest antibacterial activity. P. aeruginosa was found to be completely resistant to MF4, whereas S. aureus was found to be resistant to the MF1 isolate. The ZI of each bacterial isolate against the selected pathogenic bacterial strains is given in Table 1.
3.3. Antibiotic susceptibility profile
While screening the susceptibility profile, all the isolates posed the highest susceptibility pattern to ceftriaxone, amoxicillin, and linezolid. MF1, MF2, and MF3 exhibited intermediate susceptibility, whereas MF4 was found to be highly resistant to levofloxacin and erythromycin. The susceptibility pattern illustrates that the isolated strains are susceptible to almost all antibiotics. This means that these isolates are safe and pose no risk of transferring resistance genes into the host’s gut microbiota, as well as no pathogenic strain being released into the environment (see Table 2).
3.4. Molecular characterization
The colonies representing each of the four bacterial isolates were subjected to DNA extraction. Universal bacterial 16S rRNA gene primers were used to amplify the desired DNA fragment. The fragments were further processed by gel electrophoresis for visualization of DNA bands of the appropriate size (Figure 3).
3.5. Genetic analysis of 16S rRNA gene
The nucleotide sequences obtained after blasting on various databases and conducting phylogenetic analysis revealed their relationship and taxonomic positioning within the Bacillus genus, specifically Bacillus tequilensis (KCTC12622). The sequences of all isolates were submitted to the NCBI gene bank to obtain the accession number for each isolate (see Table 3).
Based on 16S rRNA gene sequencing, taxonomical position, and genetic analysis of each isolated bacterial strain.
To determine the similarity index with the top-hit taxon, the 16S rRNA gene sequence of each strain was submitted to the Ex-Taxon bacterial and archaeal gene database. The information on top-hit sequences and similarity indices among all four bacterial isolates reveals that all the sequences belong to the genus Bacillus and further confirms their closeness to the species tequilensis. This is supported by the data obtained from blasting in NCBI and Ez-Biocloud, which shows that MF1 (98.63%), MF2 (98.25%), MF3 (98.80%), and MF4 (98.35%) share high similarity levels, as well as through phylogenetic analysis (Figure 4). However, the analysis of similarity levels between the nucleotide sequences of the four bacterial isolates using NCBI also shows variability, with MF4 being found to be 99.78%, 99.45%, and 99.67% similar to MF1, MF2, and MF3, respectively.
Phylogenetic tree analysis of the obtained sequences and their relationship with closely related strains.
4. Discussion
The gastrointestinal (GI) tract of animals is considered a complex ecological unit that comprises a wide range of microorganisms. These microorganisms inhabit the GI tract and play a dynamic role in the health and nutrition of host animals. However, some of these microorganisms can also cause diseases. Gastrointestinal bacteria are partially known for their important role in the digestion of reptiles, herbivorous mammals, amphibians, and avian species (Godon et al., 2013). According to Sanger et al. (2011), these animals have a close resemblance in the GI tracts in terms of alimentary tract anatomy, phylogenetic relationships, and physiology.
Like other animals, fish are also known to have intestinal microflora and symbiotic relationships that aid in fish digestion. A wide range of microorganisms has been found to have a symbiotic relationship with the intestine of fish, particularly in the hindgut. According to Sanger et al. (2011), the hindgut of fish acts as a site for microbial fermentation and the production of nutritional end products. These findings suggested that the composition of fish’s intestinal microflora directly impacts digestion. However, there is currently limited understanding of the physiological role of the microbes found in the fish intestine.
So, the present study was conducted to isolate beneficial strains of bacteria from the gut of Masheer fish that may have the potential for probiotic activity. Several previous studies have been conducted to investigate probiotic bacterial strains in the fish’s gut (Balcázar et al., 2008). In the current study, four different Bacillus subspecies were isolated and characterized from the gut of Masheer fish. The isolates exhibited morphological and biochemical characteristics, including rod-shaped, motile, gram-positive, catalase and oxidase positive, and urease negative. These results are consistent with the findings of the previous study, which also characterized the isolates morphologically and biochemically (Khusro and Aarti, 2015).
The isolates were screened for probiotics aptitude using several basic steps, which included testing their tolerance to a 0.3% bile concentration at different times, acidic conditions, and their antibacterial potency against selected pathogenic bacteria comprising E. coli, S. aureus and P. aeruginosa as mentioned in a previous study (Balcázar et al., 2008; Khusro and Aarti, 2015). The current study demonstrated the isolate’s resistance to a 0.3% bile salt concentration at different time periods. The MF3 bacterial isolate showed the highest combat (7.91) in terms of colony-forming unit (cfu/ml) after three hours of incubation, followed by MF2, MF1, and MF4 with 6.34, 4.72, and 4.51 cfu/ml, respectively. After five hours of incubation, the isolates exhibited the lowest tolerance to a 0.3% bile salt concentration (Figure 1). The results of the previous study support our findings regarding significant tolerance to a 0.3% bile salt concentration (Naeem et al., 2018). Certain strains of bacteria which have the ability to hydrolyze bile salt using the bile salt hydrolyze enzyme (Erkkilä and Petäjä, 2000) have been examined in the gastrointestinal tract of animals (Maragkoudakis et al., 2006).
Figure 2 reveals that the isolated bacterial strains exhibited a wide range of tolerance to pH, ranging from 1.0 to 4.0. The growth of each isolate was slightly lower at acidic pH (1.0) and increased as the pH increased. Therefore, it was proven that the isolates can grow at acidic pH. Each isolate exhibited the maximum cfu/ml at pH 4.0. In comparison to MF1 and MF2, MF3 and MF4 showed significant tolerance in terms of cfu/ml at pH 4.0, with 6, 5, 7, and 7, respectively. In contrast, these isolates showed 1, 1, 2, and 3 cfu/ml at pH 1.0. Almost similar results were reported by Buntin et al. (2008).
The anti-pathogenic potency of each isolate was assessed using the agar well diffusion method. The table provided Table 1, details the anti-pathogenic activity of each isolate against the selected pathogenic strains. MF3 demonstrated the highest ZI when exposed to the selected pathogenic strains. Similarly, MF2 exhibited the second-largest ZI. Although MF1 and MF4 displayed strong potency against all strains, they showed complete resistance to S. aureus and P. aeruginosa, respectively. The inhibitory effects could be attributed to the presence of acid, bacteriocins-like substances, or both (Aslim et al., 2005).
Table 2 illustrates the susceptibility pattern of the isolated bacteria to the selected antibiotics. All of the isolates showed high susceptibility to antibiotics such as levofloxacin, amoxicillin, and linezolid. However, ceftriaxone and erythromycin were found to be moderately effective against MF3, but they had no efficacy against MF4. Similar results regarding the susceptibility of the isolates to antibiotics were reported in a previous study (Naeem et al., 2018).
After sequencing the 16S rRNA gene, molecular studies revealed that all four bacterial isolates were identified as subspecies of B. tequilensis (KCTC13622). The information obtained through blasting on NCBI and Ex-Taxon indicated that all four bacterial isolates showed a resemblance to B. tequilensis and were assumed to be subspecies of B. tequilensis. These findings were also validated through phylogenetic analysis, which showed similar indications of closeness to this bacterium. For example, MF1 was 98.63%, close to B. tequilensis, MF2 was 98.25%, MF3 was 98.80%, and MF4 was 98.35%. However, when the similarity level between the nucleotide sequences of the four bacterial isolates was analyzed using NCBI, variability was also observed. For instance, MF4 was found to be 99.78%, 99.45%, and 99.67% similar to MF1, MF2, and MF3, respectively. Similarly, in their study, Naeem et al. (2018) also isolated and identified B. tequilensis using 16S rRNA gene sequencing. Additionally, Khusro and Aarti (2015) isolated strains of B. tequilensis through 16S rRNA gene sequencing using the same primers as the current study.
5. Conclusion
Like other factors, probiotic strains can also influence the use of probiotics and the health of animals. In this respect, investigations of novel origins for probiotic strains will play a significant role as key findings globally. The current study is a novel study that aims to identify potential fish-origin probiotic subspecies of B. tequilensis strains that can tolerate and survive in harsh conditions of acidic pH and 0.3% bile salt concentration while also exhibiting anti-pathogenic efficacy against E. coli, S. aureus, and P. aeruginosa. Furthermore, these fish-origin probiotics can easily be consumed as fish food to avoid excessive antibiotic use. The current study also concluded that the fish’s gut microbiota is highly significant and should be identified for potential use as a fish origin to improve probiotic production in the future.
Acknowledgements
The authors highly acknowledge the staff of the laboratory of the Microbiology Department, Hazara University, Mansehra. The authors further extend their appreciation to the Research Supporting Project number (RSP2024R502), King Saud University, Riyadh, Saudi Arabia, for funding this project. The funding body has no role in designing the study.
The current study, along with the proposed procedure and methodology, was ethically examined and approved by the Institutional Bioethical Committee of Hazara University under the approval I’d number “F.No.73/HU/IBC/2023/51” and found in accordance with the ethical principles followed by this university.
References
-
ALAVANDI, S., VIJAYAN, K., SANTIAGO, T., POORNIMA, M., JITHENDRAN, K.P., ALI, S.A. and RAJAN, J.J., 2004. Evaluation of Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 on immune indices of tiger shrimp, Penaeus monodon Fish & Shellfish Immunology, vol. 17, no. 2, pp. 115-120. http://doi.org/10.1016/j.fsi.2003.11.007 PMid:15212731.
» http://doi.org/10.1016/j.fsi.2003.11.007 -
ALI, F., NIAZ, Z., SHAH, P.T., SHAKEELA, Q., UZMA, B. and AHMED, S., 2020. Antibiogram of ESBL and MBL producing Pseudomonas aeruginosa among the population of Hazara division, KPK, Pakistan. Journal of the Pakistan Medical Association, vol. 70, no. 11, pp. 1979-1984. http://doi.org/10.5455/JPMA.19089 PMid:33341844.
» http://doi.org/10.5455/JPMA.19089 -
ASLIM, B., YUKSEKDAG, Z., SARIKAYA, E. and BEYATLI, Y., 2005. Determination of the bacteriocin-like substances produced by some lactic acid bacteria isolated from Turkish dairy products. Lebensmittel-Wissenschaft + Technologie, vol. 38, no. 6, pp. 691-694. http://doi.org/10.1016/j.lwt.2004.08.001
» http://doi.org/10.1016/j.lwt.2004.08.001 -
BALCÁZAR, J.L., VENDRELL, D., DE BLAS, I., RUIZ-ZARZUELA, I., MUZQUIZ, J.L. and GIRONES, O., 2008. Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish. Aquaculture, vol. 278, no. 1-4, pp. 188-191. http://doi.org/10.1016/j.aquaculture.2008.03.014
» http://doi.org/10.1016/j.aquaculture.2008.03.014 - BUNTIN, N., CHANTHACHUM, S. and HONGPATTARAKERE, T., 2008. Screening of lactic acid bacteria from gastrointestinal tracts of marine fish for their potential use as probiotics. Songklanakarin Journal of Science and Technology, vol. 30, suppl. 1, pp. 141-148.
-
EJTAHED, H., MOHTADI-NIA, J., HOMAYOUNI-RAD, A., NIAFAR, M., ASGHARI-JAFARABADI, M., MOFID, V. and AKBARIAN-MOGHARI, A., 2011. Effect of probiotic yogurt containing Lactobacillus acidophilus and Bifidobacterium lactis on lipid profile in individuals with type 2 diabetes mellitus. Journal of Dairy Science, vol. 94, no. 7, pp. 3288-3294. http://doi.org/10.3168/jds.2010-4128 PMid:21700013.
» http://doi.org/10.3168/jds.2010-4128 -
ERKKILÄ, S. and PETÄJÄ, E., 2000. Screening of commercial meat starter cultures at low pH and in the presence of bile salts for potential probiotic use. Meat Science, vol. 55, no. 3, pp. 297-300. http://doi.org/10.1016/S0309-1740(99)00156-4 PMid:22061286.
» http://doi.org/10.1016/S0309-1740(99)00156-4 -
FOSTER-FISHMAN, P.G. and BEHRENS, T.R., 2007. Systems change reborn: rethinking our theories, methods, and efforts in human services reform and community-based change. American Journal of Community Psychology, vol. 39, no. 3-4, pp. 191-196. http://doi.org/10.1007/s10464-007-9104-5 PMid:17510793.
» http://doi.org/10.1007/s10464-007-9104-5 -
GODON, J.-J., ARCEMISBÉHÈRE, L., ESCUDIÉ, R., HARMAND, J., MIAMBI, E. and STEYER, J.-P., 2013. Overview of the oldest existing set of substrate-optimized anaerobic processes: digestive tracts. BioEnergy Research, vol. 6, no. 3, pp. 1063-1081. http://doi.org/10.1007/s12155-013-9339-y
» http://doi.org/10.1007/s12155-013-9339-y - KHUSRO, A. and AARTI, C., 2015. Discovery of polygalacturonase producing Bacillus tequilensis strain ARMATI using 16s rRNA gene sequencing. Asian Journal of Pharmaceutical and Clinical Research, vol. 8, pp. 58-62.
-
MARAGKOUDAKIS, P.A., ZOUMPOPOULOU, G., MIARIS, C., KALANTZOPOULOS, G., POT, B. and TSAKALIDOU, E., 2006. Probiotic potential of Lactobacillus strains isolated from dairy products. International Dairy Journal, vol. 16, no. 3, pp. 189-199. http://doi.org/10.1016/j.idairyj.2005.02.009
» http://doi.org/10.1016/j.idairyj.2005.02.009 -
MILLAR, B.C., JIRU, X., MOORE, J.E. and EARLE, J.A., 2000. A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material. Journal of Microbiological Methods, vol. 42, no. 2, pp. 139-147. http://doi.org/10.1016/S0167-7012(00)00174-3 PMid:11018270.
» http://doi.org/10.1016/S0167-7012(00)00174-3 -
NAEEM, M., AHMED, I., AHMED, S., AHMED, Z., RIAZ, M.N. and GHAZANFAR, S., 2018. Screening of cattle gut associated Bacillus strains for their potential use as animal probiotic. Indian Journal of Animal Research, vol. 1, no. of, pp. 6. http://doi.org/10.18805/ijar.B-948
» http://doi.org/10.18805/ijar.B-948 -
NAYAK, S.K., 2010. Role of gastrointestinal microbiota in fish. Aquaculture Research, vol. 41, no. 11, pp. 1553-1573. http://doi.org/10.1111/j.1365-2109.2010.02546.x
» http://doi.org/10.1111/j.1365-2109.2010.02546.x -
REN, G., HU, D., CHENG, E.W., VARGAS-REUS, M.A., REIP, P. and ALLAKER, R.P., 2009. Characterisation of copper oxide nanoparticles for antimicrobial applications. International Journal of Antimicrobial Agents, vol. 33, no. 6, pp. 587-590. http://doi.org/10.1016/j.ijantimicag.2008.12.004 PMid:19195845.
» http://doi.org/10.1016/j.ijantimicag.2008.12.004 -
SANGER, G.J., HOLBROOK, J.D. and ANDREWS, P.L., 2011. The translational value of rodent gastrointestinal functions: a cautionary tale. Trends in Pharmacological Sciences, vol. 32, no. 7, pp. 402-409. http://doi.org/10.1016/j.tips.2011.03.009 PMid:21531468.
» http://doi.org/10.1016/j.tips.2011.03.009 -
SCHIFFRIN, E. and BLUM, S., 2002. Interactions between the microbiota and the intestinal mucosa. European Journal of Clinical Nutrition, vol. 56, suppl. 3, pp. S60-S64. http://doi.org/10.1038/sj.ejcn.1601489 PMid:12142966.
» http://doi.org/10.1038/sj.ejcn.1601489 -
TAMURA, K., PETERSON, D., PETERSON, N., STECHER, G., NEI, M. and KUMAR, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, vol. 28, no. 10, pp. 2731-2739. http://doi.org/10.1093/molbev/msr121 PMid:21546353.
» http://doi.org/10.1093/molbev/msr121 -
ZHANG, K.M., WEXLER, A.S., ZHU, Y.F., HINDS, W.C. and SIOUTAS, C., 2004. Evolution of particle number distribution near roadways. Part II: the ‘Road-to-Ambient’process. Atmospheric Environment, vol. 38, no. 38, pp. 6655-6665. http://doi.org/10.1016/j.atmosenv.2004.06.044
» http://doi.org/10.1016/j.atmosenv.2004.06.044
Publication Dates
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Publication in this collection
22 Nov 2024 -
Date of issue
2024
History
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Received
02 Jan 2024 -
Accepted
23 Aug 2024
