The Impact of pH and Atmosphere on the Growth and Antibiotic Resistance of Pseudomonas fluorescens

Abstract

This investigation seeks to explore the impact of pH and atmospheric oxygen concentration on the growth and antibiotic resistance of Pseudomonas fluorescens. This research was conducted in order to further understand the impact of bacterial infections on patients with cystic fibrosis (CF), as CF lungs provide a slightly acidic environment that is easily colonised by the opportunistic P. aeruginosa.  P. fluorescens was used as a microbial surrogate for P. aeruginosa due to its pathogenic nature. Better understanding the effects of pH and atmospheric oxygen concentration on the growth of P. fluorescens will facilitate discussion in regards to treating P. aeruginosa in the CF lung. For the first experiment, Kings B broths at different pH levels were inoculated and incubated under different oxygen conditions, and growth was measured by the turbidity of broth cultures. To assess antibiotic resistance, the second experiment consisted of lawn plates with antibiotic disks on Kings B agar at different pH levels and oxygen conditions. Results illustrate that P. fluorescens grows best at a pH of 7 and is most susceptible to common broad-spectrum antibiotics at a pH of 7. Resistance was similar at a pH of 6 and a pH of 6.5, although it varied between antibiotics. Further research on P. aeruginosa itself is required to confirm deductions from the research, as well as to investigate a broader range of oxygen concentrations that more accurately represent the environment of the CF lung.

Introduction

Cystic Fibrosis

Cystic fibrosis (CF) is a non-infectious hereditary disease caused by a mutation to chromosome 7 of the CFTR gene. 1 in 25 Australians carry a single working copy of the gene with this mutation. However, if they do not have two copies of the mutation, they are asymptomatic and are known as carriers. Consequently, there are only roughly 70 000 – 100 000 people living with the disease^[1], and around 3% of the population are carriers.^[1] CF causes various symptoms, including a build-up of sticky mucus in the digestive tract and lungs^[2] and respiratory complications. This results in an average life expectancy of 35-40 years in developed countries, although it can be as low as 15 years in developing countries. 

Pseudomonas aeruginosa

Generally, fatalities due to CF result from external factors such as pneumonia or a Pseudomonas aeruginosa infection, with 60% of adults with CF also having a P. aeruginosa infection.^[3] P. aeruginosa is an opportunistic pathogen^[4] – meaning it becomes a problematic infection once the host is immunocompromised – that always resides in water and soil; however, it thrives in CF lungs due to the slightly acidic and reduced oxygen environment. Additionally, P. aeruginosa has developed resistance to common antibiotics^[5][6] and is able to create a biofilm that increases its resistance to broad-spectrum antibiotics.^[7]

Pseudomonas fluorescens

For this investigation, P. fluorescens was used as a microbial surrogate – used in place of the target microorganisms for safety or accessibility reasons – as P. aeruginosa is a pathogen presenting an inappropriate risk due to its identity as a known fatal pathogen with a biosafety level 2 warning.^[8] The pH of the lungs is slightly acidic due to the high concentration of CO₂ that is present in exhaled air. This CO₂ dissolves in the mucus, creating carbonic acid, produced as a result of CF to create the acidic environment favoured by P. aeruginosa.

As such, not only is the pH of the media –  as controlled by another acid – being investigated, but the impact of the oxygen concentration (and consequently the CO₂ concentration) on the growth of P. fluorescens is also a variable in this investigation.

Pyoverdine

Additionally, P. fluorescens produces a fluorescent siderophore called pyoverdine.^[2] Siderophores are compounds that bind with iron and move it through cellular membranes in microorganisms, and this pyoverdine is essential to the survival of the microorganism. Additionally, the amount of pyoverdine present, as evident by the level of fluorescence, provides a qualitative measure of microbial growth. Qualitative measures of luminosity produced from P. fluorescens have been used as secondary measures of growth for this investigation.

Relevance to current science

This research is building off significant research that has occurred in the area over decades and will be useful to medical professionals, as it will provide insight as to how pH affects the growth and antibiotic resistance of P. fluorescens, potentially aiding the development of pharmaceuticals that target the pH of areas affected by CF to limit the development of P. aeruginosa infections.

Atmosphere control

In order to create and control the atmospheric oxygen concentration, atmosphere controlling sachets in anaerobic jars were used to lower oxygen levels^[9] and cultivate on universal agar that includes antioxidants to remove oxygen from the air. Both of these techniques are capable of creating decreased oxygen conditions under which even strict anaerobes can grow, with the sachets able to control down to 0.5% O₂. ^[10] The universal agar consists of Schaedler Broth agar with additional antioxidants (Ascorbic acid and L-Glutathione) to absorb oxygen from the air and create an anaerobic environment. Additional antioxidants such as uric acid can be added to further decrease atmospheric oxygen concentration; however, that was not used for this investigation, as ascorbic acid and L-glutathione produce a sufficiently anaerobic environment, and uric acid would be an expensive addition. Universal agar, however, also provides more nutrients than common media such as trypticase soy agar (TSA).^[11][12]

Methodology

First Experiment

In order to assess the growth of P. fluorescens under varying pH and atmospheric oxygen levels, six King’s B broths were inoculated with 50µl of an aqueous suspension of P. fluorescens. This suspension had a turbidity of 0.434 Absorbance Units (Au) when measured with a spectrophotometer at A600 wavelength; Au is the standard unit for turbidity, as is calculated according to Au = 2 – Log₁₀ (% transmission). Three of the six broths were adjusted to a pH of 6 with the addition of 0.2M HCl. Two of these broths at a pH of 6 and two of the broths at a pH of 7 were incubated in a reduced oxygen environment by incubating in an anaerobic jar with a microaerophilic sachet which achieve an oxygen concentration of less than 10%. The broths were incubated at 28° C for 36 hours. After incubation, the turbidity of the broth was measured using a spectrophotometer.

Second Experiment

The antibiotic resistance of P. fluorescens was assessed under different pH levels in the second experiment. For this test, antibiotic mast rings were placed over a freshly inoculated lawn plate of P. fluorescens, which were plated on King’s B agar, and the pH was adjusted with 0.2M HCl to achieve a pH of 6 and a pH of 7. The mast ring contained various dosages of 6 common broad-spectrum antibiotics (see legend below fig 2). The plates were incubated for 36 hours at 28°C before using a ruler to measure the diameter of the zone of inhibition that was produced around the antibiotic disks. The zone of inhibition (ZOI) is used as a metric to measure antibiotic resistance and is an area on an agar plate where bacterial colonies do not grow due to the presence of an antibiotic. This indicates susceptibility to the antibiotic, whilst the lack of a ZOI suggests there is resistance to the antibiotic.

Results

The results for experiment 1 (Figure 3) show that the optimal growth condition for P. fluorescens is an aerobic atmosphere at a pH of 6, where the turbidity was measured at 1.783 Au. However, this was not consistent when considering the independent variables separately, as there was more growth at a pH of 7 in the reduced oxygen (microaerophilic) environment. Furthermore, the difference between aerobic and microaerophilic at a pH of 7 was almost negligible (turbidity difference of 0.039) versus a large difference in growth for those incubated at a pH of 6 (turbidity difference of 1.434). This indicates that the combination of the lower pH and the reduced oxygen, which would have reduced the pH as per carbonic acid,  brought the pH down to the point where there would have been a degree of denaturation of the bacteria’s enzymes, causing a significant decrease in growth. Similarly, the aerobic conditions with a pH of 6 created the least stressful environment for the bacteria; allowing the most growth. The broths at a pH of 7 were an intermediate between these two extremes.

In the second experiment (Figure 4), it is evident that there were baseline levels of susceptibility to streptomycin, sulphatriad and tetracycline. However, when the bacteria were provided less favorable conditions, such as a pH of 6, they lost resistance to ampicillin and became slightly susceptible to chloramphenicol with an 8mm² ZOI. It is important to note that these results are based on primary zones of inhibition (no growth), as there were secondary zones of inhibition (some limited growth) around some antibiotics (see Figure 2), indicating that the antibiotic has had some bacteriostatic effects (limiting reproduction), but has not been bactericidal (killing the bacteria). This indicates that there is still a degree of resistance under a pH of 6.5 from tetracycline and sulphatriad.

Discussion

The results from experiment 1 showed distinct information as to which conditions provide the best environment for P. fluorescens to thrive. The optimal growth conditions for P. fluorescens (pH 6, aerobic) supported the hypothesis. However, these optimal conditions are not consistent with one another. At a pH of 7, the bacteria grew to nearly identical levels. It is important to note that the data displayed in Fig 3 is averaged from an experiment that was run in duplicate, and there was much more variation within the data before it was averaged. One possible explanation for the lack of variation in the broths at a pH of 7  could be that the bacteria are not affected by the oxygen concentration until they are stressed to a certain level. This would explain why at a pH of 6, the microaerophilic broths were notably less turbid. It would be expected that the pH 6 aerobic broth would have a lower turbidity than the broths at a pH of 7, but it did not. The distinct difference between the broths at a pH of 6, as well as the most growth being measured at aerobic pH 6 conditions, could be that P. fluorescens grows best under slightly acidic conditions (pH 6), whereas the combination of pH 6 and the higher CO2 concentrations could mean that the pH dropped below the optimal growth level.

In order to discuss the results from the second experiment, it must be stated that the overall antibiotic susceptibility and resistance demonstrated is determined not by the size of a zone of inhibition (ZOI), but by the number of antibiotics against which resistance is demonstrated. This is because the size of a zone of inhibition is not only determined by the susceptibility of a bacteria to a particular antibiotic, but also other uncontrolled factors such as the ability of the antibiotic to diffuse through the agar. Differences in the size of the ZOI of a single antibiotic do reflect a change in resistance; however, they cannot be compared to one another, as the solubility of each antibiotic differs in the media. Consequently, the results from the second experiment did not support the hypothesis, as the most antibiotic resistance was evident at a pH of 6. Considering that  P. fluorescens is gram negative, meaning it has an outer lipid cell membrane as well as peptidoglycan as opposed to just an outer layer of thick peptidoglycan, it is possible that the higher pH makes the double-layered cell walls of the gram negative bacteria weaker, allowing the cells to be more susceptible to the bacteria. Alternatively, the stress caused by the slightly acidic environments could lead to strength when it comes to resisting antibiotics.

These results were obtained by the testing of against the independent variables; however, there is an application of this research to P. aeruginosa. The knowledge that P. fluorescens grows most effectively under aerobic conditions at a pH of 6 is concerning in regards to P. aeruginosa in the CF lung, as the CF lung is an aerobic and slightly acidic environment. It does, however, provide possible additions to antibiotics in treating P. aeruginosa, as the pH could be raised slightly to limit the growth of the bacteria, and the growth of biofilms that increase antibiotic resistance.^[7] As the bacteria are very similar, the knowledge that P. fluorescens is most resistant to common antibiotics at a pH of 7 could allow the development of antibiotics that slightly lower the pH of the environment in order for those antibiotics to be more effective against P. aeruginosa infections in the CF lung.

Key limitations of this study include the lack of repetition in order to ensure reliability. Whilst there was some repetition in experiment 1, such as duplication of the reduced atmosphere broths, there was none for the aerobic broths or plates in the second experiment. Additionally, there were limits in accuracy when measuring the ZOI in the second experiment,  as the ZOI was calculated as though it was a perfect circle (Area = π x r²), when some ZOI were irregularly shaped, meaning there may have been discrepancies in the area of the ZOI. Furthermore, it would be beneficial to conduct this research with a more precise pH range (increments of 0.1) and under strictly anaerobic conditions to determine the results. Finally, before using these results to consider treatment of P. aeruginosa infections, differences between P. fluorescens and P. aeruginosa must be considered.

These results are somewhat consistent with existing research, as several studies represent P. aeruginosa as growing best in a mostly neutral environment. In one particular study^[13], the bacteria grew best at a pH of 7 when grown at a range of a pH of 2 to a pH of 10. Unfortunately, P. fluorescens has little clinical or industrial use, and there is little research surrounding it, but the evidence that P. aeruginosa grows best at a pH of 7 has been proven in various studies.^[5][7]

Future areas of research following these results would involve either further investigating the effect of pH and atmospheric oxygen concentration on the growth and antibiotic resistance of P. flourescens by investigating a more specific pH range of 6.5-7, with 0.1 increments, or considering the impact of a microaerophilic environment. It is possible that under a microaerophilic environment at a pH of 6.8, the bacteria may be less susceptible to antibiotics, and thus it is a condition to avoid. Ultimately, testing the impact of pH and atmospheric oxygen concentration on the growth and antibiotic resistance of P. aeruginosa itself would necessary future research, as there are subtle differences between the organisms.

Conclusion

This report investigated the impact of pH and atmospheric oxygen concentration on the growth and antibiotic resistance of P. fluorescens, with the aim of determining the conditions under which it grows best and has the least antibiotic resistance. This is to consider the application of these results to the treatment of P. aeruginosa infections in cystic fibrosis patients. Results show that P. fluorescens growth was highest at a pH of 6 under aerobic conditions, and that antibiotic resistance was highest at a pH of 7. It is likely these results will be similar to those of P. aeruginosa. Ultimately, P. aeruginosa itself should be tested to determine the optimal conditions, as this would provide vital information for the treatment of P. aeruginosa infections.

Acknowledgements

I would like to thank The King\’s School, The Future Project, and Ms. Kathryn Fraser for giving me the opportunity to conduct this research, as well as Dr. Michelle Bull and Dr. Belinda Chapman of Quantal Bioscience for facilitating the project, and for all their support and guidance.

References

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About the Author

Jack Thomas, Australia

Jack is an Australian student interested in a variety of applications of the life sciences. He in keen to pursue a career in environmental science research, and has been involved in various microbiology research projects. He has also worked in pharmaceutical compounding labs, and gained work experience in various other labs. Outside of science, Jack enjoys fishing, hiking and playing the guitar.