Selective Enrichment Method Research Articles

A Foodborne Disease “Campylobacteriosis”: Global Epidemiology, Pathogenicity, Diagnosis and Treatment

Abstract: Campylobacteriosis is a foodborne disease caused by Campylobacter, which is one of the leading causative agents of bacterial gastrointestinal diseases in developed and developing countries. According to WHO, Campylobacter species infects a hundred million people yearly. The bacterium is thermotolerant, cytochrome oxidase-positive, spiral-shaped, gram-negative, and microaerophilic, by exhibiting corkscrew motility it passes through the intestines of animals and birds. It is generally transmitted through the consumption of contaminated food associated with animal and their products. The main infectious species include C. coli, C. jejuni, C. fetus and C. upsaliensis. Infection symptoms can be mild to serious depending upon the patient's age and in some cases can lead to permanent neurological disorders. Detection of Campylobacter in food, clinical and environmental samples is accomplished with the help of combinatorial usage of selective enrichment and culture methods. Currently, there is no sole viable approach for infection management because of resistance emergence. In this review article, we discuss Campylobacter epidemiology, pathogenicity, various diagnostic methods and treatment of Campylobacteriosis.

Rapid and sensitive determination of legacy and emerging per- and poly-fluoroalkyl substances with solid-phase microextraction probe coupled with mass spectrometry

This study was designed to develop a rapid and sensitive method for quantifying legacy and emerging per- and polyfluoroalkyl substances (PFASs) in environmental samples with solid-phase microextraction (SPME) coupled with mass spectrometry (MS). An innovative SPME probe was fabricated via in situ polymerization, and the probe coating was optimized with response surface methodology to maximize the fluorine–fluorine interactions and electrostatic properties and ensure high selectivity for the target PFASs with enrichment factors of 48–491. The coupled SPME and MS provided a rapid and sensitive method for analyses of PFASs, with excellent linearity (r ≥ 0.9962) over the concentration range 0.001–1 μg/L and remarkably low detection limits of 0.1–13.0 ng/L. This method was used to analyze trace PFASs in tap water, river water, and wastewater samples and proved to be a simple and efficient analytical method for selective enrichment and detection of contaminants in the environment.

Covalent organic framework aerogel for high-performance solid-phase extraction of tetracycline antibiotics: Experiment and simulated calculation on adsorption behavior

Three-dimensional sponge-architecture covalent organic frameworks (COFs)-aerogel was successfully designed and synthesized via a freeze-drying template approach, and utilized as an efficient sorbent in solid-phase extraction (SPE). A method for selective enrichment of pharmaceutical contaminants including tetracycline, chlortetracycline, methacycline and oxytetracycline in the environment and food samples was proposed by combining with high performance liquid chromatography (HPLC). To understand the adsorption mechanism, selectivity test and molecular dynamics (MD) simulated calculation were both carried out. The experimental and in-silico results demonstrated that the COFs-aerogel possessed high selectivity for contaminants with H bond acceptors/donors and good efficiency with maximum adsorption capacity up to 294.1 mg/g. The SPE-based HPLC method worked well in the range of 8–1000 ng/mL, with the need of little dose of adsorbent and sample volume while no need of spectrometer, outgoing the reported adsorbents. Under the optimized conditions, the intra-day and inter-day relative standard deviations (RSD) of repeatability were within 2.78–6.29 % and 2.44–8.42 % (n = 5). The results meet the current detection requirement for practical applications, and could be extended for further design of promising adsorbents.

Imprinting-based smart syringe: A fast, efficient, and selective approach for extraction of methyl gallate from Caesalpinia pulcherrima extract

A simple, facile, quick, highly selective, and efficient extraction protocol for a medicinal compound methyl gallate (MG) is developed for the first time using molecularly imprinted-based solid phase extraction (MISPE). Density functional theory (DFT) was employed to calculate the binding energies of the complex of MG with the functional monomer in the gas and in the implicit solvent to evaluate the best combination of the functional monomer, crosslinker, and porogen for MG. Decisively, the synthesis of a molecularly imprinted polymer (MIP) and its respective non-imprinted polymer (NIP) was conducted using methacrylamide (MAAm) as a functional monomer, trimethylolpropane trimethacrylate (TRIM) as a crosslinker, and methanol as a porogen. The polymers were characterized by SEM, FT-IR, BET, and TGA, while the adsorption properties of MG-MIP/NIP were evaluated by batch rebinding experiments. Theoretical modeling was done by Langmuir, Freundlich, Scatchard analysis, LF-isotherm, and kinetic models. The MG-MIP appeared to have a higher binding capacity towards MG compared to the corresponding NIP: 57.6 mg/g compared to only 17.6 mg/g rendering an imprinting factor 3.3. Moreover, in competitive recognition studies, the synthesized MG-MIP showed good selectivity towards MG compared to its structural analogs, confirming the presence of MG-specific imprinting sites. In competitive recognition tests and real-sample analysis, HPLC was used to determine the concentration of MG and the analogous compounds. Furthermore, the volume and elution times of the washing solvent (DI water) and elution solvent (acetonitrile) were optimized. Finally, MG-MISPE frits were successfully designed for selective enrichment of MG from Caesalpinia pulcherrima methanolic extract using a syringe, PTFE membrane, and MG-MIPs. Hence, MG-MISPE is a promising alternative of traditional extraction methods for selective extraction and enrichment of MG from complex plant matrices. The approach has potential to be applied to any compound of interest.

A Systematic Investigation of Proteoforms with N‐Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

AbstractThe N‐termini of proteins can regulate their degradation, and the same protein with different N‐termini may have distinct dynamics. Recently, it was found that N‐terminal glycine can serve as a degron recognized by two E3 ligases, but N‐terminal glycine was also reported to stabilize proteins. Here we developed a chemoenzymatic method for selective enrichment of proteoforms with N‐terminal glycine and integrated dual protease cleavage to further improve the enrichment specificity. Over 2000 unique peptides with protein N‐terminal glycine were analyzed from >1000 proteins, and most of them are previously unknown, indicating the effectiveness of the current method to capture low‐abundance proteoforms with N‐terminal glycine. The degradation rates of proteoforms with N‐terminal glycine were quantified along with those of proteins from the whole proteome. Bioinformatic analyses reveal that proteoforms with N‐terminal glycine with the fastest and slowest degradation rates have different functions and localizations. Membrane proteins with N‐terminal glycine and proteins with N‐terminal glycine from the N‐terminal methionine excision degrade more rapidly. Furthermore, the secondary structures, adjacent amino acid residues, and protease specificities for N‐terminal glycine are also vital for protein degradation. The results advance our understanding of the effects of N‐terminal glycine on protein properties and functions.

A Systematic Investigation of Proteoforms with N-Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability.

The N-termini of proteins can regulate their degradation, and the same protein with different N-termini may have distinct dynamics. Recently, it was found that N-terminal glycine can serve as a degron recognized by two E3 ligases, but N-terminal glycine was also reported to stabilize proteins. Here we developed a chemoenzymatic method for selective enrichment of proteoforms with N-terminal glycine and integrated dual protease cleavage to further improve the enrichment specificity. Over 2000 unique peptides with protein N-terminal glycine were analyzed from >1000 proteins, and most of them are previously unknown, indicating the effectiveness of the current method to capture low-abundance proteoforms with N-terminal glycine. The degradation rates of proteoforms with N-terminal glycine were quantified along with those of proteins from the whole proteome. Bioinformatic analyses reveal that proteoforms with N-terminal glycine with the fastest and slowest degradation rates have different functions and localizations. Membrane proteins with N-terminal glycine and proteins with N-terminal glycine from the N-terminal methionine excision degrade more rapidly. Furthermore, the secondary structures, adjacent amino acid residues, and protease specificities for N-terminal glycine are also vital for protein degradation. The results advance our understanding of the effects of N-terminal glycine on protein properties and functions.

Carrier with cyclodextrin and quorum sensing synergy: An efficient method for selective enrichment of anammox bacteria

High-efficiency enrichment of functional bacteria is one of the biggest technical barriers to the application of anaerobic ammonium oxidation (anammox). This study combined biofilm carriers and quorum sensing to achieve rapid biomass accumulation. A biofilm carrier with biomass adsorption and quorum sensing regulation functions was developed for the rapid start-up of the reactor and enrichment of anammox bacteria (AnAOB). The nitrogen removal rates of the three reactors were further tested, and the carrier mechanism was explored. The start-up time of the reactor with the modified carriers was 28 days, which was ∼44 % shorter than that of the reactor without carriers. After 90 days of operation, the absolute abundance of total AnAOB was 3.2 times that in the control group. The modified carrier promoted the rapid adhesion of biomass through the hydrophobic cavity of cyclodextrin and induced rapid proliferation, enhancement of activity, and extracellular protein secretion of AnAOB through quorum sensing signaling molecules. Finally, a special hydrophobic biofilm with granular sludge characteristics was formed on the modified carrier through the self-assembly of hydrophobic biomass particles. Overall, we propose a novel approach to enrich anammox bacteria involving a combination of quorum sensing and biofilm carriers.

Growth assessment of Salmonella enterica multi-serovar populations in poultry rinsates with commonly used enrichment and plating media

Isolation of Salmonella from enrichment cultures of food or environmental samples is a complicated process. Numerous factors including fitness in various selective enrichment media, relative starting concentrations in pre-enrichment, and competition among multi-serovar populations and associated natural microflora, come together to determine which serovars are identified from a given sample. A recently developed approach for assessing the relative abundance (RA) of multi-serovar Salmonella populations (CRISPR-SeroSeq or Deep Serotyping, DST) is providing new insight into how these factors impact the serovars observed, especially when different selective enrichment methods are used to identify Salmonella from a primary enrichment sample. To illustrate this, we examined Salmonella-positive poultry pre-enrichment samples through the selective enrichment process in Tetrathionate (TT) and Rappaport Vassiliadis (RVS) broths and assessed recovery of serovars with each medium. We observed the RA of serovars detected post selective enrichment varied depending on the medium used, initial concentration, and competitive fitness factors, all which could result in minority serovars in pre-enrichment becoming dominant serovars post selective enrichment. The data presented provide a greater understanding of culture biases and lays the groundwork for investigations into robust enrichment and plating media combinations for detecting Salmonella serovars of greater concern for human health.

Understanding Molecular Transfer between Aqueous Phase and Reverse Micelles and Its Application to Bioassays in Microfluidics

Abstract Micrometer-sized water-in-oil droplets (microdroplets) are attracting attention as carriers for the high-throughput parallel analysis of trace samples. By confining trace amounts of water-soluble samples or reagents within a microdroplet, reactions and detection can be performed while suppressing diffusion dilution. Although many manipulation methods for droplets, such as mixing, merging, and splitting using microfluidic channels have been reported, concentration enrichment and purification techniques for microdroplet inclusions have been limited. Our recently developed method for selective concentration enrichment of microdroplet contents focuses on molecular transport between microdroplets and reverse micelles.

Selective enrichment of licorice triterpenoid saponins from complicated samples by magnetic poly(ionic liquid)-functionalized metal-organic framework

Licorice is a frequently-used natural sweetener and herbal medicine, and triterpenoid saponins are the main sweet constituents. This study provided a novel method for selective enrichment of licorice triterpenoid saponins by Fe3O4@UiO-66-(SH)2@p[VBAI]Cl, a new magnetic poly(ionic liquid)-functionalized metal-organic framework composite. Triterpenoid saponins had strong electrostatic interactions with p[VBAI]Cl, namely poly[1-(4-vinyl)benzyl-3-alkylimidazolium chloride], and UiO-66-(SH)2 further increased the adsorption ability. Fe3O4@UiO-66-(SH)2@p[VBAI]Cl could selectively enrich triterpenoid saponins from licorice-containing commercial products and plasma/tissue samples after oral consumption, and enrichment conditions were optimized with the representative compound glycyrrhizic acid. Based on this, two quantitative analysis methods were established combined with HPLC and LC-MS analysis, respectively, which were used to efficiently determine triterpenoid saponin content in in vitro and in vivo samples. The achieved LC-MS detection sensitivity was higher than previous studies. This is the first study for selective enrichment of natural products by poly(ionic liquid)-based magnetic solid phase extraction, and the method can contribute to quality assessment and mechanistic study of licorice.

Genomic Characterization of Carbapenem-Resistant Bacteria from Beef Cattle Feedlots.

Carbapenems are considered a last resort for the treatment of multi-drug-resistant bacterial infections in humans. In this study, we investigated the occurrence of carbapenem-resistant bacteria in feedlots in Alberta, Canada. The presumptive carbapenem-resistant isolates (n = 116) recovered after ertapenem enrichment were subjected to antimicrobial susceptibility testing against 12 different antibiotics, including four carbapenems. Of these, 72% of the isolates (n = 84) showed resistance to ertapenem, while 27% of the isolates (n = 31) were resistant to at least one other carbapenem, with all except one isolate being resistant to at least two other drug classes. Of these 31 isolates, 90% were carbapenemase positive, while a subset of 36 ertapenem-only resistant isolates were carbapenemase negative. The positive isolates belonged to three genera; Pseudomonas, Acinetobacter, and Stenotrophomonas, with the majority being Pseudomonas aeruginosa (n = 20) as identified by 16S rRNA gene sequencing. Whole genome sequencing identified intrinsic carbapenem resistance genes, including blaOXA-50 and its variants (P. aeruginosa), blaOXA-265 (A. haemolyticus), blaOXA-648 (A. lwoffii), blaOXA-278 (A. junii), and blaL1 and blaL2 (S. maltophilia). The acquired carbapenem resistance gene (blaPST-2) was identified in P. saudiphocaensis and P. stutzeri. In a comparative genomic analysis, clinical P. aeruginosa clustered separately from those recovered from bovine feces. In conclusion, despite the use of selective enrichment methods, finding carbapenem-resistant bacteria within a feedlot environment was a rarity.

Nanoparticle Assembly at the Water-Oil Interface Induced by Spontaneous Emulsification for Microdroplet Immunoassay.

Micron-sized water-in-oil droplets (microdroplets) have been used for various biochemical analyses. Many studies have been reported on immunoassays using microdroplets because of their high versatility. A selective enrichment method using spontaneous emulsification was developed as a pretreatment method for analytical systems of microdroplets. In this study, a one-step immunoassay for microdroplets using nanoparticle assembly at the interface by spontaneous emulsification is proposed. At the interface of the microdroplet, with aqueous nanoparticle dispersion, it was found that nanoparticles with diameters less than 50 nm were uniformly adsorbed to the microdroplet interface as a Pickering emulsion, whereas larger nanoparticles tended to aggregate in the bulk part of the microdroplet. Based on this phenomenon, a proof of concept of the one-step immunoassay was demonstrated using rabbit IgG as the analyte. This method is expected to be a powerful tool for trace biochemical analyses.

Isolation and identification of hyaluronan-degrading bacteria from Japanese fecal microbiota.

Hyaluronan (HA) is a high-molecular-weight glycosaminoglycan and widely distributed in all connective tissues and organs with diverse biological functions. HA has been increasingly used as dietary supplements targeted to joint and skin health for humans. We here first report isolation of bacteria from human feces that are capable of degrading HA to lower molecular weight HA oligosaccharides (oligo-HAs). The bacteria were successfully isolated via a selective enrichment method, in which the serially diluted feces of healthy Japanese donors were individually incubated in an enrichment medium containing HA, followed by the isolation of candidate strains from streaked HA-containing agar plates and selection of HA-degrading strains by measuring HA using an ELISA. Subsequent genomic and biochemical assays identified the strains as Bacteroides finegoldii, B. caccae, B. thetaiotaomicron, and Fusobacterium mortiferum. Furthermore, our HPLC analysis revealed that the strains degraded HA to oligo-HAs of various lengths. Subsequent quantitative PCR assay targeting the HA degrading bacteria showed that their distribution in the Japanese donors varied. The evidence suggests that dietary HA is degraded by the human gut microbiota with individual variation to oligo-HAs components, which are more absorbable than HA, thereby exerting its beneficial effects.

Rapid Enrichment of Cupriavidus necator in Mixed Microbial Cultures Using Autotrophic Growth: Mixed Microbial Cultures for Biodegradable Polymer Production Using CO2 and Organic Wastes

Polyhydroxyalkanoate (PHA) is a biodegradable polymer that can replace petroleum-based plastics. Cupriavidus necator has been extensively utilized in industrial PHA production because the bacterium can grow and accumulate high amounts of PHA in their cells under heterotrophic and/or autotrophic growth conditions. Microbial PHA production using mixed cultures reduces the high PHA production costs and allows for the utilization of waste organics as carbon sources. However, C. necator cultivation in mixed microbial cultures has not been investigated before. In this work, we developed innovative methods for rapid and selective enrichment of C. necator in mixed cultures, utilizing the unique metabolic characteristics of C. necator (i.e., the ability to grow heterotrophically and/or autotrophically). Three enrichment methods were proposed: autotrophic-only, alternating autotrophic–heterotrophic, and heterotrophic-only cycles. These methods were examined with two wastewater sources as initial mixed mixtures. In the autotrophic-only method, the fraction of C. necator exceeded 92% of the total microbial population in only 60 h, whereas the alternating autotrophic–heterotrophic cycles showed >81.3% selection of C. necator. However, the heterotrophic-only method resulted in practically no enrichment of C. necator after five repeated feast–famine cycles. When tested for PHA accumulation, enriched cultures using the autotrophic and alternating cycles accumulated significant amounts of PHA, confirming the selective and rapid enrichment of C. necator over other microorganisms. Based on the experimental results, the autotrophic-based enrichment is a potential approach for the cost-effective cultivation of C. necator, which can reduce PHA production costs.

Attachment of potential cultivable primo-colonizing bacteria and its implications on the fate of low-density polyethylene (LDPE) plastics in the marine environment

Plastics released in the environment become suitable matrices for microbial attachment and colonization. Plastics-associated microbial communities interact with each other and are metabolically distinct from the surrounding environment. However, pioneer colonizing species and their interaction with the plastic during initial colonization are less described. Marine sediment bacteria from sites in Manila Bay were isolated via a double selective enrichment method using sterilized low-density polyethylene (LDPE) sheets as the sole carbon source. Ten isolates were identified to belong to the genera Halomonas, Bacillus, Alteromonas, Photobacterium, and Aliishimia based on 16S rRNA gene phylogeny, and majority of the taxa found exhibit a surface-associated lifestyle. Isolates were then tested for their ability to colonize polyethylene (PE) through co-incubation with LDPE sheets for 60 days. Growth of colonies in crevices, formation of cell-shaped pits, and increased roughness of the surface indicate physical deterioration. Fourier-transform infrared (FT-IR) spectroscopy revealed significant changes in the functional groups and bond indices on LDPE sheets separately co-incubated with the isolates, demonstrating that different species potentially target different substrates of the photo-oxidized polymer backbone. Understanding the activity of primo-colonizing bacteria on the plastic surface can provide insights on the possible mechanisms used to make plastic more bioavailable for other species, and their implications on the fate of plastics in the marine environment.

The 2022 Nobel Prize in Chemistry for the development of click chemistry and bioorthogonal chemistry.

The 2022 Nobel Prize in Chemistry recognized the development of biorthogonal chemical ligation reactions known as click chemistry in biomedicine. This concept has catalyzed significant progress in sensing and diagnosis, chemical biology, materials chemistry, and drug discovery and delivery. In proteomics, the ability to incorporate a click tag into proteins has propelled development of powerful new methods for selective enrichment of protein complexes that inform understanding of protein networks. It also has had a strong influence on the ability to enrich for protein post-translational modifications. This feature article summarizes the impacts of biorthogonal click chemistry on proteomics.

Mito-SiPE is a sequence-independent and PCR-free mtDNA enrichment method for accurate ultra-deep mitochondrial sequencing

The analysis of somatic variation in the mitochondrial genome requires deep sequencing of mitochondrial DNA. This is ordinarily achieved by selective enrichment methods, such as PCR amplification or probe hybridization. These methods can introduce bias and are prone to contamination by nuclear-mitochondrial sequences (NUMTs), elements that can introduce artefacts into heteroplasmy analysis. We isolated intact mitochondria using differential centrifugation and alkaline lysis and subjected purified mitochondrial DNA to a sequence-independent and PCR-free method to obtain ultra-deep (>80,000X) sequencing coverage of the mitochondrial genome. This methodology avoids false-heteroplasmy calls that occur when long-range PCR amplification is used for mitochondrial DNA enrichment. Previously published methods employing mitochondrial DNA purification did not measure mitochondrial DNA enrichment or utilise high coverage short-read sequencing. Here, we describe a protocol that yields mitochondrial DNA and have quantified the increased level of mitochondrial DNA post-enrichment in 7 different mouse tissues. This method will enable researchers to identify changes in low frequency heteroplasmy without introducing PCR biases or NUMT contamination that are incorrectly identified as heteroplasmy when long-range PCR is used.

Prevalence and antimicrobial susceptibility pattern of methicillin-resistant Staphylococcus aureus isolates from retail raw meats in Turkey

The transmission of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) through food products of animal origin may pose a public health concern. Therefore, the objectives of the present work were to determine the prevalence of S. aureus and MRSA in raw meat samples (beef, chicken, turkey, and duck) at retail level, and to determine the antimicrobial resistance profile of the MRSA isolates. Between September 2018 and January 2019, a total of 325 raw meat samples were collected from retail stores, and analysed for the presence of S. aureus and MRSA using primary enrichment method as well as using secondary selective enrichment methods for MRSA detection. All the suspected S. aureus and MRSA colonies obtained from the samples were confirmed by both phenotypic and genotypic methods. The MRSA isolates were tested against various antimicrobials, and the minimal inhibitory concentration (MIC) of vancomycin was determined. Macrolide-lincosamide-streptogramin B (MLSB) resistance phenotypes were also screened using the double-disk diffusion test. The overall prevalence of S. aureus was 57.2%, whereas the food-specific prevalence in beef, turkey, duck, and chicken samples was 75, 53, 48, and 46%, respectively. The overall prevalence of MRSA was 1.2%, whereas it was 3, 1, 0, and 0% in beef, turkey, chicken, and duck samples, respectively. All MRSA isolates were detected by the secondary selective enrichment method. These MRSA isolates had a variety of MLSB resistance phenotypes (i.e., iMLSB, cMLSB, and MS-MLSB) with vancomycin MIC values ranging between 1 - 2 µg/mL. These findings are important for developing interventions to reduce raw meat contamination with S. aureus and MRSA, and to improve public health.

Prevalence of Listeria monocytogenes in Foods of Animal Origin: Study from Assam, A North-Eastern State of India

Listeriosis is an important emerging bacterial food-borne zoonotic disease caused by Listeria monocytogenes. During the last two decades, the organism has been implicated in several food-borne disease outbreaks making it a serious public health hazard. The pathogenic strains of Listeria can cause severe illness in man after the ingestion of contaminated food products, especially the refrigerated foods of animal origin. In India, very few studies have been carried out in respect to the occurrence and incidence of L. monocytogenes. Assam, one of the eight states of Northeastern India, where approximately 79.0 percent of the total population are non-vegetarians. Chicken meat, pork, chevon and beef are exclusively taken as regular food items throughout the state. The present study describes about isolation, identification and molecular characterization of L.monocytogens from different foods of animal origin from Assam, India. A total of 750 different meat (chicken, chevon, pork and beef) and milk and their products (curd and paneer) samples were collected and analyzed for presence of Listeria. Isolation of L. monocytogenes was done from different food samples of animal origin by using selective enrichment method. PCR was employed for confirmation of the suspected cultures of Listeria targeting prsand16SrRNAgene. Upon confirmation, the isolates were further characterized based on 16S rRNA gene and phylogenetic analysis was performed. Of 750 samples, 48 (6.4%) samples were found positive. The highest rate of isolation of Listeria spp. was recorded in raw milk (8.95%) followed by curd (6.58%), chevon (5.36%), chicken (5.74%), pork (5.98%) and beef (3.75%). Molecular characterization of the isolates based on 16S rRNA gene of L.monocytogens isolated from foods of animal origin from Assam with that of other sequences reported from different parts of the world revealed that all the isolates of the present study are clustered along with other L.monocytogens isolates reported from different parts of the world, however, showing more closely related to isolates reported from China.

Selective Enrichment Method for Isolation of Efficient Phosphate Solubilizing Bacteria from Soil

ABSTRACT Isolation and screening of phosphate solubilizing bacteria (PSB) seems to become cumbersome using conventional methods, and there may be a chance to lose the more potent isolates during the screening methodology. To overcome these problems, a selective enrichment method has been developed to isolate and screen efficient PSB from diverse sources. Initially, this method includes enrichment of the phosphate solubilizers in the sample followed by selective isolation and screening. Looking into the physico-chemical data, it was revealed that the soil samples from cultivated soil were found to support growth and development of microbial life in general compared to soil samples from non-cultivated stringent environment, which was reflected in number of PSB isolated, or in some samples, PSB may not be obtained. Here, the selective enrichment method developed for isolation of PSB from stringent environmental samples yielded numerically higher number (14) of phosphate solubilizer isolates in comparison to conventional methods (4) from different soil sources. Moreover, isolates obtained following selective enrichment were found consistence for phosphate solubilization in different conditions and for different sources. In contrast to conventional isolation methodologies, the selective enrichment method consistently performed best for PSB isolation particularly from stringent habitats, i.e. mining region where conventional method/s did not perform well.