Showing papers on "Agar plate published in 2022"

Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains

Abstract: It is well-known that most of environmental microorganisms do not form colonies on agar medium despite that agar medium is the commonly used solidified medium. We previously demonstrated the negative effects of H2O2 generation during agar medium preparation on colony formation. ABSTRACT We previously showed that autoclaving in preparing agar media is one of the sources of hydrogen peroxide (H2O2) in the medium. This medium-embedded H2O2 was shown to lower the total colony count of environmental microorganisms. However, the critical concentrations of H2O2 detrimental to colony formation on the agar plate remain largely undetermined. Herein, we elucidated the specific effect of H2O2 on microbial colony formation on solid agar medium by external supplementation of varying amounts of H2O2. While common laboratory strains (often called domesticated microbes) formed colonies in the presence of high H2O2 concentrations (48.8 μM or higher), microbes from a freshwater sample demonstrated greatly decreased colony counts in the presence of 8.3 μM H2O2. This implies that environmental microbes are susceptible to much lower concentrations of H2O2 than laboratory strains. Among the emergent colonies on agar plates supplemented with different H2O2 concentrations, the relative abundance of betaproteobacterial colonies was found to be lower on plates containing higher amounts of H2O2. Further, the growth of the representative betaproteobacterial isolates was completely inhibited in the presence of 7.2 μM H2O2. Therefore, our study clearly demonstrates that low micromolar levels of H2O2 in agar plates critically affect growth of environmental microbes, and large portions of those are far more susceptible to the same than laboratory strains. IMPORTANCE It is well-known that most of environmental microorganisms do not form colonies on agar medium despite that agar medium is the commonly used solidified medium. We previously demonstrated the negative effects of H2O2 generation during agar medium preparation on colony formation. In the present study, we investigated the independent effect of H2O2 on microbial growth by adding different concentrations of H2O2 to agar medium. Our results demonstrate for the first time that even low micromolar levels of H2O2 in agar plates, that are far lower than previously recognized as significant, adversely affect colony number obtained from freshwater inoculum.

Biodegradation of Low-Density Polyethylene (LDPE) Bags by Fungi Isolated from Waste Disposal Soil

Abstract: Plastics are available in different shapes nowadays in order to enhance the living standard. But unfortunately, most of these plastics are synthetic in nature that is why they show resistance to physical and chemical degradation processes and enhance environmental hazards. The aim of the present research study was to isolate and identify beneficial fungal species from soil that have the capability to degrade plastic. Soil samples from a waste disposal site at Peshawar district were diluted and inoculated on sabouraud dextrose agar (SDA) and potato dextrose agar (PDA) for fungus isolation. After isolation, the identifications of fungal species were done using standard identification techniques such as colony morphology and microscopic examination. The isolated fungal species that were identified were Aspergillus Niger, Aspergillus flavus, Penicillium, white rot, and brown rot fungi. After isolation, a degradation experiment was conducted to evaluate the capability of fungal isolates towards degradation of plastic. For this purpose, a 2 cm2 plastic piece was treated with fungal isolates for one month in a liquid culture system. The weight loss percentage was estimated at 22.9%, 16.1%, 18.4%, and 22.7% by Aspergillus Niger, Aspergillus flavus, brown rot, and white rot, respectively, which was confirmed by the Fourier transform analysis. The obtained FTIR peaks revealed the C–H bond deformation in alkenes, ketones, and esters. It has been concluded from the study that fungal species play a significant role in the degradation of synthetic plastic which can be used in bioreactors in future studies for the degradation of complex plastic materials.

Cultivating marine bacteria under laboratory conditions: Overcoming the “unculturable” dogma

Abstract: Underexplored seawater environments may contain biological resources with potential for new biotechnological applications. Metagenomic techniques revolutionized the study of bacterial communities but culture dependent methods will still be important to help the biodiscovery of new products and enzymes from marine bacteria. In this context, we promoted the growth of bacteria from a marine rock pond by culture dependent techniques and compared the results with culture independent methods. The total number of bacteria and diversity were studied in different agar plate media during 6 weeks. Agar plate counting was of the same order of magnitude of direct microscopy counts. The highest efficiency of cultivation was 45% attained in marine agar medium. Molecular analysis revealed 10 different phyla of which only four were isolated by the culture dependent method. On the other hand, four taxonomic orders were detected by cultivation but not by the molecular technique. These include bacteria from the phyla Bacillota and Actinomycetota. Our study shows that it is possible to grow more than the traditionally considered 1% of bacteria from a seawater sample using standard agar plate techniques and laboratorial conditions. The results also demonstrate the importance of culture methods to grow bacteria not detected by molecular approaches for future biotechnological applications.

Basic Bacteriological Routines.

Abstract: In experimental bacteriology, bacteria are generally manipulated, stored, and shipped in the form of cultures. Depending on various factors, including strain genotype, storage and shipping methods, and manipulator skills, the culture may contain genetic variants or simply contaminants. It is therefore important to begin an experiment by streaking the culture on an agar plate. Streaking, a technique to disperse bacterial cells on the surface of the agar, serves the purpose of isolating individual colonies. A colony originates from a single cell and is a nearly pure culture. On rich LB medium after 24 h of incubation at 37°C, a colony of Salmonella contains ∼5 × 108 cells (about 29 generations). Streaking is also required in experiments that themselves generate single colonies as a result of selection (e.g., when constructing strains or introducing plasmids). Except in the few instances in which the selection efficiently kills all counter-selected bacteria, colonies growing on the selective plates are contaminated, sometimes heavily, with cells from the bacterial lawn. "Purifying" the colonies arising in such experiments by streaking on selective plates is therefore a mandatory step. Here, we show how this can be conveniently done using simple toothpicks. We also briefly describe the steps involved in inoculating liquid cultures, spreading plates, and replica plating.

Antimicrobial Efficacy of Morinda citrifolia, Nisin, and 2% Chlorhexidine Against Enterococcus faecalis: An In-Vitro Study

Abstract: Aim The aim of this study was to evaluate and compare the antimicrobial activity of 2% chlorhexidine gluconate (2% CHX), Morinda citrifolia (M. citrifolia), and nisin (NI) all in gel forms against Enterococcus faecalis (E. faecalis)-infected root canals. Methodology Forty single-rooted mandibular premolars extracted for orthodontic reasons were decoronated and chemomechanical preparation of the root canal was performed. After sterilization, the samples were inoculated with E. faecalis for one week and grouped according to the medicament used namely, saline as the control group (Group-A), 2% CHX (Group-B), M. citrifolia (Group-C), and NI (Group-D). After 7days of incubation, in order to evaluate the effectiveness of the intracanal medicaments on the canal wall and its radicular dentin, the specimens dentin chips were retrieved and inoculated on brain heart infusion (BHI) blood agar plates from each tube and incubated at 37°C for 24 hours to obtain bacterial colony forming unit (CFU) count. The data was statistically analyzed using one-way ANOVA test and multiple comparisons among different groups were complemented by post hoc Tukey test. Results The CFU count indicating the number of viable bacterial colonies was found to be highest in Group-A (saline). Group-B (CHX 2%) showed the least CFUs followed by Group-D (NI) and Group-C (M. citrifolia). Conclusion In an attempt to overcome the disadvantages and toxic effects of a few commercially available intracanal medicaments and irrigants, the present study was aimed at using herbal extracts to evaluate and compare their antimicrobial efficacy with the commercially available medicaments against E. faecalis. Nisin was an effective antimicrobial agent and its action was found to be comparable with CHX.

Isolation of Contaminating Bacteria from Aqueous Solution of Contact Eye Lenses

Abstract: Background: Contact lenses have been widely used as an alternative to spectacles both in developed and non-developing countries. However, under certain circumstances, adverse responses can occur during contact lens wear and several microorganisms including bacteria, fungi, and free living amoebae—can cause several eye infections. This study was aimed to isolate contaminating bacteria from eye lenses solution. Methods: The samples were collected from solution bottles of eye lenses with the help of sterile cotton tipped swabs that were pre- moistened with sterile normal saline, then sample directly cultured on solid media. All samples were inoculated on to blood agar, MacConkey’s agar were incubated at 37oC for 24-48 h, Cultures were considered negative if no growth was detected within 48 hours of incubation. Bacterial culture obtained was identified using Gram’s staining, on the basis of culture diagnosis by growing on media and performing biochemical test. Results: A total of 150 samples from aqueous solution of contact eye lenses were used to isolate bacteria from it. Out of these samples 84 bacteria were isolated from aqueous solution while 66 were without growth. The most frequent isolated bacteria were P.aeurogenosa 39(46%) followed by S. epidermidis 27 (32%) then S. aureus 12 (14%) and E. coli 6(7%) respectively which is significant result (P-Value of 0.005). Conclusion: The study revealed that contact lenses solution under investigation contains different types of bacteria and pseudomonas is more frequent bacteria. Furthermore the contact lenses with multiple use and users which not have difference types of bacteria growth.

Isolation and characterization of competitive exclusion microorganisms from animal wastes–based composts against Listeria monocytogenes

Abstract: To isolate the slow‐growing or viable but non‐culturable competitive exclusion (CE) microorganisms from composts and then verify the anti‐Listeria monocytogenes activities of those CE isolates in compost.

Inhibitory activity of an emulsifying salt polyphosphate (JOHA HBS®) used in processed cheese: An in vitro analysis of its antibacterial potential

Abstract: In the present study, the inhibitory activity of a commercial polyphosphate (JOHA®️ HBS) was tested in vitro against 21 bacterial strains. Firstly, the antimicrobial activity of JOHA®️ HBS, at different concentrations (from 0.5 to 2.0%) was evaluated in different agar and broth media (namely, Brain Heart Infusion: BHI; Nutrient: NT; Plate Count: PCA; Trypticase Soy: TSA/B), using streak assay (agar) and culture enumeration (broth). Furthermore, the bacterial inhibition of JOHA®️ HBS (at different concentrations, from 0.2% to 3.0%) was evaluated both on NT agar and broth medium, by streak assay, agar-spot method, and by spot-on-the-lawn and well-diffusion method. Finally, the JOHA®️ HBS antimicrobial activity was tested on NT agar at pH 6.3. Results of the streak assay on NT agar showed that 11 out the 21 tested strains were highly inhibited at 0.5% of JOHA®️ HBS. Interestingly, at adjusted pH levels, JOHA®️ HBS concentrations of 1.0% (w/v) were able to inhibit the same targets, confirming the antimicrobial effect of JOHA®️ HBS. This study reveals that NT medium and agar-based tests are required to properly test the inhibitory activity of polyphosphates. Furthermore, results confirmed the antimicrobial activity of JOHA®️ HBS, at low concentrations, against target bacteria of interest to the dairy industry.

Validation of the Colibrí Instrument for Automated Preparation of MALDI-TOF MS Targets for Yeast Identification

Abstract: Recently, Copan (Italy) introduced the Colibrí instrument for automated colony picking and preparation of matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) target plates. Our study aimed to validate this system for yeasts as such testing has not been performed yet and is a missing link needed to implement the system for routine use. ABSTRACT Recently, Copan (Italy) introduced the Colibrí instrument for automated colony picking and preparation of matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) target plates. Our study aimed to validate this system for yeasts as such testing has not been performed yet and is a missing link needed to implement the system for routine use. Fifty-five Candida strains were selected to evaluate the accuracy of Colibrí. For each strain, a sheep blood agar plate supplemented with X and V factors (HEM) and a Sabouraud agar plate (SAB) were inoculated and incubated using the WASPlab specimen processing system (Copan). After 18 h and 36 h of incubation, the isolates were spotted in parallel using Colibrí and manually onto MALDI-TOF target plates with the addition of formic acid and identified using MALDI-TOF mass spectrometry. The reproducibility was evaluated using ATCC reference and clinical isolate-derived strains. The cumulative percentage of acceptable identification scores (IDs) after 36 h was 91% for strains cultured on HEM plates using both Colibrí and the manual method. The SAB plates showed inferior results for both Colibrí (76%) and the manual method (78%). We observed an overall agreement of 92% at 18 h for identification of the strains on the HEM plates between Colibrí and the manual method and 94% after 36 h. For the SAB plates, the agreement was 78% after 18 h and 84% after 36 h. Apart from Candida dubliniensis and Candida tropicalis, all Candida species were identified with 100% accuracy using Colibrí on HEM plates. We observed good agreement between Colibrí and the manual reference method. These results demonstrate that Colibrí is a reliable system for MALDI-TOF target preparation for yeast identification, allowing increased standardization and less hands-on time.

Antifungal Potential of Actinomycetes Isolated from Region of Bhilai, Chhattisgarh, India

Abstract: Actinomycetes due to their unique repertoire of antimicrobial secondary metabolites can be a good source to control human pathogens. In our research plan, soil samples were collected from Bhilai, India. The strains were isolated using yeast extract-malt extract agar medium and identified based on their morphological, physiological, and biochemical characteristics. The antifungal activity of isolates was examined by the perpendicular streak plate as well as the agar well diffusion method. Out of 14 isolates, only 4 isolates (28.5%) showed antifungal activity. Further, one isolate showed the highest antifungal activity and was identified as Streptomyces antibioticus A8 based on PIB-Win software and the identification score was 0.99. Antifungal activity of new isolate Streptomyces antibioticus A8 against the T. rubrum MTCC 296, with a zone of inhibition 28±0.0 mm, whereas minimum activity was recorded against A. niger MTCC 872 with the zone of inhibition 9±0.0 mm. antifungal activity against C.albicans ATCC10231, C.albicans ATCC90028, C. albicans ATCC24433, C.albicans MTCC183, C.tropicalis MTCC184, A.alternata MTCC 1779 was recorded as 20±0.0mm, 17±0.0 mm, 17±0.0 mm, 15±0.5 mm, 14±0.0 mm and 11±0.0 mm respectively by agar well diffusion method. Further, discovering new antimicrobial-producingmicrobes probably will be helpful for uncovering novel therapeutic agents against a broad range of pathogenic organisms.

Breeding of New Strains of Gracilariopsis lemaneiformis with High Agar Content by ARTP Mutagenesis and High Osmotic Pressure Screening

Abstract: ARTP (atmospheric and room temperature plasma mutagenesis) mutagenesis was tried on G. lemaneiformis, and mutagenesis conditions were confirmed. An osmotic pressure screening program was established. Mutants were identified and characterized of relevant physiological traits. The aim of the study is to try to use ARTP mutagenesis and osmotic pressure screening for the breeding of high-agar G. lemaneiformis. Treatment time of 46 s was found to be an optimal mutagenesis time. The mutagenized spores were initially screened with 58‰ salinity artificial seawater, and then, the surviving spores were screened twice with 60‰ salinity artificial seawater in their vertical growth phase and branch growth phase, respectively. Four fast-growing and hypertonic resistance gametophytes were selected. The actual photosynthetic efficiency [Y(PSII)], photochemical quenching (qL), and non-photochemical quenching (NPQ) of four mutants were measured. The values of Y(PSII) and qL of HAGL-X3 and HAGL-X5 were higher than those of the control in the early stage of salt stress. NPQs of HAGL-X3 and HAGL-X5 were higher than control in most of the times. The growth rates of the four mutants were higher than that of the control. HAGL-X4 was the highest. The agar content was measured; HAGL-X5 displayed the highest agar content among the tested strains. HAGL-X5 was more in line with expectations, because of its high agar content and good hypertonic resistance. In this study, the mutant of G. lemaneiformis with high agar content was obtained by the procedure, which provided a certain reference for the selection of G. lemaneiformis strains with high agar content.

Diagnosis of Mycobacterium marinum infection based on photochromogenicity: a case report

Abstract: A 35-year-old immunocompetent woman from southern China went to the hand surgery clinic with a six-month history of progressive swelling in her right index finger. She had been pinched by a lobster and had received several treatments without any improvement. Pus specimens were taken from the swollen parts of her finger, and the pathology showed granulomatous inflammation. Ziehl-Neelsen staining revealed positive bacillus in the pus specimens. The bacteria grew well on Columbia blood agar. However, the MALDI-TOF MS and 16S rRNA gene sequencing were not able to distinguish between Mycobacterium marinum and Mycobacterium ulcerans because of their close genetic relationship. Photochromogenicity testing can help differentiate between these species based on the alteration in colony color after light exposure. For our patient, the colonies turned yellow after 18h of incubation in the sun, identifying the species as M. marinum. Besides surgical drainage, the patient received rifampicin and clarithromycin for three months, and her symptoms resolved without relapse after six months of follow-up.

Isolation and identification of Helicobacter pylori from raw chicken meat in Dhamar Governorate, Yemen

Abstract: Although Helicobacter pylori (H. pylori) is one of the most common bacterial pathogens of human, its natural reservoirs are still unclear. There is an increasing number of reports that document the occurrence of H. pylori in various foods. This study aimed at isolation of H. pylori from chicken meat sampled. Two hundred and sixty samples were collected randomly from slaughterhouses and markets in Dhamar Governorate, Yemen. Samples were enriched in Brain-Heart Infusion broth in microaerophilic conditions before inoculating the Camp-Blood agar and EYE agar plates. Results showed that 13.8% of samples were contaminated evidenced by H. pylori growth via traditional culture method on agar media. No significant differences between sample types (thighs and breast muscles) (p=0.353) or the sampling source (p=0.816) were observed. Autumn season was associated with increased occurrence of H. pylori. The source of H. pylori in food is still not identified. Proper cooking and good sanitation practices are highly recommended to avoid the infection. Further studies addressing the potential sources of H. pylori are highly suggested.

Effect of Culture Conditions on Fatty Acid Profiles of Bacteria and Lipopolysaccharides of the Genus Pseudomonas—GC-MS Analysis on Ionic Liquid-Based Column

Abstract: The profiling of bacterial fatty acids is a well-established technique in identifying and classifying bacteria. Cultivation conditions may affect the biosynthesis, thereby, changing the fatty acid profile in bacteria. The effect of the culture conditions on the fatty acid components of Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa polyresistant and Pseudomonas putida all are aligned to the genus Pseudomonas. The fatty acids in the lipopolysaccharides of Pseudomonas aeruginosa PAO1 were also examined. The effects of the cultivation conditions were followed by using agar and blood agar media at the characteristic temperatures, 25 °C, 37 °C and 42 °C, respectively, and an analysis was made during the 1st, 3rd and 5th day following inoculation. In addition to quantitative differences, we also experienced qualitative differences in the fatty acid profiles which detect newly appearing fatty acids, due to changes in environmental factors. The application of ionic liquid-based column unveils new possibilities for the analyses of fatty acids in GC-MS experiments for bacterial fatty acid profiling. The validation results (response linearity, limit of detection, limit of quantification, system suitability, intraday and interday repeatability and accuracy) show the high separation efficiency of the ionic liquid-based column in the analyses.

Antibacterial Properties of Non-Modified Wool, Determined and Discussed in Relation to ISO 20645:2004 Standard

Abstract: Wool is considered to possibly exhibit antibacterial properties due to the ability of wool clothing to reduce the build-up of odor, which arises from the microbial activity of skin microbiota. Indeed, when tested with a widely used agar diffusion plate test method, even wool or other textiles not treated with any antimicrobial agent can be interpreted to show certain antibacterial effects due to the lack of growth under the specimen, as instructed in ISO 20645:2004 standard. Therefore, we analyzed in detail what happens to bacterial cells in contact with untreated wool and cotton fabric placed on inoculated agar plates by counting viable cells attached to the specimens after 1 and 24 h of contact. All wool and several cotton samples showed no growth under the specimen. Nevertheless, it was shown without a doubt that neither textile material kills bacteria or inhibits cell multiplication. A reasonable explanation is that bacterial cells firmly attach to wool fibers forming a biofilm during multiplication. When the specimen was lifted off the nutrient agar surface, the cells in the form of biofilm remained attached to the wool fibers, removing the biomass and resulting in a clear, no growth zone underneath it. By imaging the textile specimens with X-ray microtomography, we concluded that the degree of attachment could be dependent on surface topography. The results indicate that certain textiles, in this case, wool, could exhibit antibacterial properties by removing excess bacteria that grow on the textile/skin interface when taken off the body.

Nonhemolytic Listeria monocytogenes—Prevalence Rate, Reasons Underlying Atypical Phenotype, and Methods for Accurate Hemolysis Assessment

Abstract: Listeria monocytogenes is a foodborne pathogen that typically presents β-hemolytic activity. However, there are literature reports indicating that L. monocytogenes strains are sometimes nonhemolytic or their zones of hemolysis are perceivable only after removal of the colonies from the agar plate. Nonhemolytic L. monocytogenes are most commonly encountered in food products, but some have also been detected in clinical samples. Usually, atypical bacteria of this species belong to serotype 1/2a. Mutations of the prfA gene sequence are the most common reason for changed phenotype, and mutations of the hly gene are the second most common cause. There are also reports that the methodology used for detecting hemolysis may influence the results. Sheep or horse blood, although most commonly used in modern studies, may not allow for the production of clear hemolytic zones on blood agar, whereas other types of blood (guinea pig, rabbit, piglet, and human) are more suitable according to some studies. Furthermore, the standard blood agar plate technique is less sensitive than its modifications such as bilayer or top-layer (overlay) techniques. The microplate technique (employing erythrocyte suspensions) is probably the most informative when assessing listerial hemolysis and is the least susceptible to subjective interpretation.

Enhanced Antibacterial Activity of Brevibacillus sp. SPR19 by Atmospheric and Room Temperature Plasma Mutagenesis (ARTP)

Abstract: Antibiotic resistance is a major health concern worldwide. In our previous study, some bacterial isolates exhibited antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). However, the production of antibacterial substances by native microorganisms is limited by biosynthetic genes. This study aimed to improve the antibacterial activity of SPR19 using atmospheric and room temperature plasma mutagenesis (ARTP). The results showed that SPR19 belonged to the Brevibacillus genus. The growth curves and production kinetics of antibacterial substances were investigated. Argon-based ARTP was applied to SPR19, and the 469 mutants were preliminarily screened using agar overlay method. The remaining 25 mutants were confirmed by agar well diffusion assay against S. aureus TISTR 517 and MRSA isolates 142, 1096, and 2468. M285 exhibited the highest activity compared to the wild-type strain (10.34–13.59%) and this mutant was stable to produce the active substances throughout 15 generations consistently. The antibacterial substances from M285 were tolerant to various conditions (heat, enzyme, surfactant, and pH) while retaining more than 90% of their activities. Therefore, Brevibacillus sp. SPR19 is a potential source of antibacterial substances. ARTP mutagenesis is a powerful method for strain improvement that can be utilized to treat MRSA infection in the future.

Isolation, Identification and Screening of Lipase Producing Fungi from the Soil Environment of Ilorin Metropolis

Abstract: This investigation was carried out to isolate, identify and screen for lipase producing fungal species present in the soil environment of Ilorin metropolis. Soil samples of approximately 200g each were collected randomly from eight different locations within the Ilorin metropolis for the investigation. Potato Dextrose Agar was used for the isolation of the fungal species by pour plate method. Six fungal species, Penicillium spp, Acremoniumspp, Mucors pp, Rhizopus stolonifer, Aspergillus nigerand Aspergillus flavuswere isolated and screened for their ability to produce lipases on tween-20 and phenol red agar. The results obtained for lipase production on tween-80 and phenol red after 5 days of incubation showed that four isolates were positive for lipase production which was indicated by diameter zone of clearance and visible precipitate of calcium monolaurate due to the deposition of calcium crystal. The result further revealed that Aspergillus niger had the highest lipase producing ability (having a diameter zone of clearance of 14 ± 0.05 mm), followed by Rhizopus stolonifer (having 10 ± 0.05 mm). Aspergillus flavus and Mucor sp had 6 ± 0.03 mm, 6± 0.01 mm respectively. Acremonium sp. and Penicillium sp. had no zone of clearance. These results demonstrate the presence of lipase producing fungi in the soil environment of Ilorin metropolis,Kwara State, and these can be harnessed locally for large scale production of the enzyme which is of value commercially in the production of leather, detergent,textiles and also as constituents of some special diets and pharmaceuticals.

Paenibacillus sp. Strain OL15 Immobilized in Agar as a Potential Bioremediator for Waste Lubricating Oil-Contaminated Soils and Insights into Soil Bacterial Communities Affected by Inoculations of the Strain and Environmental Factors

Abstract: Simple Summary Waste lubricating oil contamination is a global environmental problem. This study demonstrates the potential of using agar as an immobilization material to maintain the survival and establishment of the bioremediator. We found that the Paenibacillus sp. strain OL15 was a promising bacterial strain which tolerated high concentrations of waste lubricating oil and exhibited high degradation ability. The inoculations of the strain increased total bacterial diversity in the oil-contaminated soils. Soil pH, electrical conductivity (EC), organic matter (OM), nitrogen, potassium, manganese, iron, and zinc were the factors determining the fertility of the oil-contaminated soil environments. Our work develops and implements an efficient bioremediation system employing the promising bioremediator and immobilization procedure for degradation of waste lubricating oil in the soil environments. Abstract Waste lubricating oil is a widespread common soil pollutant. In this study, the waste lubricating oil degraders were isolated from the oil-contaminated soil. The bacterial strains OL6, OL15, and OL8, which tolerated a high concentration (10%) of waste lubricating oil, presented the degradation efficiency values (measured in culture broth) of 15.6 ± 0.6%, 15.5 ± 1%, and 14.8 ± 1%, respectively, and belonged to the genera Enterobacter, Paenibacillus, and Klebsiella, respectively. To maintain long survival, immobilization of a promising bioremediator, Paenibacillus sp. strain OL15, in agar exhibited the significantly highest number of surviving cells at the end of a 30-day incubation period, as compared to those in alginate and free cells. Remarkably, after being introduced into the soil contaminated with 10% waste lubricating oil, the strain OL15 immobilized in agar conferred the highest degradation percentage up to 45 ± 3%. Due to its merit as a promising soil pollutant degrader, we investigated the effect of an introduction of the strain OL15 on the alterations of a bacterial community in the oil-contaminated soil environments using 16S rRNA amplicon sequencing. The result revealed that the Proteobacteria, Acidobacteriota, Firmicutes, and Actinobacteriota were predominant phyla. The introduction of the strain affected the soil bacterial community structures by increasing total bacterial diversity and richness. The proportions of the genera Pseudomonas, Vibrio, Herbaspirillum, Pseudoalteromonas, Massilia, Duganella, Bacillus, Gordonia, and Sulfurospirillum were altered in response to the strain establishment. Soil pH, EC, OM, total N, P, Mg, Fe, and Zn were the major factors influencing the bacterial community compositions in the oil-contaminated soils.

Isolation, characterization and growth assessment of biodegrading chlorpyrifos-methyl Bacillus species isolated from Algerian soil

Abstract: Chlorpyrifos-methyl (CM) is a broad-spectrum organophosphate insecticide, which is widely used in pest control. In this research, the isolation, and biochemical and molecular identification of bacterial strains obtained from three soils located in northeastern Algeria were carried out, as well as the evaluation of their ability to grow in the presence of CM. Out of 48 bacterial isolates between Gram-negative and Gram-positive identified, several were able to grow on mineral agar with at least 25 mg·l–1 of CM. Four bacteria showed the best growth capacity, were identified as Bacillus sp. H1-80, Brevibacterium frigoritolerans strain WJB99 and two Bacillus sp. strains GL5. The strains were tested for their ability to grow on liquid media with CM as the sole energy and carbon source. In general, these strains showed slow but significant growth visualized by the 600 nm turbidity control, suggesting that they could be used for bioremediation applications of CM polluted soils.

Characterization of small colony variants of Klebsiella pneumoniae: Correlation with antibiotic resistance and biofilm formation

Abstract: Background: Small colony variants (SCVs) of bacterial pathogens are smaller, slow-growing variants which often pose a challenge to the clinical microbiologist in their identification and characterization. SCVs are receiving much attention in recent years due to their association with several types of chronic infections. In this study, we aimed to develop a suitable culture media for high frequency generation and stable maintenance of SCV of Klebsiella pneumoniae. We also intended to compare different phenotypic characteristics such as growth, antibiotic resistance pattern, and biofilm-forming potential of SCVs with the original parental strain. Methods: We used Mueller–Hinton agar containing the extract of clove (Syzygium aromaticum) for the generation of SCV. Antibiotic sensitivity was determined using disk diffusion method and minimum inhibitory concentration determinations using microdilution method. Biofilm formation was assessed using crystal violet dye binding assay. Results: Mueller–Hinton agar (MHA) containing clove (Syzygium aromaticum) extract (10% volume/volume; MHA-C10) supported generation of SCV from K. pneumoniae at high frequency. SCVs were smaller in colony size and grew slowly in comparison to the wild-type original strain. In addition, SCVs exhibited increased resistance to aminoglycoside group of antibiotics (gentamicin and kanamycin). Crystal violet dye binding spectrophotometric method showed increased biofilm formation potential by SCVs in comparison to their parental counterparts. Conclusions: The findings of this study show that MHA-C10 can be used as a bacterial culture media for the formation of SCV by K. pneumoniae. SCVs, thus, generated on MHS-C10 exhibited typical characteristics of SCVs.