Ex vivo development of Phasmarhabditis spp. associated with terrestrial molluscs

The success of Phasmarhabditis hermaphrodita (Schneider) Andrássy (Rhabditida: Rhabditidae) as a biological control agent of molluscs has led to a worldwide interest in phasmarhabditids. However, scant information is available on the lifecycle development of species within the genus. In the current study, the development of P. hermaphrodita, Phasmarhabditis papillosa, Phasmarhabditis bohemica and Phasmarhabditis kenyaensis were studied using ex vivo cultures, in order to improve our understanding of their biology. Infective juveniles (IJs) of each species were added to 1 g of defrosted homogenized slug cadavers of Deroceras invadens and the development monitored after inoculated IJ recovery, over a period of eight–ten days. The results demonstrated that P. bohemica had the shortest development cycle and that it was able to produce first-generation IJs after eight days, while P. hermaphrodita, P. papillosa and P. kenyaensis took ten days to form a new cohort of IJs. However, from the perspective of mass rearing, P. hermaphrodita has an advantage over the other species in that it is capable of forming self-fertilizing hermaphrodites, whereas both males and females are required for the reproduction of P. papillosa, P. bohemica and P. kenyaensis. The results of the study contribute to the knowledge of the biology of the genus and will help to establish the in vitro liquid cultures of different species of the genus.

Design and Validation of a Device for Mitigating Fluid Microgravity Effects in Biological Research in Canister Spaceflight Hardware

The major factor influencing the behavior of microbes growing in liquids in space is microgravity. We recently measured the transcriptomic response of the Gram-positive bacterium Bacillus subtilis to the microgravity environment inside the International Space Station (ISS) in spaceflight hardware called Biological Research in Canisters-Petri Dish Fixation Units (BRIC-PDFUs). In two separate experiments in the ISS, dubbed BRIC-21 and BRIC-23, we grew multiple replicates of the same B. subtilis strain in the same hardware, growth medium, and temperature with matching ground control samples (npj Micrograv. 5:1.2019, doi: 10.1038/s41526-018-0061-0). In both experiments we observed similar responses of the transcriptome to spaceflight. However, we also noted that the liquid cultures assumed a different configuration in microgravity (a toroidal shape) compared with the ground control samples (a flat disc shape), leading us to question whether the transcriptome differences we observed were a direct result of microgravity, or a secondary result of the different liquid geometries of the samples affecting, for example, oxygen availability. To mitigate the influence of microgravity on liquid geometry in BRIC canisters, we have designed an insert to replace the standard 60-mm Petri dish in BRIC-PDFU or BRIC-LED sample compartments. In this design, liquid cultures are expected to assume a more disk-like configuration regardless of gravity or its absence. We have: (i) constructed a prototype device by 3D printing; (ii) evaluated different starting materials, treatments, and coatings for their wettability (i.e., hydrophilicity) using contact angle measurements; (iii) confirmed that the device performs as designed by drop-tower testing and; (iv) performed material biocompatibility studies using liquid cultures of Bacillus subtilis and Staphylococcus aureus bacteria. Future microgravity testing of the device in the ISS is planned.

Altered Pseudomonas Strategies to Inhibit Surface Aspergillus Colonies

Pseudomonas aeruginosa and Aspergillus fumigatus infections frequently co-localize in lungs of immunocompromised patients and individuals with cystic fibrosis (CF). The antifungal activity of P. aeruginosa has been described for its filtrates. Pyoverdine and pyocyanin are the principal antifungal P. aeruginosa molecules active against A. fumigatus biofilm metabolism present in iron-limited or iron-replete planktonic P. aeruginosa culture filtrates, respectively. Using various P. aeruginosa laboratory wild-type strains (PA14, PAO1, PAK), we found antifungal activity against Aspergillus colonies on agar. Comparing 36 PA14 and 7 PAO1 mutants, we found that mutants lacking both major siderophores, pyoverdine and pyochelin, display higher antifungal activity on agar than their wild types, while quorum sensing mutants lost antifungal activity. Addition of ferric iron, but not calcium or magnesium, reduced the antifungal effects of P. aeruginosa on agar, whereas iron-poor agar enhanced antifungal effects. Antifungal activity on agar was mediated by PQS and HHQ, via MvfR. Among the MvfR downstream factors, rhamnolipids and elastase were produced in larger quantities by pyoverdine–pyochelin double mutants and showed antifungal activity on agar. In summary, antifungal factors produced by P. aeruginosa on agar differ from those produced by bacteria grown in liquid cultures, are dependent on quorum sensing, and are downregulated by the availability of ferric iron. Rhamnolipids and elastase seem to be major mediators of Pseudomonas’ antifungal activity on a solid surface.

Glycerol Stock Preparation for S. elongatus v1

Bacterial cultures in the form of agar plates or liquid cultures are not suitable for long term storage at low temperatures such as 4 degrees. Long-term storage of bacteria is best at temperatures around -80 degrees. However, agar plates and liquid cultures cannot be stored at such temperatures, as the water crystallizes in and around the bacterial cells, rupturing and killing them. Glycerol or DMSO (anti-freezing agents) is added to the media to store samples at such temperatures.

DMSO stock preparation v1

Bacterial cultures in the form of agar plates or liquid cultures are not suitable for long term storage at low temperatures such as 4oC. Long-term storage of bacteria is best at temperatures around -80oC. However, agar plates and liquid cultures cannot be stored at such temperatures, as the water crystallizes in and around the bacterial cells, rupturing and killing them. Glycerol or DMSO (anti-freezing agents) is added to the media to store samples at such temperatures.

Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria

Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology—including the tendency to clump, and “differential” culturability—and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.

ROLE OF GENE XPERT AND LIQUID CULTURE IN DIAGNOSIS OF EXTRA PULMONAR TUBERCULOSIS

INTRODUCTION: Extrapulmonary Tuberculosis (EPTB) accounts for 15- 25% of all TB cases. It is more difficult to diagnose than Pulmonary tuberculosis and often requires invasive procedures to obtain tissue and or fluid samples. Histology is time-consuming and establishing a diagnosis of TB with high specificity remains difficult. Tissue smear microscopy after special staining is often negative. Tissue culture often leads to considerable delays compromising patient care and outcomes. AIMS AND OBJECTIVES:1. To diagnose Extra Pulmonary Tuberculosis by Gene Xpert(Xpert MTB/Rif assay or CBNAAT) and Liquid Cultures. 2. To evaluate the Sensitivity and Specificity of Gene Xpert in Extra Pulmonary Tuberculosis in comparison with Liquid Culture MGIT960 system. MATERIALS AND METHODS: This retrospective cross-sectional study was carried out by reviewing all suspected extra pulmonary tuberculosis samples of 430 patients attending OPD at Institute of Respiratory Diseases, Jaipur from April 2020 to March 2021.The extrapulmonary samples (pleural fluid,CSF,pus,BAL,Ascitic fluid,Synovial fluid,Gastric aspirate,Liver aspirate) were subjected to GeneXpert and Liquid culture MGIT960 system. RESULTS: Of the 430 Extra Pulmonary Samples, The Sensitivity and Specificity of CBNAAT was 79.77% and 95.30% respectively in comparison with Liquid Culture. Out of the 430 Samples CBNAAT was Positive in 87 samples of which 71(81.60%) were Rifampicin sensitive and 16(18.39%) were Rifampicin Resistant.Out of the 430 Samples,Liquid cultures was Positive in 89 samples. CONCLUSION: Gene Xpert has a notable advantage of detecting tuberculosis within two hours which is acceptable to all clinicians to institute early treatment.CBNAAT is one of the rapid diagnostic tests available in the country and it should be routinely used under the public and private health sector effectively to detect early tuberculosis in Extra Pulmonary Samples.

Red/far-red light signals regulate the activity of the carbon-concentrating mechanism in cyanobacteria

Desiccation-tolerant cyanobacteria can survive frequent hydration/dehydration cycles likely affecting inorganic carbon (Ci) levels. It was recently shown that red/far-red light serves as signal-preparing cells toward dehydration. Here, the effects of desiccation on Ci assimilation by Leptolyngbya ohadii isolated from Israel’s Negev desert were investigated. Metabolomic investigations indicated a decline in ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation activity, and this was accelerated by far-red light. Far-red light negatively affected the Ci affinity of L. ohadii during desiccation and in liquid cultures. Similar effects were evident in the non–desiccation-tolerant cyanobacterium Synechocystis. The Synechocystis Δcph1 mutant lacking the major phytochrome exhibited reduced photosynthetic Ci affinity when exposed to far-red light, whereas the mutant ΔsbtB lacking a Ci uptake inhibitory protein lost the far-red light inhibition. Collectively, these results suggest that red/far-red light perception likely via phytochromes regulates Ci uptake by cyanobacteria and that this mechanism contributes to desiccation tolerance in strains such as L. ohadii.