Abstract
Public swimming areas such as beaches can be full of potentially harmful microorganisms, which may then classify the water as unsafe to swim in. In this study we took samples from Coogee Bay Beach and further studied them using both culture based and non-culture based testing methods. Using Illumina sequencing we identified a huge number of different species of bacteria, the majority of these being Proteobacteria, Bacteriodete, cyanobacteria and Actinobacteria. Whereas through Sanger sequencing we were able to identify a much microorganisms on a much smaller level. Through further culture based tests such as biochemical testing which included pure culturing, gram staining, oxidase texting, glucose fermentation, VP and MR testing as well as a motility test we were able to identify a single microorganism based on these tests. However results from culture and non-culture tests showed inconsistency with each other. We found evidence that Coogee Bay Beach contains infectious disease causing microorganisms, which could be potentially harmful.
Introduction
Coogee Beach is approximately 3km from the UNSW campus, which makes it a very popular destination for myself as well as many of my friends to visit. We took our sample from the water at Coogee Bay Beach, as we were interested in discovering mainly whether or not the water could really be considered “safe” for the general public to swim in. Hundreds of people from both Australia as well as overseas visit and swim this beautiful beach each week without even knowing if there are any detrimental implications on their health due to what actually is “in” the water they are swimming in.
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An article written in 2016 in The Sydney Morning Herald, noted that in the most recent State of the Beaches Annual Report 2016 Sydney Region report; the quality of the water at Coogee Beach had substantially worsened over the year of 2016 [10], Hannam. This was seen mainly when the rainfall exceeded more than 5 millimeters of rain; samples, which were taken in dry whether showed much safer, water conditions than that of when there had been heavy rain [5], Botero. Coogee was then ranked in the top 10 for Sydney’s poorest water sites. What was found in the water back in 2016 mainly consisted of faecal matter, both from humans and animals. Further reports have shown that the pollution of recreational water with faecal matter can lead to multiple different health problems due to the presence of infectious microorganisms within it. Microorganisms which have been found in beach samples include organisms such as Staphylococcus, Pseudomonas aeruginosa, Vibrio Spp., Enteric bacteria [1], Gage. With some of these microorganisms being harmful it means they may potentially lead to some health issues as a result of being exposed to them. Examples of some health issues which could occur from this water pollution includes gastrointestinal infections derived from pathogenic organisms as well as infections of the upper respiratory tract, ears, eyes, nasal cavity and skin [5], Botero.
The main purpose of this study is to find out whether the water quality of Coogee beach has either improved or further deteriorated since 2016. Also to see what type of microbes will be found in the water in 2019 compared to that of 2016 and if they are, which ones can be detrimental to an individual’s health. My team and I predict that by swimming in the water there is the potential for an individual to be exposed to or become at higher risks to health.
Materials and Methods
First of all we obtained a 600ml sample of water from Coogee Bay Beach, once isolates were obtained after a growing a sample of the water on LB20 agar plates and further pure culturing out our chosen microorganism. From our final pure sample my group and I performed a series of biochemical tests as well as non-culture tests as described in the MICRO2011 Laboratory manual 2019. These enabled us to identify what the pure culture was specifically which we had plated.
Biochemical testing
Over the time of the span of the course our specific isolate was sub-cultured onto LB20 agar plates in order to produce a pure colony. In table 1 the results of the biochemical testing performed on this pure culture are displayed. Regarding the gram staining, the testing of the isolated bacteria gave the result of gram-negative rods. This indicated that the isolated bacteria could have been one of the following Pseudomonas spp., Aeromonas spp., Vibro spp., A. hydrophilia or A. veronii. The oxidase test results showed an almost immediate change of colour on the filter paper, this reflected a positive test outcome. A negative result in the Glucose fermentation test which saw that no bubbles formed in the sample jar then confirmed that the specific microorganism that we had been culturing was part of the Pseudomonas spp. Further testing was performed following this in order to further justify that the bacteria was infact Pseudomonas spp. These included a motility test which showed a negative result, which disproves that the bacteria is a part of the Pseudomonas spp due to research showing that the Pseudomonas spp is a motile species [3], Siegrist. However our MP and VP testing showed a negative result which gives further evidence for the pure cultured bacteria to in fact be part of the Pseudomonas spp.
Discussion
Beaches are such a huge part of Australian culture, with many residents visiting and swimming in beaches all year round. With statistics showing that some beaches in Sydney have around 1 million visitors each year [4]. Many of these people swim in the beach water not fully knowing what it is really that they are swimming in. This is what my group and I aimed to find out, that being whether or not the microorganism, which we decided to pure culture, can be detrimental or not to ones health if they are exposed to it when swimming at the beach. From reports written on the water quality of Coogee Bay Beach in 2015-2016 that Coogee Beach along with Malabar Beach had been moved from a once “good” rating from 2014-2015 to a now “poor” rating [5], Botero. In saying this the samples taken from the beach only showed that in 77% of these, bacteria levels were within a limit, which showed that it was still safe to swim in [5], Botero. Most of the samples taken in this study showed that when the weather was dryer the water was safer to swim in. When there were increased rainfall levels there were increased Enterococci levels thus showing that the water would become less safe to swim in at these times of rainfall. Enterococci are organisms, which grow in high salt concentrations, hence why they are prominent in the ocean water. It is also said that they may cause clinical infections in humans [6], Fraser, which is why it could be harmful if increased levels are found in Coogee Bay Beach Water samples.
In order to see if the beach water at Coogee Bay had improved, worsened or remained the same my group and I went about culturing an organism taken from a sample of water taken from Coogee bay Beach to see if what we found was harmful or not. In our biochemical results we came to the conclusions that the type of microorganism, which we found, was part of the Pseudomona spp. Studies show that this organisms is a type of free living bacteria and can be found in natural waters [7], Mena. The particular strand of Pseudomona which is found manly in beaches is called P. aeruginosa and is known to be that cause of a wide spread of infections as well as being one of the leading cause of illness in individuals which have impaired immune systems [7], Mena. A few examples of what this bacteria is said to cause are illnesses such as endocarditis, pneumonia, urinary tract infections, meningitis and also is a leading cause of septicemia. The growth of this organism is favoured in warmer conditions, and outbreaks of folliculitis and ear infections have been reported from water slides and swimming pools [7], Mena. From reading previous reports regarding the water quality of Coogee Bay beach none of them suggested that this was a bacteria, which was found when grading the quality level of the water. The results seen here regarding the biochemical tests suggest that my group and my previous enquiries surrounding the quality of the beach water in 2019 as opposed to 2015-2016 can be said that this sample taken would be considered “poor” water quality due to the large number of infections which are a result of being exposed to the particular strand of Psedomona spp. That is most likely the one in which we believed we may have found in our sample.
Within our biochemical tests it showed that we had found Psedomona spp, however when we received the results of our Sanger Sequencing we were shocked to find that it had a different result to that of our culture testing. Our Sanger sequencing showed us that our pure cultured sample was in fact part of the Sphingomonas spp. Initially after finding this out we thought that we must have made mistakes in our biochemical testing on the basis that Sanger Sequencing is a much more accurate identification method when it comes to microbiology. However after further research and discussion we discovered that several strains of Pseudomonas have been reclassified as strains of Sphingomonas. This could be a result of previous unknown genetic relationships emerging through DNA sequencing. It is said that the genus of Sphingomonas contains around 12 species; one of these, which is widely found and distributed in both soil and water, is Pseudomonas paucimobilis [8]. This strand of Pseudomonas is contradictory to the above article, which speaks about the typical strand of this species found in water environments being P. aeruginosa. This being the results from the Sanger Sequencing however makes it a much more reliable test and source to make a conclusion from. The bacteria known as Pseudomonas paucimobili,s is a gram-negative bacillus [9], Göker, according to a report published by J Korean Neurosurgery Soc. It has been reported that this bacteria has caused a variety of infections, some of these being meningitis, septic arthritis, peritonitis, sepsis as well as septic pulmonary embolism. Thus further consolidating our previous thoughts that the water at Coogee Bay Beach is not as safe as people may think it is when it comes to swimming in it.
Analysis of Testing Methods
When it came to the various testing methods which were used when carrying out this research project it became clear that the non-culture test results were much more accurate than that of the culture test method results. The culture test methods, which included the biochemical testing, saw that there was defiantly room for human error. This was shown particularly when we got our results for our motility test back. When reading other reports it is said that the species of bacteria we had found was a predominantly motile organism, however as it shows in Table 1 the result for our motility test came in as negative. This means that there was most likely human error when performing our testing; it is also possible that there was outside contaminates, which may have gotten into the test. The other biochemical tests performed proved to be helpful in determining what the pure isolate was, however they could have been improved by repeating them more that once each time to improve the overall reliability of the experiment as a whole. The non-culture tests proved to be much more effective than the culture tests. There was not any risk of human error, which was one of the main issues when it came to biochemical testing. The Sanger Sequencing technologies proved to be fast as well as much more effective in the case that it has a lower number of targets making it easier to discover what our pure isolate was. In comparison to that of the Illumina sequencing which was better used for a large number of targets.
Through both biochemical testing as well as phylogenic analysis my group and I concluded that the final identification of the pure isolate was part of the Sphingomonas spp, more accurately and also referred to as Pseudomonas paucimobilis. As a result of this study we think that this could potentially pose risks to individuals who are regularly swimming in the water at Coogee Beach as well immunocompromised individuals. This shows us that since the reports written on the water quality of Coogee Bay Beach performed on samples taken in 2015-2016 this gives the impression that the water quality could still be considered as “poor” quality, however in a different sense than the previous studies.
Conclusion
In conclusion, the investigation of the water quality of one of Sydney’s more popular swimming destinations has revealed biochemical evidence of various different microbes. One in particular, which my group and I found the pure isolate of we also found, may pose risks to individuals, which have compromised immune systems. We decided that there should be regular samples taken of the water and processes put in place in order to prevent these levels from getting too high.
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