Tryptone Soya Agar Research Articles

Paenibacillus piri sp. nov., isolated from urban soil.

A rod-shaped, Gram-stain-positive bacterial strain, MS74T, was isolated from soil beside Itaewon road, Seoul, Republic of Korea. The strain could grow well on R2A, nutrient agar and tryptone soya agar, but not in LB agar. MS74T tolerated 3.0 % NaCl (w/v), pH 9.0 and a temperature range from 10 to 35 °C (optimal temperature, 28 °C). From the comparison of 16S rRNA gene sequence, the strain is most closely related to Paenibacillus vulneris CCUG 53270T , Paenibacillus esterisolvens CFH S0170T, Paenibacillus rigui JCM 16352T and Paenibacillus yunnanensis YN2T with similarity percentages of 96.6 %, 96.4 %, 95.9 % and 95.8 % respectively. Major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). The major menaquinone was MK-7. The fatty acids profile mainly consisted of C15 : 0 anteiso, C15 : 0 iso, C16 : 0 iso, and C16 : 0. The DNA G+C content of the isolated strain determined from the whole-genome sequence was 51 mol%. MS74T had an average nucleotide identity (ANI) value of 73.9 % and a digital DNA-DNA hybridization (dDDH) value of 20.6 % with most closely related strain, Paenibacillus vulneris CCUG 53270T. On the basis of phenotypic, chemotypic and genotypic evidence, the isolate was identified as representing a novel species of the genus Paenibacillus, for which the name Paenibacillus piri sp. nov. is proposed. The type strain is MS74T (=KACC 19385T =DSM 105496T).

Surface sampling within a pediatric ward—how multiple factors affect cleaning efficacy

The objectives of this study were to assess the number of organisms present on different surfaces within a clinical environment before and after cleaning took place, and to identify the impact of cleaning. The study involved extensive 2-week microbiological environmental monitoring of an entire ward before and after cleaning; the ward was located within a pediatric hematology-oncology ward comprised of a day unit and outpatient ward. Tryptone soya agar contact plates were used to take a total of 1,160 surface samples before and after cleaning from 55 predetermined sites. Samples were taken from representative surfaces throughout the ward representing a variety of materials, surface heights, functions, and distances from patients, as well as both high-touch and infrequently touched surfaces. After surface cleaning was undertaken within the ward, there was a significant difference between the amount of colony-forming units (CFUs) recovered before and after cleaning (P < .0001). Cleaning produced an average CFU reduction of 68% throughout the ward environment. The corridor was the most contaminated area within the ward. There were differences in the CFUs among the various areas within the ward, which were cleaned with varying efficiency. The surface material, who interacted with the surface, levels of initial contamination, perceived risk, and perceived cleanability were all found to have a varying impact on the cleaning effectiveness. To the authors' current knowledge, this is the only study to assess cleaning within a pediatric ward by taking samples directly before and after cleaning. The standard of cleaning undertaken within the ward is open for discussion, and these data highlight the need for an improved cleaning intervention and can provide insight into the multitude of factors that must be considered when designing an effective training protocol.

Microbiological Assessment of Selected Laboratories at a Local University in Malaysia

Microbiological contaminations in the laboratories create not only diagnostic issues but also pose a major health risk to lab users. This study was conducted to determine the airborne microbial contamination in seven selected laboratories (KA, KP, PB, NA, BP, CR and MB) at a local public university in Malaysia and to assess the level of contamination on the contact surfaces of the studied laboratories. Two types of sampling methods were used in this study; passive air sampling and contact surface swab sampling. The total microbial counts were determined using Tryptone Soya agar (bacterial count) and Potato Dextrose agar (fungal count). Results showed that NA laboratory had the highest level of total microbial contamination (20.33 ± 3.35 CFU/cm3 ). Most laboratories had significantly higher (p < 0.05) air fungal contamination level compared to bacterial contaminations except for PB and NA laboratories. Significant differences were observed for airborne bacterial contamination readings between sampling time (during working hours vs after hours) for all laboratories except for BP and CR. Overall, bacterial contamination was the highest for incubator door handles’ samples from MB laboratory with an average reading of 93.00 ± 1.43 CFU/cm2 whereas the highest fungal contamination level was obtained from door knobs and work benches, both from CR laboratory with an average reading of 73.33 ± 6.67 CFU/cm2 and 73.33 ± 0.58 CFU/cm2 respectively. Findings from this study could assist in monitoring the efficacy of the existing laboratory management systems namely on the good laboratory practices including aseptic techniques, care for laboratory hygiene and cross-contamination control practices by the laboratory users to ensure that the microbiological contaminations in the laboratories are minimized.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream.

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48–72 hours.

Fluviicola chungangensis sp. nov., a bacterium isolated from rice field.

A Gram-stain negative, aerobic, motile and rod-shaped novel bacterial strain, designated MAH-3T, was isolated from soil sample of a rice field. The colonies were orange pigmented, smooth, circular and 0.4-0.9mm in diameter when grown on R2A agar for 3days. Strain MAH-3T was found to grow at 10-40°C (optimum 30°C), pH 6.0-8.0 (optimum 7.0) and in the presence of 0-1.0% NaCl (optimum 0%). Cell growth occurs on R2A agar and Luria-Bertani agar, but not on nutrient agar, tryptone soya agar and MacConkey agar. Cells were positive for catalase test but negative for oxidase test. Cells were able to hydrolyze casein, gelatin, DNA and Tween 80. 16S rRNA gene sequence analysis revealed that strain MAH-3T was most closely related to the genus Fluviicola and exhibited the highest sequence similarity to Fluviicola hefeinensis MYL-8T (97.4%), Fluviicola taffensis RW262T (96.2%) and Fluviicola kyonggii CA-1T (95.6%). Strain MAH-3T had a genome size of 4,271,694bp and the genomic DNA G + C content was determined to be 41.7mol%. The genome contained 19 contigs encoded by 3,664 protein-coding genes with 34 tRNA and 4 rRNA genes. The genomic ANI value between strain MAH-3T and one of the closely related type strains, F. taffensis DSM 16823T was 76.2%. The predominant isoprenoid quinone of isolated strain MAH-3T was menaquinone-6 (MK-6). The major fatty acids were identified as C15:0 iso, C15:0 2OH and C17:0 iso 3OH. On the basis of these phenotypic, genotypic and chemotaxonomic studies and DNA-DNA hybridization results, the isolated strain MAH-3T represents a novel species, for which the name Fluviicola chungangensis sp. nov. is proposed, with MAH-3T as the type strain (= KACC 19742T = CGMCC 1.13750T).

Physical and antimicrobial properties of edible films containing Lactococcus lactis

In this study, we developed edible films designed to control the growth of Staphylococcus aureus in tryptone soya agar. We analyzed three edible film-forming substrates: sodium alginate, sodium carboxymethylcellulose, and collagen. In addition, we evaluated Lactococcus lactis, which produces bacteriocin and lactic acid. Lactococcus lactis-containing edible films were constructed via tape-casting method. Optical, mechanical, and antimicrobial properties of the edible films were measured to examine the effects of Lactococcus lactis on film matrices. Further, we determined the survival of Lactococcus lactis after the film-drying process and viability of Lactococcus lactis stored for 24 days at 4 °C. Our results indicate that incorporation of Lactococcus lactis changed the physical properties of edible films. Films containing Lactococcus lactis showed reduced gloss and transparency. There are insignificant modifications were observed in terms of tensile strength and elongation at break. At 4 °C, used to represent a low-temperature environment, the growth of Staphylococcus aureus was inhibited for 7 days in edible films populated with Lactococcus lactis. The viability of Lactococcus lactis was higher in sodium alginate/sodium carboxymethylcellulose films, and was highest in films composed of sodium alginate and methylcellulose. In summary, our study provides a new method for functional food packaging.

Treating drug-resistant wound pathogens with non-medicated dressings: an in vitro study.

To assess the in vitro antimicrobial performance of a non-medicated hydro-responsive wound dressing (HRWD) on the sequestration and killing of wound relevant microorganisms found on the World Health Organization (WHO) priority pathogens list. Suspensions of Pseudomonas aeruginosa, Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA) were placed on petri dishes. Dressings were each placed on top, incubated for 30 minutes and then removed from the inoculated petri dish. The surface of the dressings previously in contact with the bacterial suspensions were placed directly onto a tryptone soy agar (TSA) plate and incubated for 24 hours. Dressings were then removed from the TSA plate and the level of bacterial growth on the plates was assessed. Sequestered microorganism viability was assessed using LIVE/DEAD viability kits and visualisation by epifluorescence. Our results indicated that HRWDs sequester and retain Pseudomonas aeruginosa, Acinetobacter baumannii and MRSA within the dressing. Non-medicated HRWDs containing bound PHMB (polyhexamethylene biguanide, HRWD+PHMB) killed the microorganisms sequestered within the dressing matrix. These data suggest that non-medicated HRWD+PHMB is an effective against WHO priority pathogens and promoting goal of antimicrobial stewardship in wound care.

Potential effects of microbial air quality on the number of new cases of diabetes type 1 in children in two regions of Poland: a pilot study.

Aim: The aim of the study was to investigate the relationship between the concentration of psychrophilic bacteria, mesophilic bacteria and mold fungi in bioaerosols, and the number of new cases of type 1 diabetes mellitus (T1DM) in children.Methods: Air samples from the Lubelskie and Pomeranian voivodeships in Poland were collected from January 2015 to December 2016 in winter, spring, summer and autumn. Thirty-three samples were collected in the Pomeranian and 27 in the Lubelskie voivodeship. The air samples were collected on the first day of each month at 1:00 pm for 10 mins at a height of 1.5 m above the ground. The number of mesophilic bacteria was detected after 24–48 hrs incubation at 37°C on tryptone soya agar (TSA; Merck, Darmstadt, Germany). The number of psychrophilic bacteria was detected after 72 hrs incubation at 22°C on TSA. The number of fungi was detected by a 5-day long incubation at 28°C on chloramphenicol yeast glucose agar.Results: In the Lubelskie voivodeship, the mean concentration of psychrophilic bacteria was significantly higher than in the Pomeranian voivodeship (2739 vs 608 CFU/m3, respectively), the mean concentration of mesophilic bacteria was significantly higher (2493 vs 778/m3, respectively) and the concentration of fungi was significantly higher (3840 vs 688 CFU/m3, respectively). We also showed a statistically significant relationship between the number of children with recently diagnosed T1DM and the mean concentration of psychrophilic and mesophilic bacteria in the Pomeranian and Lubelskie voivodeships (P<0.001). Moreover, we found a significant relationship between the number of new cases of T1DM in children and the mean concentration of fungi in bioaerosols in the Lubelskie voivodeship (P<0.001), but not in the Pomeranian voivodeship (P=NS).Conclusion: The results of our research showed that there is a higher concentration of microbial particles in the Lublin voivodeship. Therefore, we recommend changes in climate for children (trips to the sea, mountains, etc) as often as possible.

Arthrobacter terricola sp. nov., isolated from forest soil.

A Gram-stain-positive, aerobic and rod-shaped bacterial strain, designated JH1-1T, was isolated from a forest soil sample collected in Suwon, Gyeonggi-do, Republic of Korea. Strain JH1-1T could grow at 10-35 °C (optimum, 28-30 °C), pH 4.5-8.5 and tolerated 5 % (w/v) NaCl. Strain JH1-1T was most closely related to members of the genus Arthrobacter, namely Arthrobacter alkaliphilus LC6T (98.5 % similarity), Arthrobacter methylotrophus TGAT (98.4 %), Arthrobacter ramosus CCM 1646T (97.8 %), Arthrobacter bambusae THG-GM18T (97.5 %) and Arthrobacter pokkalii P3B162T (97.3 %). The strain grew well on Reasoner's 2A agar, tryptone soya agar, nutrient agar, Mueller-Hinton agar and Luria-Bertani agar. The major polar lipid profile comprised phosphatidylglycerol, diphosphatidylglycerol, unidentified phospholipid and unidentified glycolipids. The major respiratory quinone was MK-9(H2). The main fatty acids were C15 : 0 anteiso, C15 : 0 iso, C16 : 0 iso and C17 :0 anteiso. The DNA G+C content of the isolated strain based on the whole genome sequence was 63.6 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain JH1-1T and its reference type strains ranged from 81.3 to 85.4 % and from 21.1 to 29.1 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, strain JH1-1T could be differentiated phylogenetically and phenotypically from the recognized species of the genus Arthrobacter. Therefore, strain JH1-1T is considered to represent a novel species, for which the name Arthrobacter terricola sp. nov. is proposed. The type strain is JH1-1T (=KACC 21385T=JCM 33641T).

Paenibacillus cymbidii sp. nov., isolated from Cymbidium goeringii roots.

A Gram-stain-variable, aerobic, rod-shaped, endospore-forming strain R196T) was isolated from internal tissues of roots of Cymbidium goeringii. Cells were motile with peritrichous flagella. The colonies were light pink on tryptone soya agar medium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain R196T fell into a phylogenetic cluster belonging to the genus Paenibacillus. Strain R196T was closely related to Paenibacillus cavernae C4-5T and Paenibacillus contaminans CKOBP-6T with 93.6 and 93.3% sequence similarities, respectively. The major cellular polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified phospholipids, two unidentified aminophospholipids and an unidentified aminolipid. The dominant respiratory quinone was MK-7. The main cellular fatty acids were anteiso-C15 : 0 (53.01%), C16 : 0 (13.04%) and iso-C16 : 0 (10.80%). The genome size of R196T was 9.45 Mb, containing 7617 predicted protein-coding genes, with a DNA G+C content of 57.7 mol%. Based on the results of phenotypic, chemotaxonomic and whole-genome analyses, strain R196T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus cymbidii sp. nov. is proposed. The type strain is R196T (=ACCC 61713T=KCTC 33718T).

Sphingobium chungangianum sp. nov., isolated from rhizosphere of Pinus koraiensis.

A novel Gram-staining negative, yellow-pigmented, non-motile, aerobic and rod-shaped bacterium, designated MAH-11T, was isolated from rhizosphere of Pinus koraiensis and was characterised by using a polyphasic taxonomic approach. The colonies were smooth, circular and 0.3-1.0mm in diameter when grown on R2A agar for 3days. The strain was positive for both catalase and oxidase tests. Optimum growth temperature and pH were 28-30°C and 7.0, respectively. Cell growth occurs on R2A agar, nutrient agar, Luria-Bertani agar and tryptone soya agar but not on MacConkey agar. The novel strain was found to be able to hydrolyse esculin but not casein, gelatin, starch, L-tyrosine, DNA, L-arginine, urea, Tween 20 and Tween 80. On the basis of 16S rRNA gene sequence analysis, strain MAH-11T belongs to the genus Sphingobium and is closely related to Sphingobium quisquiliarum P25T (98.1%), Sphingobium vermicomposti VC-230T (97.8%), Sphingobium mellinum WI4T (97.5%), Sphingobium barthaii KK22T (97.2%) and Sphingobium fuliginis TKPT (97.2%). In DNA-DNA hybridization tests, the DNA relatedness values between strain MAH-11T and its close phylogenetic neighbors were below 45.0%. The DNA G+C content was 64.5mol% and the predominant respiratory quinone was identified as ubiquinone-10. The major cellular fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C16:0. The DNA-DNA hybridization results in combination with chemotaxonomic and physiological data demonstrated that strain MAH-11T represents a novel species within the genus Sphingobium, for which the name Sphingobium chungangianum is proposed. The type strain is MAH-11T (= KACC 19836T = CGMCC 1.13749T).

Microbiota of different wine grape berries

The wine grape berries share a complex microbial ecology including filamentous fungi, yeasts and bacteria. The microbiota reveals different physiological characteristics and depends on the grape ripening stage and the availability of nutrients with different effect on wine production. The microbiota of grape berries (n = 12) was isolated and identified in the present study. The samples were collected in September 2018. Grape berries were obtained from Vrbovo vineyard located in Slovakia. The grape berries investigated belonged to Blue Frankish, Cabernet Sauvignon, Chardonnay, Dornfelder, Feteasca regala, Green Veltliner, Irsai Oliver, Mūller Thurgau, Pálava, Pinot Blanc, Rhinriesling and Welschriesling varieties. The microorganisms were cultivated on Malt extract agar (MEA) at 25 °C for five days in aerobically for microscopic filamentous fungi and Tryptone Soya agar (TSA) at 37 °C for 24 – 48 h aerobically for bacteria and yeasts. Total bacterial counts on different wine grape berries ranged from 2.57 ±0.09 in Chardonnay to 4.39 ±0.21 log CFU.g-1 in Pálava. Microscopic filamentous fungi count ranged from 1.18 ±0.03 in Blue Frankish to 2.60 ±0.17 log CFU.g-1 in Welschriesling. MALDI-TOF MS Biotyper mass spectrometry was used for identification of microorganisms (bacteria and yeasts) and microscopic filamentous fungi with manuals. The most identified microscopic fungal species was Alternaria sp., for yeasts Issatchenkia orientalis and Leuconostoc mesenteroides subsp. mesenteroides for bacteria.

Starter culture development using selected strains of Bacillus spp. associated with “kantong” production in Ghana

Technological properties of Bacillus spp. involved in fermenting Ceiba pentandra seeds into ‘kantong’ a condiment prepared and consumed in the northern part of Ghana were invested so as to develop starter cultures with the predominant species for commercial production of ‘kantong’. The Bacillus species which predominated 205 Bacillus strains isolated from 11 stages of ‘kantong’ production includes: Bacillus amyloliquefaciens, B. safensis, B. altitudinis, B. thuringiensis, B. pumilus, B. megaterium, B. cereus, B. circulans, B. coagulans, B. firmus, B. subtilis and B. licheniformis. These predominant species were assessed for some technological properties such as proliferation at different temperatures and pH; substrate utilization preferences including ‘kantong’ formulated media (i.e. 48 h sample, dried pellet and the final product) and inhibitory activity against 12 selected pathogenic and spoilage microorganisms. The strains proliferated at different temperatures between 10 °C and 55 °C and pH of 2 to 9. Substrate utilization preferences were Nutrient Agar with 5–9 % Sodium Chloride (NA/NaCl C, and ordinary Nutrient Broth [NB], Nutrient Agar (NA), Potato Dextrose Agar (PDA), Tryptone Soya Agar (TSA), MacConkey Agar (MCA) and ‘kantong’ formulated media. All strains exhibited inhibitory activity against one or more pathogenic and spoilage organisms. Salmonella typhimurium, Staphylococcus aureus, E. faecium and Proteus vulgaris were the most susceptible indicator microorganisms. Many strains qualified as potential candidates for selection and development as starter cultures to be used in the large scale commercial production of ‘kantong’ of consistent and acceptable organoleptic quality.

Isolation and characterization of endophytic bacteria from roots of Piper nigrum and their activities against Fusarium oxysporum and Meloidogyne incognita

Abstract. Wiratno, Syakir M, Sucipto I, Pradana AP. 2019. Isolation and characterization of endophytic bacteria from roots of Piper nigrum and their activities against Fusarium oxysporum and Meloidogyne incognita. Biodiversitas 20: 682-687. The endophytic bacteria from spices plants have potential as biocontrol agents. Nevertheless, their potential has not been explored. Pepper plant (Piper nigrum L.) is linked with promising endophytic bacteria as biocontrol agents. This study aimed to obtain isolates of endophytic bacteria from root tissues of pepper plant. The isolation of endophytic bacteria was done using surface-sterilization method and using Tryptone Soya Agar (TSA) medium. The result showed that 10 endophytic bacteria were successfully isolated. We found that a total of 9 bacteria were safe for plants and mammals and used for subsequent steps. The selected bacteria showed inhibition activity of Fusarium oxysporum (18.6 to 43.7%). Furthermore, the secondary metabolites of the endophytic bacteria also promoted lethal effects on Meloidogyne incognita (16.6 to 65.8%). The physiological activity also showed that seven isolates were able to produce chitinase, and four (4) isolates were able to produce protease. Additionally, 55.6% of isolates were also able to dissolve phosphorus and fix nitrogen. This study provided fundamental information related to the biocontrol properties of endophytic bacteria isolated from pepper plant roots.

Comparative Study on Bacterial and Fungal Loads from Siphoned and Unsiphoned Culture of African Catfish (Clarias gariepinus) Hatchlings

This study was carried out to isolate, identify and determine the microbial loads that affect the culture of hatchlings (stage whereby hatchlings absorbed their yolk) in siphoned and unsiphoned water in fish culture. The hatchlings were separated into two groups of triplicate sampling bowls and labelled siphoned and unsiphoned. Samples were analysed for presence of bacteria and fungi using standard methods. The microbial analysis of the siphoned and unsiphoned culture of hatchlings was based on Total Viable Count (TVC) and Total Coliform Count (TCC). The bacterial examination was conducted to isolate and identify bacterial isolates. Serial dilution and inoculation were carried out using sterile media on crushed hatchling samples. Water sample was analysed by inoculating on Tryptone Soy Agar (TSA). For fungal identification both crushed hatchling and water were cultured on Sabouraud Dextrose Agar (SDA), Isolates were examined macroscopically by colony shape, size, colour, and growing pattern, observed under microscope and identified with the help of fungal identification key. Bacterial identification was based on colonial, morphological and biochemical characteristics of colonies. The biochemical test were carried out on the bacterial isolates using catalase test, coagulase test, motility test, triple sugar iron test, indole test and motility test. One way anova was the statistical analysis used for all the results obtained in this experiment. The results showed that the bacterial isolates were Pseudomonas sp., Staphylococcus sp., Escherichia coli , Streptococcus sp., while the fungi include Penicillium sp., Aspergillus sp., and Mucor sp. The bacterial isolates showed trending pattern with an increase in count from the first day to the seventh day in unsiphoned as against the siphoned water sample. Conversely, the fungal isolates showed an inconsistent load value between siphoned and unsiphoned from the first day to the seventh. This study revealed that bacterial isolates are more associated than the fungal isolates in the hatchlings culture. The fungal load isolated are relatively lower and significantly different (P&lt;0.05) than the bacterial load observed. Keywords: Bacterial, Fungal, African Catfish, Culturing methods, Hatchlings DOI : 10.7176/ALST/71-05

Heat resistance of spores of 18 strains of Geobacillus stearothermophilus and impact of culturing conditions

In this study, different methods were evaluated for enumeration of spores of G. stearothermophilus, different sporulation methods were assessed for yields and wet heat resistances of obtained spores, and subsequently, the variation in heat resistances of spores was determined. Overall, tryptone soya agar (TSA) was the most suitable medium for enumeration of spores of this thermophilic bacterium. Sporulation on different media both at 55 and at 61 °C led to considerable variation in spore heat resistance. The heat resistance of spores was highest upon sporulation on medium supplemented with free ions of calcium, potassium, magnesium and manganese (CaKMgMn). For 18 different G. stearothermophilus strains that were isolated from various sources, spores were subsequently produced on nutrient agar supplemented with CaKMgMn at 55 °C. Strain ATCC 12980T, also known as 9A20, which is commonly used in steam sterilization tests was included. The survival of spores of all strains was assessed at 125 °C and 130 °C using two independent spore batches per strain. The mean D125°C for spores of the 18 strains was 1.1 min (95% PI 0.48–2.3 min) and the mean D130°C was 0.37 min (95% PI 0.17–0.82 min). For spore inactivation of these 18 strains, a z-value of 11.1 °C was estimated, resulting in an estimated D-value of 2.4 min (95% PI 1.1–5.2) at the reference temperature 121.1 °C. Based on the data sets obtained in this study, it was found that the variability in spore heat resistance could largely be attributed to strain variability and conditions used during sporulation (especially the sporulation medium); reproduction and experimental variabilities were much smaller. The established variabilities were compared with the overall variability in spore heat resistance of G. stearothermophilus based on a meta-analysis of reported D-values. The data presented indicate that strain variability and history of sporulation each account for approximately half of the overall variability observed with respect to the heat resistance of spores of G. stearothermophilus. The findings presented in this study allow for optimal recovery of G. stearothermophilus spores from foods and a better understanding of factors that determine the heat resistance properties of spores of G. stearothermophilus. Moreover, this study once more underlines the limited effects of heat treatments used in the food industry on inactivation of spores of this bacterium.

Biological Synthesis of Ginsenoside Rd Using Paenibacillus horti sp. nov. Isolated from Vegetable Garden.

A Gram-stain positive, aerobic, motile, rod-shaped, and ginsenoside Rd producing novel bacterial strain, designated as MAH-16T, was isolated from soil sample of a vegetable garden and was characterized by using a polyphasic approach. The colonies were beige color, smooth, circular, and 0.3-0.7mm in diameter when grown on tryptone soya agar for 3 days. Strain MAH-16T can grow at 20-40°C temperature, at pH 5.0-7.0 and at 0-1% NaCl. Cell growth occurs on nutrient agar, R2A agar, tryptone soya agar, and Luria-Bertani agar but not on MacConkey agar. The strain was positive for both catalase and oxidase test. The novel strain rapidly synthesized ginsenoside Rd from major ginsenoside Rb1. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Paenibacillus and was most closely related to Paenibacillus barengoltzii SAFN-016T (97.1%), Paenibacillus faecis 656.84T (96.7%), and Paenibacillus konsidensis LBYT (96.2%). In DNA-DNA hybridization tests, the DNA relatedness between strain MAH-16T and its closest phylogenetic neighbor was below 45.0%. The genomic DNA G + C content of isolated strain was determined to be 52.0mol% and the predominant isoprenoid quinine was menaquinone-7 (MK-7). The major fatty acids were identified as anteiso-C15:0. The genetic characteristics in combination with chemotaxonomic and physiological data demonstrated that strain MAH-16T represented a novel species within the genus Paenibacillus, for which the name Paenibacillus horti sp. nov. is proposed, with MAH-16T as the type strain (=KACC 19299T = CGMCC1.16487T).

Simultaneous growth, survival and death: The trimodal behavior of Salmonella cells under osmotic stress giving rise to “Phoenix phenomenon”

Time-lapse microscopy methods were used to monitor growth, survival and death of Salmonella enterica serotype Agona individual cells on solid laboratory medium (tryptone soy agar) in the presence of various salt concentrations (0.5%, 3.5%, 4.5% and 5.7% NaCl). The results showed a highly heterogeneous behavior. As NaCl concentration increased, the distribution of the first division time was shifted to higher values and became wider. The mean first division time increased from 1.8 h at 0.5% NaCl to 5.48 h, 16.2 h, and 35.9 h at 3.5%, 4.5% and 5.7% NaCl, respectively. The concentration of NaCl in the growth medium also affected the ability of the cells to divide. The percentage of cells able to grow decreased from 88.9% at 0.5% NaCl to 66.5%, 32.8%, and 6.9% at 3.5%, 4.5% and 5.7% NaCl, respectively. In the latter case (5.7% NaCl), 74 cells out of 406 cells tested (18%) died with mean time to death 5.03 h and standard deviation 6.70 h. To investigate the effect of the behavior of individual cells on the dynamics of the whole population, simulation analysis was used. The simulation results showed that the simultaneous growth, survival and death of cells observed under osmotic stress can lead to a total population behavior known as the “Phoenix” phenomenon. The simulation findings were confirmed by validation experiments using both viable counts and time lapse microscopy. The results of the present study show the high heterogeneity of individual cell responses and the complexity in the behavior of microbial populations at conditions approaching the boundaries of growth.

Microvirga rosea sp. nov.: a nanoparticle producing bacterium isolated from soil of rose garden.

A Gram-stain negative, aerobic, non-motile, and rod-shaped novel bacterial strain, designated MAH-2T, was isolated from a soil sample of rose garden and was characterized using a polyphasic approach. The colonies were light pink color, smooth, circular and 0.2-0.6mm in diameter when grown on nutrient agar for 3 days. Strain MAH-2T grows at 15-40°C (optimum growth temperature 30°C), at pH 5.0-7.0 (optimum growth pH 6.5) and at 0-2% NaCl (optimum 0-0.5%). Cell growth occurs on nutrient agar and R2A agar but not on tryptone soya agar, luria-bertani agar and MacConkey agar. The strain was positive for both catalase and oxidase tests. The strain was able to synthesis of silver nanoparticles. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Microvirga and was most closely related to Microvirga soli R491T (96.7% sequence similarity), Microvirga subterranea Fail4T (96.4%), Microvirga guangxiensis 25BT (96.0%) and Microvirga aerophila 5420S-12T (95.9%). The genomic DNA G + C content of isolated strain was determined to be 62.5mol% and the predominant isoprenoid quinone was Q-10. The major fatty acids were identified as summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c) and C19:0 cyclo ω8c. On the basis of these phenotypic, genotypic, and chemotaxonomic studies and DNA-DNA hybridization results, the isolated strain MAH-2T represents a novel species, for which the name Microvirga rosea sp. nov. is proposed, with MAH-2T as the type strain (= KACC 19290T = CGMCC1.16488T).

A comparative study of the wild and mutated heavy metal resistant Klebsiella variicola generated for cadmium bioremediation

ABSTRACTTen indigenous heavy metals resistant bacteria were isolated from the discharged effluent of Biological Sciences building at Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria. The bacterial strains were isolated using enrichment method in tryptone soya agar (TSA) supplemented with 100 mg/L of Pb, Cd, As, and Ni. All the isolated bacteria showed multiple tolerances to the heavy metals; however, strain GBB 220, which showed a maximum tolerable concentrations (MTCs) of 2000, 1200, 4700, and 1000 mg/L for Pb, Cd, As, and Ni, respectively, was selected for further studies. The bacterium was identified by 16S rRNA sequences as Klebsiella variicola and was subjected to mutational enhancement by acridine orange and ethidium bromide. Eight mutants were recovered, strains K. variicola MutAa–Ad showed improvement in their MTCs of 2500, 2200, and 5000 mg/L for Pb, Cd, and As whereas K. variicola MutEa–Ed had same MTCs as the wild type except for cadmium which increased from 1200 to 1500 mg/L. The antibiotic susceptibility patterns showed that all the strains of K. variicola had multiple resistances to some of the antibiotics. The K. variicola mutants had their optimum pH at 5 and 8 while the wild type had optimum pH at 7 and 8. The cadmium removal efficiency of 97.9% at pH 7, 97.7% at pH 8, and 99.4% at pH 7 was observed for K. variicola Wt, K. variicola Mut Eb, and K. variicola Mut Ac, respectively. These findings suggested that the artificially mutated strains of K. variicola may be applied to remove cadmium from polluted environment.