Pure Bacterial Cultures Research Articles

Quantification of nitrogen cycle functional genes from viable archaea and bacteria in paddy soil.

One of the main challenges of culture-independent soil microbiology is distinguishing the microbial community's viable fraction from dead matter. Propidium monoazide (PMA) binds the DNA of dead cells, preventing its amplification. This dye could represent a robust means to overcome the drawbacks of other selective methods, such as ribonucleic acid-based analyses. We quantified functional genes from viable archaea and bacteria in soil by combining the use of PMA and quantitative polymerase chain reaction. Four N-cycle-related functional genes (bacterial and archaeal ammonia monooxygenase, nitrate reductase, and nitrite reductase) were successfully quantified from the living fraction of bacteria and archaea of a paddy soil. The protocol was also tested with pure bacterial cultures and soils with different physical and chemical properties. The experiment results revealed a contrasting impact of mineral and organic fertilizers on the abundance of microbial genes related to the N-cycle in paddy soil.

Efficiency of Lactiplantibacillus plantarum JT-PN39 and Paenibacillus motobuensis JT-A29 for Fermented Coffee Applications and Fermented Coffee Characteristics.

To develop a process for low-cost and ecologically friendly coffee fermentation, civet gut bacteria were isolated and screened to be used for fermentation. Among 223 isolates from civet feces, two bacteria exhibited strong protease, amylase, lipase, pectinase, and cellulase activities. By analyzing 16S rDNA phylogeny, those bacteria were identified to be Lactiplantibacillus plantarum JT-PN39 (LP) and Paenibacillus motobuensis JT-A29 (PM), where their potency (pure or mixed bacterial culture) for fermenting 5 L of arabica parchment coffee in 48-72 h was further determined. To characterize the role of bacteria in coffee fermentation, growth and pH were also determined. For mixed starter culture conditions, the growth of PM was not detected after 36 h of fermentation due to the low acid conditions generated by LP. Coffee quality was evaluated using a cupping test, and LP-fermented coffee expressed a higher cupping score, with a main fruity and sour flavor, and a dominant caramel-honey-like aroma. Antioxidant and anti-foodborne pathogenic bacteria activity, including total phenolic compounds of PM and LP fermented coffee extracts, was significantly higher than those of ordinary coffee. In addition, LP-fermented coffee expressed the highest antibacterial and antioxidant activities among the fermented coffee. The toxicity test was examined in the murine macrophage RAW 264.7 cell, and all fermented coffee revealed 80-90% cell variability, which means that the fermentation process does not generate any toxicity. In addition, qualifications of non-volatile and volatile compounds in fermented coffee were examined by LC-MS and GC-MS to discriminate the bacterial role during the process by PCA plot. The flavors of fermented coffee, including volatile and non-volatile compounds, were totally different between the non-fermented and fermented conditions. Moreover, the PCA plot showed slightly different flavors among fermentations with different starter cultures. For both the cupping test and biological activities, this study suggests that LP has potential for health benefits in coffee fermentation.

Isolation and screening of keratinolytic bacteria from feather dumping soil near in Lucknow and Kanpur city, North region of Indian

Background Keratinases are proteolytic enzymes that have the capability of degrading insoluble keratin substrates. LS4, LS5, and KS3 were newly isolated strains showing ability to hydrolyze keratins and keratin based products. Objective In this study, different bacterial strains were isolated from soil, screened for protease and keratinase production, and the best isolates were identified. Methods Serial dilution and plating method were used for isolation of pure bacterial culture. Isolated, 48 strains were screened, and their feather degrading ability on feather meal agar plates and keratin azure-based agar. The proteolytic activity of the isolates was also screened on casein agar. Results Three strains; LS4, LS5, and KS3 shows keratinolytic activity that reveal the keratinase enzyme on medium; feather meal agar, keratin azure agar, and casein agar. Among all the three strains (LS4, LS5, and KS3), KS3 show largest inhibition zone that show maximum enzymatic activity. For its characterization 16S RNA was performed, sequence KS3 was submitted to GenBank and was identified as Bacillus tropicus KS3. Newly isolated strain B. tropicus KS3 showed the keratinase production and highest keratinase activity (35.06 ± 2.5 U/ml). B. tropicus KS3 also show the highest degradation of feather efficiently showed 84% of whole chicken feather biodegradation at 37 °C within 15 days among all the isolated strains. Conclusion Keratinase enzyme possesses a wide range of potential applications in the bioremediation of feather waste and bio-fertilizer in agricultural land. The industry demands a better source of this enzyme due to its multivariate application and huge market demand. In the present study, a novel keratinase-producing bacteria was isolated and characterized as Bacillus tropicus KS3 from poultry waste contamination soil.

Moisture-induced electrical power generation with waste activated sludge

Moisture-induced power generation from pure bacterial cultures has been demonstrated, but the feasibility of extending this renewable energy harvesting method to mixed bacterial cells from environmental samples is uncertain. Herein, waste active sludge (WAS) from wastewater treatment reactors was used to fabricate whole-cell film-based moisture-induced electricity generators (MEGs). Electrical power could be harvested from all four representative WAS samples, which were obtained from wastewater treatment reactors or plants treating liquid-ammonia mercerization, municipal, beer brewery, and printing-dyeing wastewaters. The highest performing MEGs were obtained from anammox activated sludge with an open circuit voltage of 0.45 ± 0.02 V and a maximum power density of 5.24 μW/cm2, which outperformed those MEGs with pure bacterial cultures. Physicochemical characterizations combined with partial least-squares path modeling suggested that the amount of microbial biomass and hydrophilicity of the as-prepared WAS films were the most important factors in determining the performance of the WAS-based MEGs. Extracellular polymer substances were essential in creating a water gradient inside the WAS, which was a crucial mechanism for generating electricity from MEGs based on WAS. This study not only provides a new approach for developing materials for moisture-induced energy harvesting but also presents a novel avenue for resource utilization of WAS.

Isolation and characterization of Rhodococcus sp. GG1 for metabolic degradation of chloroxylenol

The coronavirus disease 2019 (COVID-19) pandemic has significantly increased the demand of disinfectant use. Chloroxylenol (para-chloro-meta-xylenol, PCMX) as the major antimicrobial ingredient of disinfectant has been widely detected in water environments, with identified toxicity and potential risk. The assessment of PCMX in domestic wastewater of Macau Special Administrative Region (SAR) showed a positive correlation between PCMX concentration and population density. An indigenous PCMX degrader, identified as Rhodococcus sp. GG1, was isolated and found capable of completely degrading PCMX (50 mg L−1) within 36 h. The growth kinetics followed Haldane's inhibition model, with maximum specific growth rate, half-saturation constant, and inhibition constant of 0.38 h−1, 7.64 mg L−1, and 68.08 mg L−1, respectively. The degradation performance was enhanced by optimizing culture conditions, while the presence of additional carbon source stimulated strain GG1 to alleviate inhibition from high concentrations of PCMX. In addition, strain GG1 showed good environmental adaptability, degrading PCMX efficiently in different environmental aqueous matrices. A potential degradation pathway was identified, with 2,6-dimethylhydroquinone as a major intermediate metabolite. Cytochrome P450 (CYP450) was found to play a key role in dechlorinating PCMX via hydroxylation and also catalyzed the hydroxylated dechlorination of other halo-phenolic contaminants through co-metabolism. This study characterizes an aerobic bacterial pure culture capable of degrading PCMX metabolically, which could be promising in effective bioremediation of PCMX-contaminated sites and in treatment of PCMX-containing waste streams.

Vaginal bacteria of healthy cats during different stages of their oestrous cycle

Little is known about vaginal and uterine bacteria in clinically healthy cats and their correlation with different stages of the oestrus cycle. The differences in vaginal bacterial flora between household and stray queens remains unknown. The aim of this study was to investigate the occurrence of vaginal and uterine bacteria in clinically healthy household and stray queens and to correlate culture findings with specific stages of the oestrous cycle. Vaginal and uterine samples from 40 clinically healthy queens were collected for isolation of bacteria and cytological examination. Bacteria were isolated from 31 vaginal swabs (77.5%) from stray (16/20; 80%) and household (15/20; 75%) cats. The isolates were more frequently detected in pure culture (18/31; 58%) than in mixed cultures (13/31; 41.9%). Streptococcus spp. was the most commonly identified bacteria (n = 16; 51.6%), followed by coagulase negative Staphylococcus spp. (n = 15; 48.4%) and E. coli (n = 12; 38.7%). A mixed bacterial culture of E. coli and Streptococcus spp. was commonly detected (50%), mainly in households (66.7%), whereas a mixed culture of Staphylococcus spp. and Streptococcus spp. (41.6%) was commonly isolated from stray cats (60%). The frequency of isolation of pure or mixed bacterial cultures and the isolates did not vary significantly during the different stages of the oestrus cycle. All uterine samples tested were negative for bacteria. This study identified the most common bacteria in the vagina of clinically healthy cats. The isolation of vaginal bacteria in pure or mixed cultures should be considered as normal finding. The stage of the oestrous cycle apparently does not affect vaginal bacterial flora. Vaginal bacteria may differ between stray and households cats. No bacteria can be isolated from the uterus of clinically healthy cats.

Bio‑hydrogen and VFA production from steel mill gases using pure and mixed bacterial cultures

A major source of CO2 emissions is the flaring of steel mill gas. This work demonstrated the enrichment of carboxydotrophic bacteria for converting steel mill gas into volatile fatty acids and H2, via gas fermentation. Several combinations of pure and mixed anaerobic cultures were used as inoculum in 0.5-L reactors, operated at 30 and 60 °C. The process was then scaled up in a 4-L membrane bioreactor, operated for 20 days, at 48 °C. The results showed that the enriched microbiomes can oxidize CO completely to produce H2/H+ which is subsequently used to fix the CO2. At 30 °C, a mixture of acetate, isobutyrate and propionate was obtained while H2 and acetate were the main products at 60 °C. The highest CO conversion and H2 production rate observed in the membrane bioreactor were 29 and 28 mL/LR/h, respectively. The taxonomic diversity of the bacterial community increased and the dominant species was Pseudomonas.

Utilization of a New Hundred-Genomes Pipeline to Design a Rapid Duplex LAMP Detection Assay for Xanthomas euvesicatoria and X. vesicatoria in Tomato.

Xanthomonas euvesicatoria (Xe) and X. vesicatoria (Xv) are two economically important causal agents of bacterial spot (BS) of tomato and pepper. Management of BS in the field requires rapid and accurate detection. This work therefore aimed to develop a pipeline to design a simple, fast, and reliable assay for the detection of Xe and Xv by Loop-Mediated Isothermal Amplification (LAMP). A total of 109 publicly available whole genomic sequences of 24 different species of bacterial pathogens were used to design primers that would amplify the DNA of the two target species. Laboratory testing of the assay was performed on pure bacterial cultures and artificially infected plants, and amplification was conducted with both a sophisticated laboratory instrument and a simple mobile platform. The testing of the assay confirmed its specificity with a sensitivity reaching 1 pg μL-1 for both pathogens with an assay duration of 40 minutes on a mobile detection platform. Our diagnostics development pipeline enables the easy and fast design of a reliable detection assay in the genomics age. By validating the pipeline with Xe and Xv pathogens, we have simultaneously developed an assay with high specificity, sensitivity, and speed, which will allow it to be deployed, contributing to successful management of BS.

Susceptibility of Campylobacter jejuni to Stressors in Agrifood Systems and Induction of a Viable-but-Nonculturable State.

Many bacteria can become viable but nonculturable (VBNC) in response to stressors commonly identified in agrifood systems. Campylobacter is able to enter the VBNC state to evade unfavorable environmental conditions, but how food processing can induce Campylobacter jejuni to enter this state and the potential role of foods in inducing the VBNC state in C. jejuni remains largely unknown. In this study, the culturability and viability of C. jejuni cells were investigated under chlorine treatment (25 ppm), aerobic stress (atmospheric condition), and low-temperature (4°C) conditions that mimicked food processing. In addition, the behaviors of C. jejuni cells in ultrahigh-temperature (UHT) and pasteurized milk were also monitored during refrigerated storage. The numbers of viable and culturable C. jejuni cells in both the pure bacterial culture and food matrices were separately determined by propidium monoazide (PMA)-quantitative PCR (qPCR) and plating assay. The C. jejuni cells lost their culturability but partially retained their viability (1% to 10%) once mixed with chlorine. In comparison, ~10% of C. jejuni cells were induced to enter the VBNC state after 24 h and 20 days under aerobic and low-temperature conditions, respectively. The viability of the C. jejuni cells remained stable during the induction process in UHT (>10%) and pasteurized (>10%) milk. The number of culturable C. jejuni cells decreased quickly in pasteurized milk, but culturable cells could still be detected in the end (day 21). In contrast, the number of culturable C. jejuni cells slowly decreased, and they became undetectable after >42 days in UHT milk. The C. jejuni cells responded differently to various stress conditions and survived in high numbers in the VBNC state in agrifood systems. IMPORTANCE The VBNC state of pathogens can pose risks to food safety and public health because the pathogens cannot be detected using conventional microbiological culture-based methods but can resuscitate under favorable conditions to develop virulence. As a leading cause of human gastroenteritis worldwide, C. jejuni can enter the VBNC state to survive in the environment and food-processing chain with high prevalence. In this study, the effect of food-processing conditions and food products on the development of VBNC state in C. jejuni was investigated, providing a better understanding of the interaction between C. jejuni and the agroecosystem. The knowledge elicited from this study can aid in developing novel intervention strategies to reduce the food safety risks associated with this microbe.

Scedosporium/Lomentospora Species Induce the Production of Siderophores by Pseudomonas aeruginosa in a Cystic Fibrosis Mimic Environment

Over the last years, the interkingdom microbial interactions concerning bacteria and fungi cohabiting and/or responsible for human pathologies have been investigated. In this context, the Gram-negative bacterium Pseudomonas aeruginosa and fungal species belonging to the Scedosporium/Lomentospora genera are widespread, multidrug-resistant, emergent, opportunistic pathogens that are usually co-isolated in patients with cystic fibrosis. The available literature reports that P. aeruginosa can inhibit the in vitro growth of Scedosporium/Lomentospora species; however, the complex mechanisms behind this phenomenon are mostly unknown. In the present work, we have explored the inhibitory effect of bioactive molecules secreted by P. aeruginosa (3 mucoid and 3 non-mucoid strains) on S. apiospermum (n = 6 strains), S. minutisporum (n = 3), S. aurantiacum (n = 6) and L. prolificans (n = 6) under cultivation in a cystic fibrosis mimic environment. It is relevant to highlight that all bacterial and fungal strains used in the present study were recovered from cystic fibrosis patients. The growth of Scedosporium/Lomentospora species was negatively affected by the direct interaction with either mucoid or non-mucoid strains of P. aeruginosa. Moreover, the fungal growth was inhibited by the conditioned supernatants obtained from bacteria-fungi co-cultivations and by the conditioned supernatants from the bacterial pure cultures. The interaction with fungal cells induced the production of pyoverdine and pyochelin, 2 well-known siderophores, in 4/6 clinical strains of P. aeruginosa. The inhibitory effects of these four bacterial strains and their secreted molecules on fungal cells were partially reduced with the addition of 5-flucytosine, a classical repressor of pyoverdine and pyochelin production. In sum, our results demonstrated that distinct clinical strains of P. aeruginosa can behave differently towards Scedosporium/Lomentospora species, even when isolated from the same cystic fibrosis patient. Additionally, the production of siderophores by P. aeruginosa was induced when co-cultivated with Scedosporium/Lomentospora species, indicating competition for iron and deprivation of this essential nutrient, leading to fungal growth inhibition.

Isolation and Cultivation of Human Gut Microorganisms: A Review.

Microbial resources from the human gut may find use in various applications, such as empirical research on the microbiome, the development of probiotic products, and bacteriotherapy. Due to the development of "culturomics", the number of pure bacterial cultures obtained from the human gut has significantly increased since 2012. However, there is still a considerable number of human gut microbes to be isolated and cultured. Thus, to improve the efficiency of obtaining microbial resources from the human gut, some constraints of the current methods, such as labor burden, culture condition, and microbial targetability, still need to be optimized. Here, we overview the general knowledge and recent development of culturomics for human gut microorganisms. Furthermore, we discuss the optimization of several parts of culturomics including sample collection, sample processing, isolation, and cultivation, which may improve the current strategies.

Multiplex polymerase spiral reaction for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are common food-borne pahogens that cause food poisoning in humans. In this study, we developed a method for the simultaneous determination of S. typhimurium and S. aureus based on multiplex polymerase spiral reaction (m-PSR) and melting curve analysis. Two pairs of primers were designed specifically to target the conserved invA gene of S. typhimurium and nuc gene of S. aureus, and the nucleic acid amplification reaction was achieved under isothermal conditions in the same reaction tube for 40 min at 61 °C, melting curve analysis of the amplification product was carried out. The distinct mean melting temperature allowed simultaneous differentiation of the two target bacteria in the m-PSR assay. The limit of detection of S. typhimurium and S. aureus that could be detected simultaneously was 4.1 × 10−4 ng genomic DNA and 2 × 101 CFU/mL pure bacterial culture. Based on this method, analysis of artificially contaminated samples showed excellent sensitivity and specificity consistent with those of pure bacterial cultures. This method is rapid, simultaneous and promises to be a useful tool for the detection of food-borne pathogens in the food industry.

High-throughput microbial culturomics using automation and machine learning

Pure bacterial cultures remain essential for detailed experimental and mechanistic studies in microbiome research, and traditional methods to isolate individual bacteria from complex microbial ecosystems are labor-intensive, difficult-to-scale and lack phenotype–genotype integration. Here we describe an open-source high-throughput robotic strain isolation platform for the rapid generation of isolates on demand. We develop a machine learning approach that leverages colony morphology and genomic data to maximize the diversity of microbes isolated and enable targeted picking of specific genera. Application of this platform on fecal samples from 20 humans yields personalized gut microbiome biobanks totaling 26,997 isolates that represented >80% of all abundant taxa. Spatial analysis on >100,000 visually captured colonies reveals cogrowth patterns between Ruminococcaceae, Bacteroidaceae, Coriobacteriaceae and Bifidobacteriaceae families that suggest important microbial interactions. Comparative analysis of 1,197 high-quality genomes from these biobanks shows interesting intra- and interpersonal strain evolution, selection and horizontal gene transfer. This culturomics framework should empower new research efforts to systematize the collection and quantitative analysis of imaging-based phenotypes with high-resolution genomics data for many emerging microbiome studies.

Fluorescent spectroscopy paired with parallel factor analysis for quantitative monitoring of phenanthrene biodegradation and metabolite formation

Polycyclic aromatic hydrocarbons (PAHs) are a class of environmental contaminants released into the environment from both natural and anthropogenic sources that are associated with carcinogenic, mutagenic, and teratogenic health effects. Many remediation strategies for the treatment of PAH contaminated material, including bioremediation, can lead to the formation of toxic transformation products. Analytical techniques for PAHs and PAH transformation products often require extensive sample preparation including solvent extraction and concentration, chromatographic separation, and mass spectrometry to identify and quantify compounds of interest. Excitation-emission matrix (EEM) fluorescent spectroscopy paired with parallel factor analysis (PARAFAC) is an approach for analyzing PAHs that eliminates the need for extensive sample preparation and separation techniques before analysis. However, this technique has rarely been applied to monitoring PAH biotransformation and formation of PAH metabolites. The objectives of this research were to compare an established targeted analytical method to two-dimensional fluorescent spectroscopy and combined EEM-PARAFAC methods to monitor phenanthrene degradation by a bacterial pure culture, Mycobacterium Strain ELW1, identify and quantify phenanthrene transformation products, and derive kinetic constants for phenanthrene degradation and metabolite formation. Both phenanthrene and its primary transformation product, trans-9,10-dihydroxy-9,10-dihydrophenanthrene, were identified and quantified with the EEM-PARAFAC method. The value of the EEM-PARAFAC method was demonstrated in the superiority of sensitivity and accuracy of quantification to two-dimensional fluorescent spectroscopy. Quantification of targets and derivation of kinetic constants using the EEM-PARAFAC method were validated with an established gas chromatography-mass spectrometry (GC-MS) method. To the authors’ knowledge, this is the first study to use an EEM-PARAFAC method to monitor, identify, and quantify both PAH biodegradation and PAH metabolite formation by a bacterial pure culture.

Evaluation of Xpert Carba-R for detecting carbapenemase-producing organisms in South Africa.

This study evaluated the performance of the Xpert Carba-R assay for detecting the five common carbapenemases in carbapenemase-producing organisms in Johannesburg, South Africa between April 2021 and September 2021. The assay demonstrated 98% sensitivity and 97% specificity. It was also able to detect all the carbapenemases in double carbapenemase producers, as well as carbapenemases in non-fermenter organisms. The Xpert Carba-R assay, therefore, allows the rapid (< 1 h) and accurate identification of the common carbapenemases in pure bacterial cultures and rectal swabs. This assay can aid in the timeous institution of appropriate treatment and infection prevention and control measures.

Antimicrobial Potential of Cocos nucifera (Coconut) Oil on Bacterial Isolates.

This study investigates the in vitro antibacterial activity of coconut oil on selected clinical and pure bacterial isolates. Clinical samples were isolated from the people of Ras Al Khaimah, United Arab Emirates. Biochemical examination of the microorganisms was done according to standard methods. Pure bacterial cultures were provided from LTA srl Italia. In this research work, an effort has been made to highlight the valuable properties of Cocos nucifera oil, in order to rationalize the use of coconut oil against bacteria. Experiments were performed by agar well diffusion method. Ciprofloxacin was used as a standard antibiotic. The assay of antibacterial activity of clinical isolate of Streptococcus species showed the highest susceptibility to coconut oil while Escherichia coli had the least. This study endorses the use of coconut oil as therapeutic agent since it contains lauric acid which is bactericidal. The utilization of coconut oil should be promoted as a functional food and the use of coconut seed flesh in our diets should be encouraged for health-supporting functions. Further studies should be done on the oil and its derivatives both in vitro and in vivo to unveil their mechanism of action.

Bacteria in the produced water and wastewater samples from the oil industry

Today, studying the diversity of microbial communities associated with samples of highly mineralized oil industry waters is expanding our knowledge of the ecology of polyextremophilic microorganisms. During this work, samples of produced water and wastewater from the oil industry were thoroughly analyzed. The analyzed waters were characterized by very high concentrations of Na+, Ca2+, Mg2+, and Cl– ions. Furthermore, enriched and pure bacterial cultures from oilfield waters were obtained. Additionally, enriched cultures were analyzed using high-throughput sequencing of the bacterial 16S rRNA gene on the Illumina platform. Among the representatives of the studied bacterial communities, members of the genera Halomonas, Marinobacter, Modicisalibacter, Bacillus, Clostridium, Prauserella, and Rubrobacter were identified. They can be considered for various biotechnological applications.

Перспективы использования микробных препаратов для снижения окислительного стресса сельскохозяйственных растений

Anthropogenic factors expose agricultural plants to abiotic and biotic stresses, one of which is oxidative stress. Oxidative stress changes cell metabolism, as well as inhibits plant growth and development. Microbial treatment is an environmentally safe method of oxidative stress prevention. The research objective was to study the antioxidant activity of microflora native to coal dumps in order to combat the oxidative stress in crops.&#x0D; The study featured microorganisms isolated from technogenically disturbed soils. Pure bacterial cultures were isolated by deep inoculation on beef-extract agar. A set of experiments made it possible to define the cultural, morphological, and biochemical properties of cell walls. The antioxidant activity and the amount of indole-3-acetic acid were determined on a spectrophotometer using the ABTS reagent and the Salkowski reagent, respectively. The isolated microorganisms were identified on a Vitek 2 Compact device. The biocompatibility of strains was tested by dripping, while the increase in biomass was measured using a spectrophotometer.&#x0D; The study revealed ten microbial strains with antioxidant activity ranging from 67.21 ± 3.08 to 91.05 ± 4.17%. The amount of indole-3-acetic acid varied from 8.91 ± 0.32 to 15.24 ± 0.69 mg/mL. The list of microorganisms included Klebsiella oxytoca, Enterobacter aerogenes, Pseudomonas putida, and Bacillus megaterium. The consortium of P. putida and E. aerogenes demonstrated the best results in antioxidant activity, indole-3-acetic acid, and biomass. Its ratio was 2:1 (94.53 ± 4.28%; 15.23 ± 0.56 mg/mL), while the optical density was 0.51 ± 0.02. Extra 2% glycine increased the antioxidant activity by 2.34%, compared to the control. Extra 0.5% L-tryptophan increased the amount of indole-3-acetic acid by 3.12 mg/mL and the antioxidant activity by 2.88%.&#x0D; The research proved the antioxidant activity of strains isolated from microflora native to coal dumps. The consortium of P. putida and E. aerogenes (2:1) demonstrated the best results. Further research will define its ability to reduce oxidative stress in plants.

Overcoming Antibiotic Resistance with Novel Paradigms of Antibiotic Selection.

Conventional antimicrobial susceptibility tests, including phenotypic and genotypic methods, are insufficiently accurate and frequently fail to identify effective antibiotics. These methods predominantly select therapies based on the antibiotic response of only the lead bacterial pathogen within pure bacterial culture. However, this neglects the fact that, in the majority of human infections, the lead bacterial pathogens are present as a part of multispecies communities that modulate the response of these lead pathogens to antibiotics and that multiple pathogens can contribute to the infection simultaneously. This discrepancy is a major cause of the failure of antimicrobial susceptibility tests to detect antibiotics that are effective in vivo. This review article provides a comprehensive overview of the factors that are missed by conventional antimicrobial susceptibility tests and it explains how accounting for these methods can aid the development of novel diagnostic approaches.

Thermophilic helicase-dependent amplification-based CRISPR/Cas12a system: Detection of stx2 in Escherichia coli O157:H7 by controlling primer dimers

Backgrounds: To overcome the limitation of polymerase chain reaction (PCR), isothermal amplification methods such as thermophilic helicase-dependent amplification (tHDA) have been developed. However, formation of primer dimer due to the single amplification temperature are major problems of tHDA. When cross-dimerization of forward and reverse primer occurred, false-positive results can be found on the lateral flow assay (LFA) which is one of the major detection methods widely used as a point of care diagnosis. Therefore, specific method of detecting only the target amplicon is required. ResultsIn this study, a tHDA-based CRISPR/Cas12a system was developed to detect low levels of Escherichia coli O157:H7 in fresh salad mix without the false-positive results produced by primer dimers. For the comparison of the effect in eliminating false-positive results by CRISPR/Cas12a system, LFA was also evaluated. The tHDA-based CRISPR/Cas12a system detected as low as 101 CFU/mL E. coli O157:H7 in bacterial pure culture. In LFA false-positive results were produced due to the primer dimer, whereas the primer dimer produced by tHDA was not detected in the CRISPR/Cas12a system. These results indicated that the CRISPR/Cas12a system eliminated the formation of primer dimer. In fresh salad mix, the tHDA-based CRISPR/Cas12a system combined with the filter concentration method detected 103 CFU/g E. coli O157:H7. ConclusionThis study was the first to amplify stx2 of E. coli O157:H7 with tHDA as an isothermal amplification method and detected the amplicon without false-positive results by combining tHDA with CRISPR/Cas12a. Therefore, this study showed great potential for detecting low levels of E. coli O157:H7 present in fresh salad mix.