Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

Background Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains. Results We constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, a maximum titer of 142.32 mg/L was reached by the use of an optimized medium for ectoine production containing 6% NaCl and 0.05 μM of tungsten without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date. Conclusions Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production. Graphical Abstract

A comparative study for production of germ tube in Candida albicans of various pulmonary samples, by different methods in a tertiary care hospital of south west Rajasthan

Background: Systemic candidiasis is associated with a high crude mortality rate, even with first line antifungal therapy. C. albicans is the predominant cause of invasive fungal diseases which is a serious public health issue. The main objective was to assess the reliability of different media for germ tube production in Candida albicans isolated from various clinically diagnosed pulmonary samples.Methods: All Candida isolates were identified and speciated by conventional methods such as Gram’s staining, germ tube test, chlamydospore formation on corn meal agar, sugar fermentation test, sugar assimilation test, and growth on Hi-chrome candida agar.Results: Out of 108 clinical isolates of Candida albicans, 5 different methods were used for germ tube production. Pooled human sera showed 93/108 (86.1%) was the most sensitive method wherein YEPD (yeast extract peptone dextrose) broth 91/108 (84.7%) was the reliable and easy method for detection of germ tube, followed by trypticase soy broth 81/108 (81.4%); peptone water 80/108 (74.7%) and 2% sucrose 71/108 (65.7%).Conclusions: YPED broth is found to be a better serum free substrate and subsequently for the presumptive differentiation of C. albicans from non-albicans candida (NAC), without the extensive time required for the preparation and testing of pooled human serum. Furthermore, this medium is commercially available, more stable, effective, and is not bio hazardous.

Speciation of candida species isolated in clinical samples in a tertiary health care centre in Northern India

The purpose of this study is to isolate, identify and specification of various the Candida species from various clinical samples in a tertiary care hospital, and to characterize various the isolated Candida species. A study was conducted on people of different age groups from January 2019 to December 2019. Candida species isolated from different patients by using Potassium Hydroxide mount and processed by BacTalert 3D (Biomerieux) automated blood culture system. Further culture identification of Candida species were done on Sabouraud dextrose agar (SDA). Speciation of Candida was done using Germ tube test, CHROM agar Candida Medium, Cornmeal agar, Sugar Fermentation test and Sugar Assimilation test.In our study was the most common species isolated, among non albicans Candida i.e. 21 (38.9%); 19(35.2%) of was the most common followed by 9(16.7%) ofand 5(9.3%) of . Maximum number of Candida isolates were obtained from NICU i.e. 27(50.0%) followed by 11 from Med (20.3%), 7 from E/W (13.0%), 2 from BICU (3.7%), 2 from Skin (3.7%), 1 from PICU (1.9%), and 1 from R/R (1.9%).Our study showed that is the most common isolates species. Among , was found to be the most common isolate followed by . Children less than 1 year are most affected with maximum number of Candida species were obtained from NICU department. HiChrom Candida is proven to be more useful as differential agar.

Isolation, identification and biochemical characterization of lactic acid bacteria from selected yogurt samples

This research was conducted to study the types of lactic acid bacteria (LAB) present in selected yogurts available in the local market of Bangladesh. For this purpose, nine different yogurt samples were collected (viz. MV, Mw, Pst, Psr, Bik, Bog, WF, Kw and Nab) and cultured in the selective MRS agar media for enumerating LAB colony. Out of 9 samples, colony forming LAB were found in 6 samples and the population ranged from 1.0×104 to 9.5×105 cfu/ml. Catalase negative and Gram’s positive colonies were initially identified as LAB. Then the isolates were purified by subsequent culturing in MRS broth and MRS agar media. Biochemical properties of selected colonies were evaluated by performing gas production from glucose, growth at different temperatures (10ºC, 15ºC and 45ºC), growth at different NaCl concentrations (2, 4 and 6.5% NaCl) and sugar fermentation tests (lactose, sorbitol, salicin, trehalose, melibiose, sucrose, mannitol, melezitose, maltose, galactose, glucose, arabinose, raffinose and ribose). According to the tests stated above, a total of five different species of LAB were identified from 6 samples. The isolate Lactobacillus lactis was identified in MV, PSr and Bog yogurt, while Lactobacillus bulgaricus was found in MV and Bik yogurt. The species Leuconostoc cremoris, a avor producing bacteria, was found in six yogurt samples. On the other hand, Lactobacillus acidophilus and Lactobacillus helveticus was found only in Pst and Psr samples, respectively. Bang. J. Livs. Res. Vol. 27 (1&2), 2020: P. 64-72

Biochemical property analysis of native probiotic isolates from selective poultry

Isolation and identification of probiotic bacteria are the prerequisites for their safer use in the food and feed industry. The objectives of the present study were the isolation of probiotic bacteria from the selective gastrointestinal tract of poultry obtained from Khulna and Barisal Divisions, and their identification based on bacterial morphological characterization and biochemical property analysis. Ten potential native probiotics were isolated from the poultry gastrointestinal tract and assayed for their morphological, physiological and biochemical properties. It was observed that, all the isolates were rod-shaped, gram-positive, endospore-negative, catalase-negative, non-motile and were able to ferment particular sugars which are an indicator for typical probiotic bacteria. The sugar fermentation pattern, ability to survive and growth in inhibitory substances like 1-4% NaCl, 0.3% bile salt as well as their ability to grow in different temperatures and pH levels ensured the presumptive identification of the lactic acid bacteria. All the ten isolates exhibited a clear zone of inhibition when they were grown with five enteric pathogens which are indicative of their antimicrobial activity. Ten isolates were assayed for their susceptibility to eight antibiotics using the disc diffusion method. All the isolates were resistant to tetracycline and nalidixic acid. Further research regarding molecular characterization and identification of specific genes using different technologies may open the door to utilize these isolates in different probiotic-based inventions. Bang. J. Livs. Res. Vol. 27 (1&2), 2020: P. 39-54

Non-Candida albicans Candida Species: Virulence Factors and Species Identification in India

Background and Purpose: The predominant cause of candidiasis was Candida albicans which has recently changed to non-Candida albicans Candida (NCAC) (i.e., Candida spp. other than the C. albicans). The NCAC spp., earlier considered non-pathogenic or minimally virulent, are now considered a primary cause of morbidity and mortality in immunocompromised individuals. Given the NCAC spp.has become more common in clinical cases, this study aimed to determine the prevalence of NCAC spp. in different clinical specimens and assess a few of their virulence factors. Materials and Methods: Routine samples for bacterial culture and sensitivity that showed colony characteristics, like Candida on Blood Agar and microscopic features resembling Candida spp., were processed further. Candida isolates underwent tests for chlamydospore formation and biochemical tests, including sugar fermentation and sugar assimilation tests. These were grown at 42oC, and their colony color was identified using HiCrome™ Candida Differential Agar (HiMedia Laboratories Pvt. Ltd., Mumbai,India), HiCandidaTM Identification Kit (HiMedia Laboratories Pvt. Ltd., Mumbai, India),and VITEK-2® Compact (Biomérieux, France). Virulence factors, such as adherence to buccal epithelial cells (ABEC), biofilm formation, hemolytic activity, and production of coagulase enzyme were also tested. Results: Mean age of the patients was 38.46 years with a male-female ratio of 1.36:1. In total, 137 Candida isolates were recovered; 45.3%, 19.7%, and 13.9% of the isolates were isolated from urine, vaginal swabs, and oropharyngeal swabs, respectively. Moreover, 55(40.1%) isolates were those of C. albicans and 82 (59.9%) isolates belonged to NCAC spp.,with C. tropicalis (23.4%) contributing highest among NCAC species. Furthermore, C. albicans (3; 50%) was the most common spp. in cases of candidemia. Haemolysin production (85.5%) and ABEC (78.2%) were the major virulence factors in C. albicans. C.tropicalis (59.4%) and C. dubliniensis (50%) showed maximum ABEC. Biofilm forming capacity was higher in C. tropicalis (78.1%) than C. albicans (67%). Conclusion: Results of this study suggest varied prevalence and virulence based on geographical locations, even within a subcontinent. It clearly indicates the emergence of the NCAC spp. and their predominance in different body fluids. Identification of Candida to the spp. level should become a routine in all laboratories.

Enhanced Production of Ectoine From Methane Using Metabolically Engineered Methylomicrobium Alcaliphilum 20Z

Background: Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains.Results: We constructed a genetically tractable M. alcaliphilum 20Z with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, the use of an optimized medium for ectoine production, containing 6% NaCl and 0.05 µM tungsten, gave ectoine yields of up to 142.32 mg/L without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date.Conclusions: Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production.