Mersey Kidney First

The Role of Bacterial and Host Factors in Persistent Bacterial Infections in Complicated Urinary Tract Infections

Investigating host-pathogen interactions in urinary tract infections caused by Pseudomonas aeruginosa

Supervisors:

Dr Jo Fothergill, Institute of Infection and Global Health, University of Liverpool
Dr Rachel Floyd, Institute of Translational Medicine, University of Liverpool

Investigator:
John Newman

First year progress:

P. aeruginosa in chronic infections can be very diverse and show different levels of adaptation to their environment. Our hypothesis is that P. aeruginosa populations in complicated UTIs display both diversity and intracellular invasion that makes infections more difficult to treat. These processes are aided by host factors, particularly steroid hormones, leading to persistence and antibiotic resistance. This study will form the basis for improving microbiological diagnostic methods and treatment strategies for clinicians and patients. The three key aims of our 3 year study are as follows:
1. To characterise urinary tract isolates of P. aeruginosa from populations during chronic infection to determine whether populations display phenotypic diversity and adaptation.
2. To elucidate the mechanisms by which hormones have a direct effect on the characteristics of clinically relevant P. aeruginosa in an in vitro model of biofilm formation that closely resembles human urine.
3. To determine whether clinical isolates are able to cause intracellular invasion of host cells using a unique and physiologically relevant differentiated human cell model and determine the impact of host factors such as hormones on this process.
Studies in the first year were focused towards addressing Aim 1 of our project synopsis whereby 11 samples of uropathogenic Pseudomonas aeruginosa were collected from the Royal Liverpool University Hospital to be used as representative isolates for the remainder of the study. From each of these 11 samples we isolated 40 unique bacterial colonies and stored them in our freezer to enable us to continue studies beyond the duration of the current grant. From 5 of these samples, all 40 isolates were tested for their ability to produce different substances or survive different conditions. The tests conducted were:
• Pyocyanin assays (Pyocyanin is a product that P. aeruginosa produces which can cause severe damage to human tissue in infections)
• Hypermutability assays (These show how rapidly the bacteria can adapt to new environments)
• Minimal growth assays (These show whether the bacteria are capable of growing in a low nutrient environment, such as the urinary tract)
• Antimicrobial susceptibility tests (ASTs) (These show how susceptible the bacteria are to different types of antibiotics)
The aim of these tests was to see whether there is diversity between the bacterial isolates from each sample and to generate a general profile for the clinical isolates that we have collected. Pyocyanin production was highly variable across the different samples but was generally high (Figure 1). This could mean that pyocyanin is important for P. aeruginosa to successfully infect the urinary tract. None of the isolates appeared to be hypermutators and all were able to grow on minimal media. This suggests that P. aeruginosa does not rely on spontaneous mutations or diversification to thrive in the urinary tract. Some of the samples showed resistance to certain antibiotics but none showed particularly remarkable multidrug resistance (Figure 2). This result may highlight that current test of antibiotic resistance may not capture how resistant Pseudomonas can really be when it forms a biofilm in the urinary tract.
A test was also performed to generate a genetic fingerprint for each isolate across the 5 samples. These genetic fingerprints are a simple method that highlighted that each of the 5 samples were made up of a genetically different strain of Pseudomonas. Also, the test showed that the 40 isolates collected within each of the 5 samples was genetically very similar. This suggests that each UTI that was sampled was caused by a single strain of Pseudomonas rather than multiple genetically different strains. In the next 3 months, I plan to sequence all of the DNA from 10 of my clinical isolates. This will allow us to analyse any possible genetic differences in far greater detail and may help to identify which genes help Pseudomonas to cause UTIs.
John was able to present his work at the 16th International Pseudomonas conference in September in the form of a poster. The poster received good reception from the delegates that took interest in it and also allowed John to meet a potential collaborator in the US with experience researching catheter associated UTIs in a mouse model. This will enable us to expand the studies in the future into a more translational model of infection.
Outside of the lab John has written a review paper on uropathogenic Pseudomonas which was published in FEMS Microbiology Letters (Newman et al. 2017). The last (and only) review of research on Pseudomonas in urinary tract infections was written in 2009, therefore John’s review collated all the progress that has been made by researchers in the subsequent 8 years to highlight gaps in our understanding.
John’s research has also contributed to a paper on the effect of hormones and artificial urine media on clinical isolates of Pseudomonas that has been submitted to Nature Scientific Reports. The manuscript is currently awaiting a decision from the Editor following revision. It is anticipated this paper will be published in the next few weeks, should we be successful.
References
Newman et al. 2017 FEMS Microbiology Letters 2017; 364(15)


Research Partners
Liverpool University The Royal Liverpool and Broadgreen University Hospital
Supported by
Mitchell Charlesworth