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Extraction And Isolation Of Bacteria

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Abstract

Water is known to be a popular habitat for many pathogens which pose severe risks to surrounding communities. In this study, a sample of water from a Sydney creek was analysed using culture-dependent and culture-independent methods. The use of metagenomics targeting the 16s SSU rRNA revealed the presence of over 90,479 bacterial species in our sample, including potentially pathogenic Staphylococcus, Clostridium and Legionella. Further analysis was conducted by culturing our sample until we produced a pure isolate colony. Using this isolate, various biochemical tests were performed and our culture was discovered to be Micrococcus luteus. Though the Micrococcus family isn’t known widely as being dangerous, Micrococcus luteus has been proven to be an opportunistic pathogen. Current research is noticeably conducted in largely clinical setting, and having proven its presence in a local water source, this study aims to signify the importance of understanding the danger such bacteria poses on local communities, and the necessity of continually measuring water quality.

Introduction

Water is a great environment for microbial growth, and has been known as harbouring various deadly microbes which cause diseases such as cholera, dysentery and typhoid fever. Even in developed countries such as the United States of America, it has been reported that every year “560,000 people suffer from severe water borne diseases, and 7.1 million suffer from mild to moderate infections”, the result of which is an “estimated 12,000 deaths a year” [1]. This exemplifies the importance of continually measuring the levels of bacteria in water, as well as the methods currently in place to prevent the transmission of microbial pathogens to ensure that the health of surrounding communities are not compromised. The water sample tested was taken from a stagnant pool of water on a bed of soil at Cattai Creek in Kellyville, and was particularly interesting due to it being downstream from a sewerage disinfection plant. Cattai Creek is a largely rural water body located in east New South Wales, and is surrounded by an increasingly urbanised area. It is a vital water source for the community, and leads into the Hawkesbury-Nepean river which supplies 97% of the fresh drinking water for more than 4.8 million people living in and around Sydney [2]. Knowing this, it is worrying that water quality at Cattai Creek has been continuously degraded with a 115% rise in nitrogen levels every year since 1992 [3]. Having seen the issues caused by insufficient water filtration methods in the 1998 Sydney Water crisis, which involved the release of unsafe levels of Cryptosporidium and Giardia into Metropolitan Sydney’s water supply [4], it was of particular interest whether or not the water sample would contain microbes such as Salmonella, Shigella and E. coli which are typically found in sewerage samples.

In order to successfully isolate and identify a microorganism present in the sample, it was decided that a variety of techniques that encapsulated both culture-dependent and culture-independent methods was the best approach. It is well known that partnering traditional processes “more intimately with informatics” can lead to an “increase in the accuracy, timeliness, and completeness of microbiology testing while decreasing the laboratory workload” [5}. The culture-dependent methods included biochemical tests and Sanger sequencing coupled alongside phylogenetic analysis, whereas the culture-independent method was the new-age Illumina sequencing of the 16s SSU rRNA. In using traditional microbiological methods and new sequencing methods, it was visible that the traditional culture-dependent methods provided results almost instantly in a majority of cases (the biochemical tests), whereas the Illumina sequencing required significant time to be put in in order to produce meaningful analysis. In the end however, results from both were combined as a means of identifying the bacteria present in the environmental microbiome, and in the subculture.

Micrococcus luteus is a gram-positive cocci belonging to the family Micrococcaceaea. It was first discovered by Alexander Fleming is 1928 and is often found in soil and water, but has also been isolated from various foods such as cheese and milk. Current knowledge dictates that is an opportunistic pathogen responsible for nosocomial infections, and may also be responsible for septic shock in immunocompromised patients [6]. Considering the environment the sample was taken from, it is not surprising that Micrococcus luteus was present, but it’s presence should still be taken into consideration now that it is clearly present in local environments outside of clinical settings, which is where most studies concerning it are produced.

Materials and methods

The environmental sample was retrieved aseptically using a water bottle and was kept at room temperature. Once brought into the lab, it was filtered and inoculated as per the MICRO2011 lab manual [7].

The sample was inoculated aseptically onto 2 agar plates (Luria agar and M9), and the culture obtained incubated and isolated as per the MICRO2011 lab manual [7]. Once a pure culture was produced, biochemical tests, gram staining and microscopy, and Sanger sequencing took place as outlined in the MICRO2011 lab manual [7].

Having being filtered, the sample was treated as per the MICRO2011 lab manual [7] and the culture-independent method conducted.

Culture-dependent

Biochemical tests included catalase test, mannitol fermentation and glucose fermentation. These were used to determine the characteristics of the microbial metabolism.

Sanger sequencing uses the 16s SSU rRNA from the pure culture isolate to classify organisms taxonomically. The DNA was extracted and purified as instructed in the MICRO2011 lab manual [7], before undergoing PCR amplification and being sent to the Ramaciotti Centre for Genomics for further analysis.

Bioinformatics analysis: The results given to us from the Ramaciotti Centre’s analysis were used to find similar sequences through the NCBI Basic Local Alignment Search Tool (BLAST). These sequences were then combined to form a phylogenetic tree using the MEGA 7 (Molecular Environmental Genetic Analysis) program.

Cultivation-independent

DNA extraction and Illumina sequencing: This method involves sequencing the entire environmental microbiome. The sample was collected and prepared as per the MICRO2011 lab manual [7], and the DNA extract sent to the Ramaciotti Centre for Genomics for further analysis. The results were provided to us through Krona’s metagenomic visualisations.

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Results

Culture-dependent methods

Biochemical tests: Throughout the entirety of the project, our sample was sub-cultured onto Luria Broth agar plates to produce a pure isolate. Once obtained, this pure isolate underwent various biochemical tests as per the flowchart on page 131 of the MICRO2011 lab manual [7]. The results for these biochemical tests are summarised on Table 1 and Figure 1.

Sanger sequencing – phylogenetic testing: Using the computer system MEGA 7, our Sanger sequencing results for the V3-V5 regions of the 16s SSU rRNA were analysed. Using the NCBI Basic Local Alignment Search Tool (BLAST), our sequence was compared to those of other closely related and more distantly related organisms, allowing us to create a phylogenetic tree demonstrating the relationships between the taxa. The phylogenetic tree constructed can be seen in Figure 2, and the percentage identifies and E-values of the sequences in Table 2.

Culture-independent methods

Environmental microbiome analysis using Illumina: The data received from the Ramaciotti Centre for Genomics was presented to us in the form of two KRONA pie graph’s (Figure 3 and Figure 4 respectively) which identified that our sample contained 90479 bacteria across 4120 species.

Discussion

Water is the most important chemical in the world, and is necessary to support the existence of all life forms. This is not limited to just humans and other larger animals, but also a significant variety of micro-organisms, many of which are highly pathogenic. This paper aimed to understand the composition of an environmental microbiome of a water sample. This was done using both culture-dependent and culture-independent methods, allowing for a characterisation the microbiome whilst also extracting and isolating a single bacterium. In doing so, it was possible to determine the quality of the water sample, and the necessity of ensuring the maintained quality of local water bodies.

This study was conducted through a combination of biochemical tests, phylogenetic analysis using Sanger sequencing and a combination of MEGA 7 and the NCBI BLAST, as well as Illumina sequencing. Using both culture-dependent and culture-independent methods, It was possible to successfully isolate and identify the Actinobacteria Micrococcus luteus. In doing so, it was determined to be a gram-positive cocci bacteria with circular yellow colonies. The biochemical tests also determined its ability to produce catalase, and inability to ferment mannitol and glucose as noted in Table 1.

Although Micrococcus luteus Is often found in soil and water [8], it’s presence in a local water sample should still be a cause for concern. Past studies conducted have indicated the potential for Micrococcus luteus to act as an opportunistic pathogen, causing septic shock in immunocompromised hosts, while also causing skin and nosocomial infections [8]. However, many of the studies on Micrococcus luteus have only addressed it’s risk in a clinical setting, and thus have failed to address the danger it poses in residential communities. The most recent study that aimed to address this was conducted by Kooken et.al in 2011, yet this study was only able to confirm its presence in indoor air samples [9]. Knowing the danger that Micrococcus luteus poses, the identification of it in a heavily relied upon water sample should cement the importance of both conducting more studies on Micrococcus luteus in non-clinical settings, while also ensuring that maintaining water quality in the environment is a priority.

The biochemical tests used were mostly useful due to their convenience and inexpensive nature. The catalase and gram staining tests produced results during the lab, and the mannitol and glucose fermentation only took one week. These cultivation-based methods were also useful as they produced results that were clearly visible and could be used to complete the identification flowchart provided in the MICRO2011 lab manual [7]. Although there was success in the use of agar plates with varying compositions, such as a custom Luria broth with 0.2 g of tryptone, 0.15 g yeast extract and 0.2 g sodium, as well as a pre-prepared plate of M9 agar (minimal salt, with glucose as a carbon and energy source), studies have shown other growth mediums can be more effective in allowing novel bacteria from samples to grow in lab petri dishes. A study conducted by Takami et.al in 2005 showed that novel bacterial species affiliated with Actinobacteria can be successfully cultivated using gellan gum as opposed to regular agar plates [10]. Since Micrococcaceaea only made up 0.08% of the total bacterial diversity, it was quite lucky that Micrococcus luteus was isolated from the sample. However, for future studies, the use of gellan gum would increase the chances of ensuring the bacteria isolated is from a group with a small percentage of representation in the sample.

The phylogenetic analysis conducting using Sanger sequencing demonstrated that Micrococcus luteus is quite closely related to various strains of Staphylococcus, namely Staphylococcus epidermis and Staphylococcus capitis as shown in Table 2. Both of these Staphylococcus genera are part of the normal flora of the human scalp, face, neck and ears [11], and so it is not surprising they are closely related to Micrococcus luteus which is also found on human skin. However, the attempt at Sanger sequencing did not go entirely according to plan as our DNA extract was unfortunately misplaced. This meant that the Micrococcus luteus sequence had to be input directly from the NCBI database, and so the “lab-sequenced gene” present in the phylogenetic tree was discovered to have also been Staphylococcus capitis. This makes the tree produced rather confusing as it places Micrococcus luteus in-between different Staphylococcus genera, which should not be the case. For future studies, extra care would be taken to ensure the DNA extract is not lost in order to produce a fully correct tree.

The results provided from Illumina sequencing confirmed the presence of Micrococcus in our sample, while also indicating the presence of several other potentially dangerous bacterial species.. A search revealed the presence of Staphylococcus, Listeria and Legionella. The presence of Staphylococcus would typically raise a cause for concern, however as the phylogenetic analysis indicated, the Staphylococcae present would most likely be strains such as Staphylococcus capitis and Staphylococcus epidermis which are not particularly dangerous. On the other hand, the presence of Listeria and Legionella, which are highly pathogenic and cause Listeria and Legionnaire’s disease respectively is definitely worrying. The presence of Legionella is particularly worrying as it is typically found in faeces, and the water sample was in close proximity to a sewerage disinfection plant. This hints that the current measures used at that facility are not working as well as they should be. The Illumina sequencing has no doubt produced vital information about the environment microbiome in detail that culture-dependent techniques would not be able to. The only issues with this technique is the time taken to obtain results and the need to send the sample to a centre in order to conduct the analysis.

Although it proved successful in fulfilling the aim, various limitations constrained the breadth of the investigation. Isolating a single colony from the sample did provide some meaningful information about its degrading quality, yet this would have been more reliable and accurate if many different colonies were isolated. The environmental microbiome was shown in Figure 6 to have been incredibly vast, and being able to isolate any of the other bacterium present would provide further insight into the quality of the sample and water body. This was largely unable to occur due to the time constraints. The labs were conducted once a week over three hours which was enough to complete the investigation, but did create pressure to finish on time, especially when roadblocks such as losing our DNA extract occurred.

For the future, dedicating more time to the investigation would ensure that it is conducted to the highest standard, and would prevent issues such as the misplacement of the DNA extract mentioned above. Furthermore, using water from more than just one area would provide a stronger understanding about the need to increase water quality measures in Sydney, especially if those other areas are also found to contain pathogenic bacteria. Additionally, identifying more than a single, isolated colony would provide exceedingly valuable information about the quality of the water sample and the other bacterium present.

In conducting this study, the microbiome of an environment from a water sample has been characterised using various culture-dependent and culture-independent techniques, and the presence of potentially harmful Micrococcus luteus made clear. As a consequence, the necessity of maintaining the quality of local water samples including Cattai Creek in Sydney is made exceptionally clear due to the risk posed by such bacteria on communities that surround and rely on the water body as a source.

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Extraction And Isolation Of Bacteria. (2022, February 21). Edubirdie. Retrieved November 27, 2022, from https://edubirdie.com/examples/extraction-and-isolation-of-bacteria/
“Extraction And Isolation Of Bacteria.” Edubirdie, 21 Feb. 2022, edubirdie.com/examples/extraction-and-isolation-of-bacteria/
Extraction And Isolation Of Bacteria. [online]. Available at: <https://edubirdie.com/examples/extraction-and-isolation-of-bacteria/> [Accessed 27 Nov. 2022].
Extraction And Isolation Of Bacteria [Internet]. Edubirdie. 2022 Feb 21 [cited 2022 Nov 27]. Available from: https://edubirdie.com/examples/extraction-and-isolation-of-bacteria/
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