When infecting mice with bacteria, the dosage needs to carefully considered, especially if attempting to establish a chronic infection. Too few bacteria, and a sufficiently robust infection will not develop. Too many bacteria, and the mice may succumb to bacterial pneumonia. We first attempted to establish an optimum dose of infection by infecting five day old BALB/c mice with doses of PAO1ranging from 1 103 to 1 107 CFU. The mice
were euthanized two days post inoculation, the lungs harvested and the CFU remaining in
Table 6.2 Observational assessment of animal behaviour in response to intranasal application of bacteria
Assessment Criteria Observations
Viscosity of inoculum Inoculum containing higher doses of bacteria were more
viscous Efficacy of intranasal
application of bacteria
Some pups reflexively inhale the complete inoculum while others initially exhaled before re-inhaling.
Signs of distress No major indicators of distress upon application of
inoculum. Some pups rested for a few minutes before freely moving around
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the lungs was enumerated by qPCR of the bacterial 16srRNA gene (psd7). The CFU was calculated by comparing the cycle threshold (CT) of psd7 expression in lung tissue with a
standard curve relating CT to PAO1 CFU (Figure 6.1). A linear relationship was observed (R2
= 0.9693) allowing us to relate CT of psd7 expression to number of CFU. The CFU
enumerated in each mouse are displayed in Figure 6.2, along with the mean CFU (n = 5–8) of each treatment dose.
No dose dependent effects were observed relating to bacterial load of infected mice. In addition, none of the mice had enough CFU in the lungs to be considered to have an infection as decided by our arbitrary cut-off of >103 CFU/ml. In fact, for between 2-4 mice in each
group of 5-8 mice we found no detectible P. aeruginosa in their lungs. The mice that received the highest dose of P. aeruginosa, 1 107 CFU, were the most capable of clearing
the infection with 4/5 mice having no detectible P. aeruginosa in their lungs two days post infection.
In order to determine if establishment of chronic infection is possible using this model, five day old mice were infected with 1 108 CFU of PAO1. This dose was used because the lower
doses were unable to cause infection with >105 CFU/mL that persisted two days post
infection, and did not have any obvious detrimental effects on the animals. PAO1 CFU in the lungs was enumerated by qPCR from lungs harvested from the mice 12 hours, five days and 12 days post infection. The number of CFU in the lungs of each mouse and the mean CFU (n = 6–9) in the lungs of mice for each time point are displayed in Figure 6.3. At the earliest
Figure 6.1 Relationship between qPCR cycle threshold (CT) of psd7 expression and log10 PAO1
CFU. Cycle threshold of psd7 expression of RNA extracted from PAO1 cultures compared to the number of CFU in those cultures. n = 2
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Figure 6.2 Bacterial load of P. aeruginosa infected mice lungs two days post infection. Five day old neonatal mice infected with a range of CFU’s of PAO1 and bacterial load two days post infection of individual calculated by qPCR. Results expressed as log10 CFU recovered from whole lung. Mean CFU
recovered indicated by horizontal line, while error bars represent S.E.M. Significant differences assessed using Kruskal–Wallis non-parametric test with Dunn’s post-test. n = 5–8. Mice with no detectible P. aeruginosa shown on logarithmic scale as 1.
Figure 6.3 Bacterial load of P. aeruginosa infected mice lungs over 12 days. Five day oldneonatal mice infected with 1 108 CFU of PAO1 and bacterial load 12 hours, five days and 12 days post infection
determined by qPCR. Results expressed as log10 CFU recovered from whole lung. Mean CFU recovered
indicated by horizontal line, while error bars represent S.E.M. Significant differences assessed using Kruskal–Wallis non-parametric test with Dunn’s post-test.* p < 0.05.n = 6–9. Mice with no detectible P. aeruginosa shown on logarithmic scale as 1.
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time point, 12 hours post infection, the P. aeruginosa load was highest, with 1/6 mice having >103 CFU in the lung. However, the mice cleared most of the bacteria within this short time
period suggesting that an immediate acute infection was not established. The numbers of CFU detected in the lungs decreased significantly (p < 0.05) by day five to less than <102
CFU. The number of CFU detected in the lungs by day 12 post infection remained similar, demonstrating that small numbers of bacteria were persisting in the lungs.
However, 3/ 9 mice had completely cleared the infection. Though the number of P. aeruginosa detected at the later time points were inconsistent, and orders of magnitude lower than the infection dose, the persistence of low levels of bacteria is consistent with establishment of a low-level chronic infection in 30% of the mice.
To investigate whether a virulent infection had been established, we measured the expression of a number of bacterial QS and virulence genes summarized in Table 6.3. We used qPCR to quantify gene expression in the mouse lung tissue from two mice from each group with the highest reported bacteria loads. However, there was no detectable
expression of the virulence genes by qPCR.
Table 6.3 Expression of bacterial virulence and QS genes in two mice from each group with highest bacterial carriage by qPCR
Genes Role Expression
lasI QS signal (C12HSL-HSL inducer) synthesis
No detectable expression
lasB Elastase B synthesis No detectable
expression
rhlI QS signal (C4-HSL inducer) synthesis No detectable expression
algD Alginate biosynthesis No detectable expression
pqsA Pseudomonas quinolone signal synthesis No detectable expression
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6.4 DISCUSSION
Establishment of an intranasal chronic P. aeruginosa infection model will enable the in vivo
investigation of rhPON2 as a novel anti-Pseudomonal therapy. The intranasal method of P. aeruginosa bacterial delivery to the neonatal mouse lung investigated in this chapter enabled the development of a low-level chronic infection. Chronic infections are characterized by surviving animals having a stable bacterial load, often with
intrapulmonary replication, in the airway, typically for at least two weeks. In our study we saw persistence of a relatively small numbers of bacteria (102–103 CFU) 12 days post
infection. This rate of PAO1 carriage in the mouse lungs is on the lower end of the scale of carriage rates reported by other authors who have established chronic P. aeruginosa
infections in BALB/c mice. Hoffmann and colleagues intratracheally challenged 12–14 week old BALB/c with 5 107 CFU of a mucoid P. aeruginosa strain and recovered between 103
and 109 CFU/mouse seven days post infection and between 105 and 107 CFU/mouse 13 days
post infection [410]. Lazenby and colleagues intratracheally challenged 8–12 week old BALB/c with 1 106 CFU PAO1 and the investigators recovered between 103–104 CFU/g
lung tissue 24 hours post challenge and ~ 104 CFU/g lung tissue 10 days post challenge
(average weight of 8–12 week mouse lung 200 mg–400 mg) in their study. [362]. We were however unable to establish whether this level of infection is physiologically relevant, as there was no detectable expression of Pseudomonas virulence and QS genes. This inability to detect expression of Pseudomonas virulence and QS genes may be a result of the low
bacterial numbers. Without histopathological analysis of the lungs, it is difficult to determine whether this level of bacteria can be considered to be pathological.
It must also be noted that the mice were monitored for signs of distress and none was evident. In comparable studies, intratracheal challenges with the higher doses of PAO1, such as those used in our study (108), have normally resulted in neutrophilic pneumonia,
bacteraemia and death within the first 24–48 hours in neonates [402, 404, 410]. The fact that all the mice survived suggests the bacteria were unable to produce sufficient virulence to harm the mice. Our mice were challenged with 108 CFU of PAO1 but 12 hours post
infection only ~103 CFU were detected. It is plausible that the majority of the bacteria were
cleared mechanically by the mice before any infection was initiated. Intranasal infections have generally displayed a lower effectiveness at delivering bacteria to the lungs than more invasive methods such intratracheal infection or a tracheotomy [414]. This raises the prospect that our delivery method for bacteria may need to be further developed to reduce
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the mechanical clearance of the bacteria perhaps through application of smaller volume of, but multiple doses of bacterial suspensions.
The low bacterial loads in the mice poses some intriguing questions. Tang and colleagues were successfully able to establish an acute Pseudomonas infection in BALB/c neonates. Upon application of 1 105 CFU, 12/15 mice had increased CFU 24 hours post infection. By
96 hours, no bacteria remained in the lungs [402]. Morissette and colleagues infected a number of inbred mouse strains with low doses (104 CFU) of P. aeruginosa and found
BALB/c to be resistant to infection based on both their mortality and bacterial load post infection [409]. This suggests that the BALB/c strain has high resistance to P. aeruginosa
infection. The authors considered the C57BL/6 mouse strain to be of an intermediate phenotype while the DBA/2 strain was the most susceptible to infection. Tam and colleagues investigated the susceptibility of BALB/c and C57BL/6 mice to chronic P. aeruginosa and observed two thirds of their BALB/c mice cleared the infection by 28 days post inoculation [415]. However, almost 80% of the C57BL/6 mice had persistent infections. These differences have been attributed to the differing intrinsic inflammatory and immune response in the two strains, particularly the lower tumour necrosis factor- gene
expression and protein secretion [416, 417], increased nitric oxide production [417], and reduced proliferative responses from lung T cells [408]. Sapru and colleagues reported a considerably stronger immune response in C57BL/6 mice when compared to the BALB/c mice, with a higher count of polymorphonuclear cells in the bronchoalveolar lavage fluid and this correlated with increased susceptibility to infection [417]. An exaggerated
inflammatory response may thus predispose the C57BL/6 mouse to infection and also more accurately mimic the CF disease pathophysiology. It is therefore possible that the strain of mouse selected for this study may have contributed to the inability to establish chronic high-level colonization of the lungs by P. aeruginosa and that this could be overcome by using an alternate mouse strain. It may also be still possible to use the current mouse strain but prime the immune system using microbial ligands such as LPS, flagellin [418, 419] or protein from pili [402] to stimulate the immune response. We could also adopt a strategy whereby we initially challenge the mice with an intermediate dose (106 CFU/mL) before re-
challenging the mice with a higher dose (108) 24 hours later.
Given the low levels of bacteria persisting in the lungs in this study, the use of a PCR based method to quantify bacteria is validated. Numerous studies have investigated the suitability of real-time quantitative PCR to quantify bacterial load. Provided sensitive methods are used to detect the bacteria, such as quantifying high expression genes like 16S ribosomal
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RNA, as in this study, bacteria can be reliably enumerated down to a single colony forming unit [276]. While there are studies that indicate that PCR may be less sensitive than conventional culture methods [420], most studies generally agree that PCR is as good as if not more sensitive than culture based methods for enumerating bacteria [276, 421-423]. The inability to detect the expression of QS and virulence genes explored in this study can most likely be attributed to the low levels of bacteria present in the mouse lungs.
A promising development in imaging technology has been the advent of in vivo imaging of mice infected with a GFP- or luciferase-tagged P. aeruginosa. This enables the real-time, sensitive monitoring of infection in mice without the need to use multiple groups to study multiple treatment conditions, thereby reducing inter-animal variations [424, 425]. There is the added advantage of the ability to monitor the progression of bacteria from the nares into the lower lung, and give a good indication of where the mice establish the infection. Further studies will be required to develop a suitable intranasal murine model of P.
aeruginosa infection. This study has provided some important insight into how this could be achievable. Such a model would have numerous advantages, particularly the ability to rapidly check the effectiveness of novel therapies such as rhPON2 and better understand the pathophysiology of chronic infection.
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7.1 INTRODUCTION
This thesis investigates human PON2 as a quorum sensing inhibitor of P. aeruginosa. The inhibition of QS is of particular interest because conventional antibiotic therapy is
ineffective against chronic P. aeruginosa infections. The efficacy of these antibiotics might be rescued using a therapeutic approach that combines conventional antibiotic therapy with anti-virulence therapy. Anti-virulence therapies have the advantage of targeting the
pathogenicity of bacteria in an infection setting without necessarily affecting viability. With attenuated virulence, the bacteria are unable to cause pathological infections, allowing the host immune response to respond effectively. In addition, because anti-virulence therapies do not affect microbial growth, the general consensus amongst researchers is that such therapies are less likely to exert selective pressures towards resistance development [426]. The C12HSL QS molecule is a potent signalling molecule produced by P. aeruginosa during infection. C12HSL induces the production of many bacterial host-damaging virulence factors and is involved in biofilm formation by this organism. C12HSL also has important roles in conditioning host cells for infection and even at low concentrations induces host-cell immune pathways, the unfolded-protein stress-responses, as well as intra-cellular calcium signalling. Consequently, C12HSL represents a potential target for novel anti-virulence therapies. The results described in thesis demonstrate the potential usefulness of human PON2 as an anti-Pseudomonal therapy, with the added advantage of abrogating the negative effects of QS signalling molecules on host-cells.
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