Delineating Kocuria turfanensis 2M4 as a credible PGPR: a novel IAA-producing bacteria isolated from saline desert

Indole-3-acetic acid (IAA)-producing bacteria Kocuria turfanensis strain 2M4 was isolated from the rhizospheric soil of halotolerant plant Suaeda fruticosa from a unique saline desert of Little Rann of Kutch, Gujarat, India. Rhizobacteria was bright orange pigmented, gram-positive, coccoid, non-endospore forming, and aerobic in nature. 16S rRNA gene sequence analysis showed that 2M4 isolate matched best with type strain of K. turfanensis HO-9042^T. Isolate optimally produced 38 µg ml^?1 IAA when growth medium was supplemented with 600 µg ml^?1 of L-tryptophan. Thin layer chromatography and Fourier transform infrared spectroscopy analysis were performed to corroborate IAA production. To characterize rhizobacterial isolate as a plant growth-promoting bacteria, it was tested for phosphate solubilization where it solubilized maximum 12 µg ml^?1 phosphate in presence of fructose, produced 53% siderophore units under iron-free minimal MM9 medium and produced 1.8 µmol ml^?1 ammonia in peptone water broth. Plant growth promotion by test isolate was studied on groundnut (Arachis hypogaea L.) under non-saline and saline soil. There was increase by 18% in total plant length and 30% in fresh biomass observed under non-saline control soil. Under saline soil, test isolate showed 17% increase in total length of the plant and 13% increase in fresh biomass.     

Peptones from diverse sources: pivotal determinants of bacterial growth dynamics

Aims: In view of the major problems encountered by microbiologists in obtaining reproducible data on growth dynamics in complex media, we studied the effects of different peptones made from different biological sources and produced by numerous manufacturers. Methods and Results: Peptones (including casein, gelatin, meat, soy and yeast) were assessed as a constituent of the pre‐enrichment broth buffered peptone water (BPW). Generation times (g) and yields of Salmonella serovar Typhimurium were significantly affected by the type of peptone employed with yeast peptones generating yields of 7·04 × 10⁹ CFU ml^?1 and gelatin peptones producing 0·81 × 10⁹ CFU ml^?1. Medium sterilization was also found to have significant effects (P = 0·000) upon subsequent bacterial growth. Filter sterilization of BPW media produced lower generation times compared with those obtained after sterilization by autoclaving. Finally, it was observed that some peptones which produced good growth when inoculated with healthy organisms, showed relatively poor growth when inocula were sublethally injured by heating. Conclusions: Variation in peptone as a constituent of BPW has a significant effect on growth and enumeration of bacteria. Significance and Impact of the Study: Increased consideration with respect to culture media may significantly improve bacterial growth and experimental reproducibility.     

Influence of media and temperature on bacteriocin production by<Emphasis Type="Italic"> Bacillus cereus</Emphasis> 8A during batch cultivation

Cerein 8A is a bacteriocin produced by the soil bacterium Bacillus cereus 8A, isolated from native woodlands of Brazil. The influence of temperature and media on the growth of B. cereus 8A and the production of this bacteriocin was studied during batch cultivation. Maximum activity was detected by cultivation in brain/heart infusion broth, reaching 3200 activity units ml^–1. Bacteriocin was also produced in peptone, MRS, Mueller–Hinton and nutrient broth, while no activity was observed during cultivation in thioglycollate or tryptic soy broth. Temperature had a strong influence on bacteriocin production, which was higher at 30 °C than at 25 °C. An important decrease in bacteriocin activity was observed at 37 °C. The relationship between growth and specific production rates, as a function of the temperature, showed different kinetics of production and there were several peaks in the specific production rates during growth. Bacteriocin was produced at the stationary phase, indicating it is synthesized as a secondary metabolite.     

Addition of a surfactant to tryptic soy broth allows growth of a lactic acid bacteria food antimicrobial, Escherichia coli O157:H7, and Salmonella enterica

Aims: This study aimed to determine the survival and growth of Escherichia coli O157:H7 and Salmonella enterica subsp. enterica in a medium supporting the growth of a Lactic Acid Bacteria (LAB) food antimicrobial culture. Methods and Results: Foodborne pathogens and LAB were cultured individually in tryptic soy broth (TSB), tryptic soy broth supplemented with one g l^?1 Tween 80^® (TSB‐T80), and de Man, Rogosa and Sharpe (MRS) broth. Growth of E. coli O157:H7 and Salmonella was similar in TSB and TSB‐T80 but was significantly less in MRS. Conversely, LAB growth was similar in MRS and TSB‐T80 but was significantly less in TSB. Conclusions: Supplementation of TSB with Tween 80^® allows growth of LAB to levels similar to that observed with MRS but does not inhibit the growth of E. coli O157:H7 and Salmonella. We present the formulation of a medium useful in studies useful for evaluating competitive inhibition of foodborne pathogens by LAB in vitro. Significance and Impact of the Study: This study reports the utility of TSB‐T80 for the completion of in vitro competitive inhibition assays incorporating a Lactic Acid Bacteria food safety culture.     

Effects of thymol and diphenyliodonium chloride against Campylobacter spp. during pure and mixed culture in vitro

Aims: To determine if the purported deaminase inhibitors diphenyliodonium chloride (DIC) and thymol reduce the growth and survivability of Campylobacter. Methods and Results: Growth rates of Campylobacter jejuni and Camp. coli were reduced compared to unsupplemented controls during culture in Muellar–Hinton broth supplemented with 0·25 μmol DIC or thymol ml^?1 but not with 0·01 μmol monensin ml^?1 or 1% ethanol. Recovery of Camp. jejuni and Camp. coli was reduced >5 log₁₀ CFU from controls after 24 h pure culture in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Similarly, each test Campylobacter strain was reduced >3 log₁₀ CFU from controls after 24 h mixed culture with porcine faecal microbes in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Treatments with 0·25 μmol thymol ml^?1, 0·01 μmol monensin ml^?1 or 1% ethanol were less effective. Ammonia production during culture or incubation of cell lysates was reduced by 0·25 or 1·0 μmol DIC ml^?1 but only intermittently reduced, if at all, by the other treatments. Conclusions: Diphenyliodonium chloride and thymol reduced growth, survivability and ammonia production of Camp. jejuni and Camp. coli. Significance and Impact of the Study: Results suggest a potential physiological characteristic that may be exploited to develop interventions.     

Evaluation of waste chicken feathers as peptone source for bacterial growth

Aims: Peptones are one of the most expensive constituents of microbial media. This study was undertaken to prepare the peptone from waste chicken feathers through a new process. Methods and Results: The chemical analysis of chicken feather peptone (CFP) was performed. The ability of CFP to support the growth of the three test bacteria in liquid and agar media was comparable to those of three commercial peptones [tryptone peptone (TP), fish peptone and protease peptone (PP)]. Conclusions: CFP was found to be rich in ash (42·1 g 100 g^?1), protein (55·8 g 100 g^?1) and mineral contents. The maximum biomass yield (3·13 g l^?1) and colony number (83 × 10⁸ CFU ml^?1) for bacterium Bacillus subtilis were attained with CFP. The maximum biomass yields and colony numbers for Lactobacillus delbrueckii ssp. bulgaricus and Escherichia coli were reached in TP medium. Second high biomass yield (2·64 g l^?1) and colony number (75 × 10⁸ CFU ml^?1) for E. coli were achieved using CFP. Third high biomass yield (1·29 g l^?1) and colony number (90 × 10⁷ CFU ml^?1) for Lact. delbrueckii ssp. bulgaricus were obtained in CFP medium. Significance and Impact of the Study: Usability of waste chicken feathers as substrate for bacteria was investigated for the first time in the present study. The peptone may be used in industrial fermentations for production of antibiotics, organic acids, enzymes and biopolymer. It may be also used in clinical microbiology. A new chemical process was developed for peptone preparation. This process may be also employed for peptone preparation from other organic materials, especially fibrose protein‐containing materials.     

Plant growth promoting potentials of Pseudomonas spp. strain OG isolated from marine water

Abstract

Bacterium Pseudomonas spp. olive green (OG) was isolated from marine water, yet, it was characterized as plant growth promoting bacterium (PGPB). Multiple plant growth promoting traits of OG isolate were determined in vitro. It was tested positive for Indole-3-acetic acid (IAA) production with 29 µg ml^?1 of IAA yield, phosphate solubilization with 34 µg ml^?1 solubilization of Tri-calcium-phosphate and it showed maximum of 32 µg ml^?1 of ammonia production. OG isolate was affirming siderophore production, hydrocyanic acid (HCN) production and catalase production. 16S rRNA gene sequence comparison was used to identify the isolate which showed its closest neighbor to be Pseudomonas fluoroscens strain BCPBMS-1. Efficacy of this PGPB was tested on the seedling growth of two test plants chickpea and green gram. Both the test plants treated with OG-based talc bioformulation showed significant growth promotion. Chickpea showed enhanced overall fresh biomass by 24%, overall dry biomass by 27% was observed after 15 days of seeded in pots. Green gram showed enhanced overall dry biomass by 28% was observed after 10 days of seeded in pots.     

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life‐threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 10⁸ pfu ml^?1 of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (10⁶ and 10² cfu ml^?1). In contrast, broth inoculated with 10⁴ phage and 10² bacteria per ml first showed normal bacterial growth until reaching a cell titre of 10⁵ cfu ml^?1. Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml^?1. Phages could be produced with titres of 10¹⁰ pfu ml^?1 in broth culture, but they were not stable upon freeze‐drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.     

Inhibitory activity of natural antimicrobial compounds alone or in combination with nisin against Enterobacter sakazakii

Aim: To determine the antimicrobial activity of natural organic compounds alone and in combination with nisin on the growth of Enterobacter sakazakii in laboratory media. Methods and Results: The minimum inhibitory concentrations (MIC) of five natural organic compounds were determined, and their effects in combination with nisin were evaluated by comparing treatment with each natural organic compound alone and in combination with 25 mg ml^?1 nisin in tryptic soy broth. Among the tested natural organic compounds, the MIC of carvacrol and thymol was 1·25 mmol l^?1 and showed the strongest inhibitory activity against E. sakazakii, whereas the MIC of cinnamic acid was higher than 5 mmol l^?1, and therefore showed the weakest inhibitory activity. However, the combination of each compound with nisin did not result in the enhancement of their antimicrobial activities except when nisin was combined with diacetyl. Conclusions: The order of inhibition attributed to natural organic compounds was carvacrol = thymol > eugenol > diacetyl > cinnamic acid, and only the combination of diacetyl and nisin showed a synergistic effect of inhibiting the growth of E. sakazakii. Significance and Impact of the Study: This study shows the potential of natural organic compounds for controlling E. sakazakii.     

Optimum Culture Conditions for Production of Exfoliative Toxin by Staphylococcus hyicus

Optimum culture conditions for the production of exfoliative toxin by Staphylococcus hyicus (shET) were examined. High shET activity was obtained from the culture filtrate of HI and TY broth inoculated with S. hyicus. The pH in these two media ranged from 7 to 8.5 during bacterial culture, while the lowest pH in TS and BHI broth was less than 6. shET activity in the culture filtrate from TY broth inoculated with 10⁷ CFU of S. hyicus per ml was higher than that in TY broth inoculated with 10⁶ and 10⁸ CFU of bacteria per ml. When shET activity in the culture filtrate was measured under various shaking conditions, the culture filtrate shaken at 75 oscillations per min had the highest shET activity of the five shaking conditions. shET activity of the culture filtrate of TY broth to which protease inhibitor had been added was the same as that of TY broth without inhibitor. shET activity in a shaking culture in an Erlenmeyer flask was also the same as that in sac culture and that in shaking culture using a shaking (Sakaguchi) flask. shET activity in TY broth supplemented with 100 mM glucose was significantly lower than that in TY broth without glucose. Based on the above results, the optimum culture conditions for the production of shET were as follows: inoculation of 3 × 10⁹ CFU of S. hyicus strain P-1 into 300 ml of TY broth in a 2,000-ml Erlenmeyer flask, and incubation at 37 C with shaking at 75 oscillations per min. Then shET activity of the culture filtrate under appropriate culture conditions was measured after various incubation periods. shET activity was detected 6 hr after inoculation, reached the maximum (2⁵³ exfoliative unit/0.1 ml) at 16 hr and decreased between 20 and 48 hr. Thus, the optimum incubation period was determined to be 16 hr. Then the optimum concentration of ammonium sulfate for isolation of shET from the culture filtrate under appropriate culture conditions was examined. The greatest shET activity was obtained from the fraction salted out with 90% saturated ammonium sulfate. Thus, the optimum concentration of ammonium sulfate for the isolation of shET was determined to be 90% saturation.     

Application of experimental designs to optimize medium composition for production of thermostable lipase/esterase by Geobacillus thermodenitrificans AZ1

Thirty two morphologically different bacterial were isolated from different soil samples and screened for their ability to produce lipolytic enzymes. Among all isolates, the isolate coded AZ1 was selected due to its high potency to produce lipase at elevated temperature up to 65 °C. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Geobacillus thermodenitrificans. The effect of ten culture variable on lipase production was evaluated by implementing Plackett–Burman statistical design. d-sucrose, peptone and soy bean flour were the most significant variables affecting lipase production. A pre-optimized medium based on this experiment yielded an enzyme activity of 260 U min^?1 ml^?1. For further optimization, a fourteen trials’ multi-factorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The tested variables, namely: d-sucrose (X₁); peptone (X₂) and soy bean flour (X₃) were examined, each at three different levels coded ?1, 0, +1. The optimal levels of the three components were founded to be (g/L): d-sucrose, 6.56; peptone, 6.35; and soy bean flour, 6.92, with a predicted activity of approximately 610 U min^?1 ml^?1. According to the results of the Plackett–Burman and Box–Behnken designs the following medium composition is expected to be optimum (g/L): d-sucrose 6.56, peptone 6.35, soy bean flour 6.92, CaCl₂ 0.02, Y.E. 2.5, K₂HPO₄ 1.0, MgSO₄.7H₂O 0.2 and Fe₂ (SO₄)₃ 0.02; pH, 8; cultivation temperature 55 °C and incubation time 24 h, the enzyme activity measured in the medium was approximately 593 U min^?1 ml^?1.     

Submerged Cultivation of a Fungal Mutant Strain Humicola Lutea 120-5 Producing Acid Proteinases

The optimal parameters for the cultivation in 10-l fermenters of a mutant strain Humicola lutea 120-5 were established:temperature 30°C, inoculum size 6%, inoculum age 24 h, aeration rate 0,6 vol/vol · min, medium agitation 620 rpm and cultivation time 72 h. A maximal proteolytic activity of 2000 µg tyrosine liberated from 2%casein ml^?1 culture filtrate min^?1 at pH 3.0 and 40°C was obtained under the fixed conditions. α-Amylase biosynthesis during the cultivation of H. lutea 120-5 was observed but it was insignificant to the 72nd h. It is demonstrated that starch can be used as alternative to glucose carbon source. It is proved that the mutant strain H.lutea 120-5 produced two acid proteinases.     

A novel alkaline, thermostable, protease-free lipase from Pseudomonas sp.

An extracellular, alkali-tolerant, thermostable lipase was from a Pseudomonas sp. It had optimal activity at 65 °C and retained 75% of its activity at 65 °C for 90 min. The pH optimum was 9.6 and it retained more than 70% activity between pH 5 and 9 for 2 h. The culture broth was free of protease and, at 30 °C, the culture filtrate retained all the activity for at least 7 days, without any stabilizer. In shake flask culture, addition of groundnut oil (3 g l^–1) towards the end of growth phase increased the activity from 4 U ml^–1 to 8 ml^–1.     

Effects of jackbean lectin (ConA) on the feeding behaviour and kinetics of intoxication of the pea aphid, Acyrthosiphon pisum

Mannose‐binding lectins were shown to be useful in creating transgenic plants resistant to insects, including many phloem‐feeding Hemiptera. Before these plants can be used extensively, it is important to understand how these lectins exert their toxic effects on the target organisms. We investigated the feeding alterations induced by presenting the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), with a diet containing the lectin from Canavalia ensiformis (ConA). A series of behavioural experiments were carried out to detect potential sensory mediation of lectin activity. Choice tests performed with a 400 µg ml^?1 ConA diet (3.7 µm of native tetramer) showed that A. pisum quickly rejected the ConA diet, but that this reaction was not typical of a sensory‐mediated phagodeterrent effect. In addition, the aphids did not develop a conditioned taste aversion to the lectin. Diet uptake was evaluated using a radioactive tracer (¹⁴C‐methylated inulin), and showed depression of ingestion only after 16 h at 200 µg ml^?1 or after 8 h at 400 µg ml^?1 ConA. This effect was reversible under our test conditions. No evidence was obtained for early detection of the lectin, even by intoxicated aphids. An electrical penetration graph technique was adapted to artificial diets and provided short‐term continuous analysis on feeding/probing events. At the 400 µg ml^?1 level, adults were affected and had reduced ingestion durations as early as in the first 4 h of contact, but experienced an adaptation to the behavioural alterations induced by lectin feeding. Overall, feeding deterrency following exposure to mannose lectins appeared to be a consequence of intoxication, and not due to a sensory mediated process.     

In vitro degradation of wheat straw by anaerobic fungi from small ruminants

Abstract

Anaerobic ruminal fungi may play an active role in fibre degradation as evidenced by the production of different fibrolytic enzymes in culture filtrate. In the present study, 16 anaerobic fungal strains were isolated from ruminal and faecal samples of sheep and goats. Based on their morphological characteristics they were identified as species of Anaeromyces, Orpinomyces, Piromyces and Neocallimastix. Isolated Neocallimastix sp. from goat rumen showed a maximum activity of CMCase (47.9 mIU ml^?1) and filter paper cellulase (48.3 mIU ml^?1), while Anaeromyces sp. from sheep rumen showed a maximum xylanolytic activity (48.3 mIU ml^?1). The cellobiase activity for all the isolates ranged from 178.0 – 182.7 mIU ml^?1. Based on the enzymatic activities, isolated Anaeromyces sp. from sheep rumen and Neocallimastix sp. from goat rumen were selected for their potential of in vitro fibre degradation. The highest in vitro digestibility of NDF (23.2%) and DM (34.4%) was shown for Neocallimastix sp. from goat rumen, as compared to the digestibility of NDF and DM in the control group of 17.5 and 25.0%, respectively.     

Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar ‘Villafranca’) cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml^?1, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml^?1 and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin‐induced NO production was sensitive to sodium azide and N^G‐monomethyl‐l ‐arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.     

Antimicrobial activity of Coniothyrium minitans and its macrolide antibiotic macrosphelide A

Aims: Assessment of antimicrobial activity of the mycoparasite Coniothyrium minitans and its macrolide antibiotic macrosphelide A. Methods and Results: Thirteen isolates of C. minitans were tested for ability to inhibit a number of filamentous fungi, yeasts, oomycetes and bacteria in agar based tests. Activity was found against some ascomycetes, basidiomycetes, oomycetes and Gram‐positive bacteria, but not against zygomycetes, yeasts or Gram‐negative bacteria tested. Six C. minitans isolates (Conio, Contans, IVT1, CM/AP/3118, B279/1, A1/327/1) were found to produce macrosphelide A in liquid culture and no other antibiotics were detected. On agar, macrosphelide A inhibited growth of some ascomycetes, basidiomycetes, oomycetes and all four Gram‐positive bacteria tested, including the medically important Staphylococcus aureus with a minimum inhibitory concentration of ≤500 μg ml^?1. There was no inhibition observed against the yeasts and Gram‐negative bacteria when macrosphelide A was tested at 700 μg ml^?1. Conclusions: The spectrum and level of activity of macrosphelide A produced by C. minitans against micro‐organisms are extended markedly compared to previous reports. Significance and Impact of the Study: Macrosphelide A was effective against Staph. aureus. Further study on the control of this bacterium is merited in view of the development of antibiotic resistance.     

Diversity and characterization of oxytetracycline-resistant bacteria associated with non-native species, white-leg shrimp (Litopenaeus vannamei), and native species, black tiger shrimp (Penaeus monodon), intensively cultured in Thailand

Aims: This study aimed at surveying prevalence of oxytetracycline (OTC)‐resistant bacteria in the white‐leg shrimp Litopenaeus vannamei, and the black tiger shrimp Penaeus monodon, intensively cultured in Thailand. We investigated the phylogenetic diversity of the bacterial isolates, as well as the minimum inhibitory concentration (MIC) of OTC, the occurrence of major OTC‐resistant genes and multiple‐antibiotic resistance in the isolates. Methods and Results: Shrimps were collected from culture ponds, and the homogenates of whole bodies were plated on tryptic soy agar supplemented with or without OTC. Percentages of OTC‐resistant bacteria were 0·3–52·1% in white‐leg samples and 0·008–22·3% in black tiger samples. Analyses of 16S rDNA sequences indicated that most OTC‐resistant isolates were closely related to Aeromonas spp. and Lactococcus garvieae. MICs of OTC were 4–128 μg ml^?1 in the OTC‐resistant aeromonads and 128–256 μg ml^?1 in OTC‐resistant L. garvieae. OTC resistance was found to be conferred by the genes tet(A), tet(C), tet(D), tet(E), tet(M) and tet(S), detected either singly or in pairs. No resistance to ceftazidime, imipenem or chloramphenicol was observed in any isolate. Conclusions: Both species of shrimp are associated with OTC‐resistant bacteria, occasionally at high densities exceeding 10⁶ cfu g^?1. The associated bacteria, predominantly Lactococcus and Aeromonas genera, are potential pathogens and are reservoirs of a variety of OTC‐resistant genes. Significance and Impact of the Study: Cultured shrimps can be vehicle to carry OTC‐resistant bacteria to domestic and foreign consumers via the food chain. Very low populations of OTC‐resistant bacteria observed in the several ponds suggest that levels of the resistant bacteria are artificially high and should be reduced in farmed shrimps.