Bacterial communities associated with the biofilm formation in Pangong Tso Lake

Pangong Tso Lake is situated in the Himalayan Plateau on both sides of India/China border. This high-altitude lake has an oligotrophic environment with extremes of temperature and exposure to UV radiation. The water of the Pangong Tso is generally very clear. The sediments, including the pebbles and small rocks, did not show any biofilm or microbial mats formation in the past.  However, human activities have increased tremendously near this lake, which might lead to disturbance of this lake ecosystem.  The presence of biofilms in a small area near the shore of the Pangong Tso next to the Maan village was observed by researchers.

Pangong Tso lake

Researchers at NCMR-NCCS Pune were curious to understand the bacterial communities associated with the Pangong Tso lake sediment, water and biofilms. Researchers also studied the metabolic potential of the bacterial community. They used amplicon sequencing of the particular region of 16S rRNA gene and other different tools for this study.  Based on the previous findings on biofilm bacterial communities, researchers hypothesized that the biofilm bacterial communities at Pangong Tso Lake consist of phototrophs and chemotrophs. They also hypothesized that the diversity of the biofilms community is different from suspended water and sediments, where biofilm formation was not observed.

Researchers collected sediment and microbial biofilms sample from the Pangong Tso Lake. Analysis of physio-chemical parameters of water was done. The Calcium and Magnesium chloride contents of water were analyzed .The dissolved Chloride content of water, Sulphate concentration, Nitrate nitrogen and Ammonium nitrogen estimation was done using different techniques. DNA extraction was done from water, sediment and microbial biofilm samples. Different bioinformatics and statistic tools were used in the study. The metabolic potential of the microbial community was predicted using functional prediction tool Tax4Fun and the relative abundance of highly abundant genes involved in different functions were compared across the biofilm, sediment and the water sample.

Overall a total of 6,682,012 raw sequences were generated in the study. Proteobacteria was the most dominant and diverse phylum followed by Bacteroidetes, Acidobacteria, Planctomycetes, Actinobacteria, Firmicutes, Verrucomicrobia, Chlorofexi and Gemmatimonadetes. Significant differences were observed in the microbial diversity of water with sediment and microbial biofilm samples. The water sample was least diverse in comparison to the microbial biofilm and sediment samples. Among the top 50 bacterial genera, which constitutes about 50% of the entire microbiome, Loktanella was highly abundant in the water sample, Rhizobium in sediment samples, and Planktosalinus and Aliidomarina were in biofilm samples. The relative abundance of Proteobacteria was the highest in the water. A sharp decline was observed in the relative abundance of Proteobacteria in sediment and biofilm samples.

Loktanella constitutes nearly half of the total bacterial communities in the water sample, while Loktanella represented less than 1% in the biofilm and sediment samples. Differences were observed in the relative abundance of bacterial taxa across the biofilm and the sediment samples at the phylum and genus based on the Welch t test. Bacterial phyla Verrucomicrobia, Deinococcus-Thermus and Cyanobacteria were explicitly enriched in the biofilm samples. The abundance of Planktosalinus, Aliidiomarina, Halomonas, Predibacter, Paracoccus, and Hyphomonas was significantly high in the microbial mat, whereas Enterobacter and Mesorhizobium were highly abundant in the sediment samples. In addition to this higher abundance of Flavobacterium, Pseudomonas, Luteolibacter, Dyadobacter, Chryseobacterium, Halomonas, Stenotrophomonas, Hyphomonas, Enterobacter, Peredibacter, Acinetobacter, Arenibacter and Exiguobacterium was also recorded across the samples.

A total of 49 pathways were highly abundant, with more than 0.5% mean relative abundance. The pathways related to different functions like peptidases, porphyrin and chlorophyll metabolism, glycoxylate and dicarboxylate metabolism, chaperones and folding catalysts, DNA repair and recombination proteins, pyruvate metabolism, nitrogen metabolism, propanoate metabolism, cysteine and methionine metabolism, butanoate metabolism, transcription machinery, prokaryotic defense system, alanine, aspartate and glutamate metabolism, and homologous recombination were highly abundant in the biofilm samples.

The less diverse bacterial communities in microbial biofilm in comparison to sediments indicated the enrichment of a specific group of bacteria. Stratification of Cyanobacteria (primary producer), sulfate-reducing/ oxidizing bacteria and anoxygenic phototrophic bacteria in the hypersaline microbial mat, took place according to the micro-gradient of oxygen, sulfide, and light which selectively allows the specific bacteria to colonize. The higher abundance of Cyanobacteria in the biofilm samples in comparison to sediment and water sample supported the hypothesis on the establishment of primary producers in the biofilm samples. Sediment samples were the most diverse in comparison to water and microbial biofilm samples, which represents both rare and abundant taxa in the sample. The less diverse bacterial communities in microbial biofilm in comparison to sediments indicated the enrichment of a specific group of bacteria.

To conclude, significant differences were observed in the bacterial diversity in the lake water, sediment, and microbial biofilm samples. Enrichment of specific phyla like Verrucomicrobia, Deinococcus-Thermus, and Cyanobacteria in the microbial biofilm samples indicated the development of saprophytic and photosynthetic communities, which is an important succession event in this high-altitude lake. The predictive analysis of potential functions of these communities also supported the observation as the genes involved in porphyrin and chlorophyll metabolism, glyoxylate and dicarboxylate metabolism, DNA repair and recombination proteins were enriched in the microbial biofilm samples.


MALDI-TOF Mass Spectrophotometry (MS): Emerging technique in Microbial Ecology

-By Kranti Karande

Microorganisms have been identified by their biochemical properties and using techniques like 16S rRNA sequencing for more than a century. Recently, MALDI-TOF Mass Spectrometry (MS) has become a key technique for microbial identification due to its rapid and reliable performance.

MALDI-TOF MS facility at National Centre for Microbial Resource, National Centre for Cell Science Pune

Mass Spectrometry is an important tool for the detection of atoms or molecules in a chemical complex. A mass spectrometer works by turning atoms into ions. Ions get separated when they are passed through electric field and magnetic field respectively. A spectrum is generated determining types of atoms present in the sample.

MALDI-TOF MS technique has a variety of applications in biology including intact mass determination, peptide mas fingerprinting, MALDI imaging. This method is widely used for the identification of DNA and proteins, but recent advances in this technique have incorporated use in microbiology for pathogen detection, which is contributing immensely to research in microbiology.

Dr. Praveen Rahi from NCMR-NCCS Pune and Dr. Parag Vaishampayan from the California Institute of Technology, USA, recently published an editorial detailing the recent advancements and challenges in MALDI-TOF MS for Microbial Ecology applications.

This technique is in the early stage of development for the direct identification of microorganisms in positive blood cultures, detection of drug resistance factors and for bacterial function assessment. Recently, researchers identified Brucella infection from positive blood culture using this technique which is a great breakthrough in MALDI-TOF MS application. Researchers are also trying to develop this technique for studying antimicrobial resistance in bacteria and fungi.

In one of the recent publications, data from MALDI-TOF spectra of intact protein and specialized metabolite spectra from bacterial cells grown on agar were combined to study the link between microbial identities and their potential environmental functions.

This technique is becoming a fast and effective method for filtering out same species strains and genetically identical clones. Until now, identification of same species strains was done with the help of techniques like dendrograms. Now MALDI-TOF MS is used for identifying identical strains as it provides accurate results with rapid analysis.

With the help of MALDI-TOF MS technique medical researchers revealed a potential clonal route of transmission of Enterobacter spp. This research is a significant source of information on the spread of Enterobacter, an evolving pathogen for its ability to acquire antimicrobial resistance factors and the role of MALDI-TOF MS in microbiological surveillance of diseases.

Dr. Praveen Rahi

While discussing about MALDI-TOF MS applications with Dr. Praveen Rahi, he commented that, “MALDI-TOF MS will be ‘new microscope’ for microbiologists in the era of ‘culturomics’, which include large-scale cultivation of microorganisms and require high-throughput identification tools. Right now the commercial MALDI-TOF MS spectral databases primarily include profiles of microorganisms of clinical relevance, and researchers across the globe are making efforts to generate spectral profiles for microbes according to their research interests. However, most of these efforts are being made in isolations, which might lead to erroneous database entries. There is urgent need to develop universal SOPs for MALDI-TOF MS profile generation, and subsequent curation and validation of newly generated database profiles.”

Though this technique has emerged as a key player in microbial identification and microbial ecology research, but there are many fine tuning steps required to improve this technique.

One study noted that only 35% of species level identifications done using MALDI Biotyper correlated with those detected by 16S rRNA gene sequences, suggesting a poor species level detection by this technique. Insufficient coverage of bacterial species in the repositories was proposed as the primary reason for this difference. The lack of a public repository to send new spectral references created by researchers further worsens this issue. The major challenge is to find the solution for same.

During the identification of the collection of microorganisms isolated from cell phone surfaces, researchers observed that the efficiency of MALDI-TOF MS spectrum filtering can be achieved by improving the methods of sample processing and enriching the spectral database leading to better and highly reliable results for MALDI-TOF MS microbial identification.

If we find feasible solutions for the challenges in this technique, this technique has the potential to make an immense contribution to the field of microbial ecology.

NCMR-NCCS Pune provide services for microbial identification using MALDI-TOF MS.