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A dose response model for the inhalation route of exposure to P.

aeruginosa

Kara Dean Graduate Student , Jade Mitchell Ph.D. Professor

PII: S2352-3522(20)30021-9
DOI: https://doi.org/10.1016/j.mran.2020.100115
Reference: MRAN 100115

To appear in: Microbial Risk Analysis

Received date: 17 July 2019

Revised date: 25 March 2020
Accepted date: 1 April 2020

Please cite this article as: Kara Dean Graduate Student , Jade Mitchell Ph.D. Professor , A dose re-
sponse model for the inhalation route of exposure to P. aeruginosa, Microbial Risk Analysis (2020),
doi: https://doi.org/10.1016/j.mran.2020.100115

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Highlights:

 P. aeruginosa poses a potential human health risk via the inhalation exposure route
 The multi-hit dose response model significantly fit available data
 Single-hit theory may not capture P. aeruginosa behavior in immunocompetent hosts
Title: A dose response model for the inhalation route of exposure to P.
aeruginosa
Authors: Kara Dean1, Jade Mitchell2, Ph.D.
1
Graduate Student, Department of Biosystems and Agricultural Engineering, Michigan State University,
524 S. Shaw Ln, East Lansing, MI 48823. Email: deankara@msu.edu
2
Professor, Department of Biosystems and Agricultural Engineering, Michigan State University, 524 S.
Shaw Ln, East Lansing, MI 48823 (corresponding author). Email: jade@msu.edu

ABSTRACT

This study develops a dose response model for Pseudomonas aeruginosa for the

inhalation route of exposure using pre-existing data. P. aeruginosa is an opportunistic pathogen

capable of causing community and hospital-acquired lung infections. As such, a dose response

model for this route of exposure is needed to assess risks posed by the inhalation of aerosols

from showers, humidifiers, or hot tubs contaminated with P. aeruginosa. Single-hit theory

models traditionally used for dose response modeling did not provide significant fits to the

limited available data. The multi-hit dose response model operates under the cooperativity theory

and did provide a significant fit, suggesting that a single P. aeruginosa bacterium is not

sufficient to initiate a pulmonary infection in an immunocompetent host. A hierarchical Bayesian

analysis was used to benchmark this model against additional dose response experiments and

results further suggested that the multi-hit model may better represent the dose response behavior

for this exposure route. The best fitting model has an LD50 of 2,588,047 Colony Forming Units.

This model can be used to quantify risk in inhalation exposure scenarios, however, due to the

limited amount of primary data, it is especially important for any future risk assessment to

analyze the impact that using different dose response models may have on the final risk estimates

and recommended control measures.

Keywords: P. aeruginosa, multi-hit, dose response, inhalation, aerosols

Funding Source: Partial support for research was provided under Assistance Agreement No.
R836890 awarded by the U.S. Environmental Protection Agency to Purdue and Michigan State
University. This work has not been formally reviewed by the EPA. The views expressed in this
document are solely those of the authors and do not necessarily reflect those of the Agency.
INTRODUCTION

P. aeruginosa is a gram-negative bacterium associated with acute and chronic respiratory

infections. It is a common cause of nosocomial, ventilator-associated, and community-acquired

pneumonia, and immunocompromised hosts and patients with cystic fibrosis are at a higher risk

of infection (Driscoll et al. 2007; Sadikot et al. 2005). Although less common, P. aeruginosa can

also cause lung infections in immunocompetent hosts, and previous infections in healthy

individuals have reported a mortality rate of 33% (Hatchette et al. 2000; Sadikot et al. 2005). In

the United States, 51,000 healthcare-associated infections and about 440 deaths are caused by P.

aeruginosa annually (Azam & Khan 2019; CDC 2013; Moradeli et al. 2017). P. aeruginosa is

recognized worldwide as a public health risk and the bacterium is becoming more difficult to

treat, as it is naturally resistant to a number of antibiotics (Moradeli et al. 2017). In the U.S.

about 13% of severe healthcare-associated P. aeruginosa infections are caused by multidrug

resistant strains (Moradeli et al. 2017; CDC 2013).

P. aeruginosa can exist and thrive in a range of natural and built environments, and water

has been implicated directly or indirectly in most of the investigated outbreaks (Bedard et al.

2016). Water-related sites like taps and showers and moist, humid environments like respiratory

therapy equipment are the most likely to be colonized in healthcare settings (Kerr & Snelling

2009). P. aeruginosa develops biofilms that aid in its production of virulence factors and

persistence in the environment, and biofilms in engineered water systems can become an ideal

long-term habitat for this opportunistic pathogen (Sadikot et al. 2005; Wingender & Flemming

2011). P. aeruginosa colonizes premise plumbing systems and point-of-use devices like

showerheads and faucets, and the threat this poses to the user is uncertain. It is possible that

during a showering event the pathogen may be aerosolized and inhaled. In order to be able to

1
understand the risk of a respiratory infection from P. aeruginosa in such an exposure scenario, it

is first necessary to have an understanding of the dose response relationship. Dose response

models characterize the relationship between the dose of a pathogen and the probability of an

adverse health outcome in the exposed population, such as infection, illness, or death (Haas et al.

2014). A dose response model for the inhalation route of exposure of P. aeruginosa has not

previously been developed but would be valuable in facilitating future risk assessments and to

support studies that determine if there is a need for remediation of premise plumbing.

Opportunistic pathogens such as P. aeruginosa, Legionella pneumophila, and

Mycobacterium avium are able to persist and grow within premise plumbing and distribution

systems and all three have been associated with infections from exposure through premise

plumbing (Falkinham et al. 2015). Unlike L. pneumophila and M. avium, P. aeruginosa is not

currently on the Environmental Protection Agency’s Contaminant Candidate Lists (CCL) (U.S.

EPA 1998, 2005, 2009, 2016). Both L. pneumophila and M. avium pose inhalation risks and to

facilitate risk assessments that can quantify the risk they pose to human health, these pathogens

have had inhalation dose response models previously fit (Hamilton et al. 2017; Armstrong &

Haas 2007). An inhalation model for P. aeruginosa is a noted gap and this study aims to fit a

dose response model to pre-existing data. Such a model could be used to provide a greater

understanding of the threat posed by opportunistic pathogens like P. aeruginosa in premise

plumbing systems and is needed to protect human health.

METHODS

Dose Response Data

2
 A review of the literature was conducted to find a dose response study that simulated an

inhalation exposure, ran three or more dosing groups, and documented the positive and negative

responses from each dose. A study conducted by Ojielo et al. (2003) evaluated the risk of

pulmonary infection after bone marrow transplantation in mice. The researchers first evaluated

the course of P. aeruginosa pneumonia in normal hosts by intratracheally inoculating groups of

10 wild-type, specific pathogen-free B6D2F1/J mice with seven graded doses of P. aeruginosa.

P. aeruginosa PAO1 frozen stock was grown in 10 mL of tryptic soy broth at 37°C. The trachea

was exposed in a sterile fashion and a 26-gauge needle was used to administer the inoculum

intratracheally. The positive endpoint response was death (Ojielo et al. 2003). The seven dosing

groups and the respective responses are shown in Table 1.

Table 1: Dose Response Data for P. aeruginosa from Ojielo et al. (2003)

Dose (CFU) Positive Response Negative Response

80,000 0 10

400,000 0 10

900,000 0 10

2,000,000 2 5

3,000,000 6 4

4,500,000 10 0

8,000,000 10 0

Ojielo et al. (2003) was the only data set found in the literature that met the

aforementioned criteria, however 13 other studies (16 experiments) were identified that

administered one or two doses of P. aeruginosa through the intranasal or intratracheal route to

mimic an inhalation exposure. The animal species, pathogen strain, dosages, and response

percentages are outlined for each study in Table 2. All of the studies in Table 2 had death as the

positive endpoint response. For the remainder of the paper, the Ojielo et al. (2003) data set in
3
Table 1 is referred to as Experiment 1, and the 16 additional experiments are numbered from 2-

17, as shown in Table 2. From these 13 studies, there were 13 experiments that recorded a

response for a single dose and three experiments that recorded responses for two doses (shaded

in grey in Table 2). Although these estimates could not be modeled like the data in Table 1, the

data were evaluated in conjunction with the Table 1 data through other methods to benchmark

and analyze the results of fitting the data in Table 1, given the limited data available for

modelers.

Table 2: Additional Dose Response Data for P. aeruginosa

Exposure Positive
Experiment Animal Strain Dose (CFU) Resource
Route Response (%)
C3 +/+ (C57BL/6 Mueller et al.
2 PA103 Intranasal 100,000
mice) 0.222 2004
Factor B +/+ Mueller et al.
3 PA103 Intranasal 100,000
(C57BL/6 mice) 0.125 2004
C4 +/+ (C57BL/6 Mueller et al.
4 PA103 Intranasal 100,000
mice) 0.222 2004
Wolbeling et al.
5 C57BL/6JZtm mice TBCF10839 Intratracheal 600,000
0 2011
Kenawy et al.
6 MASP-2 mice KR420 Intranasal 1,000,000
0.182 2011
C3 +/+ (C57BL/6 Mueller et al.
7 PA243 Intranasal 3,000,000
mice) 0.273 2004
Morello et al.
8 Balb/c mice CHA Intranasal 3,000,000
1 2011
Factor B +/+ Mueller et al.
9 PA243 Intranasal 3,000,000
(C57BL/6 mice) 0.273 2004
Balb/c mice PAK Intranasal 5,000,000 Debarbieux et
10 0
al. 2010
Balb/c mice PAK Intranasal 15,000,000 1
PAK/PA01/ Hassan et al.
11 Balb/c mice Intranasal 10,000,000
DM125/DM126 2017
1
Balloy et al.
12 C57/BL6 mice PAK Intratracheal 15,000,000 2007; Ramphal
0.5 et al. 2005
13 C57BL6/J CHA Intranasal 20,000,000 0.78 Faure et al. 2014

14 CD-1 mice AC869 Intranasal 1,610 0 George et al.

CD-1 mice AC869 Intranasal 27,000,000 1991
0.5
de Souza Morias
15 Swiss Webster mice PA14 Intranasal 80,000,000
0.714 2014
El-Aziz et al.
16 Balb/c mice PA9 Intranasal 100,000,000
1 2019
17 Congenic B6.129P2- van Heeckern et
PA M57-15 Intranasal 100,000,000
Cftrtm1Unc 0.167 al.

4
 Congenic B6.129P2-
PA M57-15 Intranasal 1,000,000,000
Cftrtm1Unc 1

Briefly, Experiments 2, 3, 4, 7, and 9 are data from a study of P. aeruginosa pulmonary

infection pathogenisis in which Mueller et al. (2004) intranasally inoculated mice with the

PA103 and PA243 strains. Wolbeling et al. (2011) intratracheally inoculated C57BL/6J mice

with the TCVF10839 strain and did not obersve any mortality (Experiment 5) and Kenawy et al.

(2012) intranasally inoculated MASP +/+ (wild-type) mice with 1x106 CFU of P. aeruginosa KR

420 and observed a 18.2% response (Experiment 6). In Experiment 8, Morello et al. (2011)

intranasally inoculated a multidrug resistant mucoid strain of P. aeruginosa and observed 100%

mortality in Balb/c mice. DeBarbieux et al. (2010) used the bioluminescent PAK strain of P.

aeruginosa to also study Balb/c mice (Experiment 10). Within a study conducted by Hassan et

al. (2017), control Balb/c mice were intranasally inoculated with 1.0x107 CFU of the PAO1,

PAK, DM125, and DM126 strains of P. aeruginosa, and 100% mortality occurred in all cases, as

listed in Table 2 as Experiment 11.

Balloy et al. (2007) used the PAK strain to challenge C57/Bl6 mice intratracheally and

calculated the dose where a 50% response was observed to be 1.5x107 CFU (Experiment 12).

Ramphal et al. (2005) also determined that a 50% response for the same mice species, strain, and

exposure route was 1.5x107 CFU. Faure et al. (2014) inoculated C57Bl/6J mice intranasally with

a mucoid strain CHA and saw a 78% mortality response (Experiment 13). George et al. (1991)

also studied CD-1 mice but with doses of the AC869 strain (Experiment 14). Studying potential

vaccine systems, de Souza Morais et al. (2018) intranasally inoculated Swiss Webster mice with

the P. aeruginosa strain PA14. The mortality response of the controls from this study are

recorded as Experiment 15 in Table 2. Whilst evaluating the efficacy of bacteriophage treatment,

5
El-Aziz et al. (2019) infected Balb/c mice with the PA9 strain of P. aeruginosa intranasally and

treated one group with PBS (Experiment 16). Finally, Van Heeckern et al. (2006) studied the

response of mice to free-living mucoid P. aeruginosa by intranasally inoculating the P.

aeruginosa strain PA M57-15. The mortality of the wild-type mice are recorded in Table 2 as

Experiment 17.

Model Fitting

Using the statistical programming language, “R”, and a previously developed code that

uses maximum likelihood estimation (MLE) methods as outlined in Haas et al. (2014), the

exponential and beta-Poisson models were fit to the dose response data in Table 1 (R Core Team,

2019; Weir et al. 2017). The exponential model (Equation 1) determines the probability of a

response, P(d), based on the dose, d, and the parameter, k, which represents the likelihood that a

single organism survives to initiate infection.

( ) Eq. 1

The approximate form of the beta-Poisson model (Equation 2) determines the probability

of response based on the α parameter, which dictates scale, and the median infective dose, N50.

The endpoint of response for the dose response data being considered is death, and as such the

N50 is equal to the LD50, the median lethal dose.

⁄
( ) ( ) ( ) Eq. 2

Both the exponential and exact beta-Poisson models are “single-hit” models; they operate

under the assumption that just a single organism, kmin equal to 1, is needed to initiate infection

(Haas et al. 2014). The cooperativity theory assumes that for some pathogens a kmin greater than

6
1 may be necessary to initiate infection (Haas et al. 2014). This difference in underlying

assumptions results in the multi-hit dose response model (Equation 3). The multi-hit dose

response model is represented by the incomplete gamma function (Haas et al. 2014). As it

follows the gamma probability distribution, the multi-hit model can be coded in R using the

pgamma() function from the stats package. It is coded as pgamma(x, a), where x is the dose, d,

multiplied by the probability that the pathogen survives to initiate infection, k. The kmin value is

a. Note, when kmin is equal to one, the probabilities output by the multi-hit model are equivalent

to that of the exponential model.

( ) ( ) Eq. 3

The multi-hit model was fit iteratively, with the kmin parameter fixed at values ranging

from 1-187 and the k parameter determined using the MLE methods as described above (Haas et

al. 2014). Both parameters could not be solved for using MLE methods simultaneously because

they are inherently correlated and the kmin value should be an integer, as it represents a number of

pathogens.

Model Selection

Goodness of fit was determined by comparing the optimized deviance of the model to the

χ2 distribution with the degrees of freedom equal to the number of parameters of the model

subtracted from the number of doses (Haas et al. 2014). The null hypothesis, that the model

provides an acceptable fit, is rejected if the deviance value exceeds the critical χ2 value. The best

fitting model was determined by comparing the difference in deviances between the models to

the critical χ2 value at one degree of freedom. Confidence bands were determined by

bootstrapping the data.

7
 Benchmarking

The previously outlined methods for model fitting and selection were applied to the data

in Table 1. Since the data sets with one or two point estimates could not be modeled directly or

pooled within the classical framework, a hierarchical Bayesian analysis of the data in Tables 1

and 2 was completed. The Bayesian framework allows for the incorporation of dose response

data sets that are otherwise uninformative within the classical framework (Mitchell-Blackwood

et al. 2011). This model was used to benchmark the model predictions from the frequentist or

classical statistic approach. Under the Bayesian framework, inferences about a parameter, θ,

given data, , are drawn from the posterior distribution, ( | ), which is calculated with the

likelihood, ( | ), and the prior distribution, ( ), as shown in Equation 4 (Gelman et al.

2014). The prior distribution allows for the incorporation of prior knowledge of the parameter

behavior. Using broad and non-informed prior distributions minimizes the impact of the prior

knowledge on the resulting posterior distributions (Gelman & Hill 2006).

( ) ( | )
( | ) Eq. 4
∫ ( ) ( | )

Another benefit of a Bayesian analysis is the ability to look at data hierarchically. This is

important for dose response modeling because ultimately the goal is to be able to apply the

model and interpret the results across different animal species and pathogen strains. Under this

framework, it is assumed that dose response relationship from one experiment to the next is

related via a joint probability model for the parameters (Gelman et al. 2014). For example, Table

1 and Table 2 together yield 17 individual “experiments.” Although each experiment may have

its own best fitting dose response parameter(s), a hierarchical Bayesian analysis views each of

these best fitting parameters as a sample from a common population distribution. This common

8
distribution is described by hyperparameters (Gelman et al. 2014). The hyperdistibution can be

analyzed to draw conclusions about unobserved dose response data and to produce a

generalizable model. The relationship between the hyperdistribution and the individual model

parameters for each experiment is represented in Figure 1.

Figure 1: The relationship between the individual k parameters as described by the hyperdistribution

Both the multi-hit (Equation 3) and exponential model (Equation 1) were fit to the 17

experiments using this framework and the hyperdistributions for the k parameter in each model

were compared. Analyzing the distributions described by the hyperparameters gave further

insight into the uncertainty of the dose response relationship for P. aeruginosa between

experiments, strains, and hosts. The hierarchical Bayesian analysis was completed using R and

Markov chain Monte Carlo methods. A random walk Metropolis-Hastings algorithm (Gelman et

al. 2014) was written for each model, with uninformative, lognormal priors on the mean of the k

parameter (µlnk) and the standard deviation of the k parameter (σlnk). These relationships are

shown as Equations 5 and 6.

µlnk ~N(-15, 3) Eq. 5

σlnk ~ N(2, 1) Eq. 6

9
 Starting values and priors were modified to understand their influence on the posterior

distribution inferences. The algorithm was run sequentially with the final values for the

parameters from the first run becoming the starting values for the second, etc. to evaluate

convergence. The algorithm was also run in parallel with three different sets of starting values to

evaluate convergence. Trace plots and density plots were used to visually assess chain mixing

and convergence. Estimated potential scale reduction factors (R) were also calculated using the

gelman.diag() function in R to confirm that 10,000 runs was a sufficient number of iterations

(R<1.1) (Gelman et al. 2014; Plummer et al. 2006). Additionally, the Bayesian estimate of the

model fit to the data in Table 1 was compared to the corresponding MLE estimate as method of

validation.

RESULTS

Model Fitting

The exponential model fit the dose response data with a deviance of 16.88, which is

greater than the χ2 value at 5 degrees of freedom (11.1). The beta-Poisson model had a deviance

of 16.88 compared to the critical χ2 value of 9.49 for 4 degrees of freedom. The exponential

model was a better fit than the beta-Poisson, however neither model was a statistically good fit.

The fit statistics for both models are shown in Table 3.

Table 3: Conventional Dose Response Model Fit Statistics for P. aeruginosa

Model Deviance Δ DF Better Fit Parameters LD50

Exponential 16.88 5 11.1

beta- 0.001 3.84 Exponential k= 3.22 x10-7 2,150,065

16.88 4 9.49
Poisson

10
 The multi-hit dose response model fit the data with a deviance of 1.09, which is below

the critical χ2 value of 11.1. The parameters for the best fitting model were a k value of 4.12x10-6

and a kmin of 11. The fit statistics are shown in Table 4. The multi-hit model is depicted in Figure

2 with 95% and 99% confidence bands. Figure 3 is a histogram of the k parameter estimates after

bootstrapping. The model was fit for kmin values ranging from 1 to 187 to ensure the optimal fit

statistics were identified. After a kmin of 187, the numerical search algorithm was no longer able

to find an optimum k value. Figure 4 illustrates the minimum deviance value for each iteration

with the lowest deviance and respective kmin value identified with a star.

Figure 2: The multi-hit dose response model with 95 and 99% confidence bands; Figure 3: Histogram of the k

parameter estimates after bootstrapping

11
 Figure 4: The kmin value and minimum deviance from each iteration of fitting the multi-hit model, illustrating the

optimal solution at kmin =11

Table 4: Multi-hit Dose Response Model Fit Statistics for P. aeruginosa

Model Deviance Δ DF Best Fit Parameters LD50

Multi-hit 1.09 15.69 4 9.49 3.84 Multihit k= 4.12x10-6 2,588,047

kmin=11

Benchmarking

A hierarchical Bayesian analysis was conducted for the 17 experiments in Tables 1 and 2

for the best fitting multi-hit model (Equation 3) with a set kmin of 11 and for the more

traditionally used exponential model (Equation 1). The exponential model was included in this

analysis for comparison purposes, with the goal of understanding if the multi-hit model was

more representative of the P. aeruginosa dose response relationship generally or only for the

Ojielo et al. (2003) data set. Figure 5 shows the doses and responses for the data in Tables 1 and

2. The triangles are the seven points from the Ojielo et al. (2003) data and the circles are the

additional estimates from Table 2. Figure 5 clearly demonstrates that the P. aeruginosa animal

data for mortality responses to inhalation exposures vary across different experiments, host

species, and pathogen strains.

12
 Figure 5: Dose response data for inhalation exposures to P. aeruginosa

Figure 6 shows the 17 individual multi-hit models fit to the data in Figure 5. The black

dashed and dotted curves represent the 2.5%, 97.5%, and median estimates of the µlnk

hyperparameter for the multi-hit model. The seventeen k parameters varied from median values

as low as 7.9x10-8 to as high as 8.1x10-5. The k parameter for the Ojielo et al. (2003) experiment

had a median value of 4.1x10-6, similar to the MLE estimate (Table 4). The median value for

µlnk was 3.1 x10-6. All of the data points are captured by one or more of the 17 curves and the

95% confidence band for µlnk (1.0x10-6 to 1.0x10-5) captures six of the 17 curves, indicating

higher confidence for those parameter estimates, and approximately 18 of the 26 data points. The

trace plots for the hyperparameters showed high variation and no obvious trends, indicating

convergence was quickly achieved. The density plots demonstrated consistent parameter

estimates and all estimated R point values were less than 1.003, also indicating convergence.

13
 Figure 6: Plot of the 17 individually fit multi-hit models and the multi-hit model fit with the
median and 95% confidence band of µlnk

Figure 7 shows the 17 different best fitting exponential models fit to the data in Figure 4.

The black dashed and dotted curves represent the 2.5%, 97.5% and median estimate of µlnk

hyperparameter for the exponential model. The 17 k parameters varied from median values as

low as 4.3x10-9 to as high as 1.9x10-6. The median k parameter for the Ojielo et al. (2003)

experiment was 3.2x10-7, similar to the 3.22x10-7 from the MLE fit (Table 3). The median

estimate of µlnk was 1.4x10-7. The 95% confidence band for the µlnk hyperparameter (5.1x10-8 to

1.4x10-7) captured six of the 17 curves, indicating a higher level of confidence for those

parameter estimates, and approximately 12 of the 26 data points. The trace plots, density plots

and R values (<1.005) indicate convergence.

14
 Figure 7: Plot of the 17 individually fit exponential models and exponential model fit with the
median and 95% confidence bands of µlnk

The summary statistics for both the multi-hit and exponential hierarchical Bayesian

analyses are shown in Table 5. Both analyses achieved convergence, however the k parameter

estimates for the 13 experiments with only a single data point were less stable than those for the

experiments with two or seven data points, as expected. The estimates for the k parameters for

the experiments with only a 0% or 100% response were the least stable. The 17 multi-hit curves

in Figure 6 capture all of the 24 observed data points and the 17 exponential models in Figure 7

clearly exclude at least three of the data points. The 95% confidence band of the µlnk

hyperparameter for the multi-hit model also captures more of the observed data points than the

exponential model.

Table 5: Parameter Estimates from the Hierarchical Bayesian Analysis

Multi-hit Model Estimates Exponential Model Estimates

15
Parameter 2.50% Median Mean 97.50% 2.50% Median Mean 97.50%
k1 3.4x10-6 4.1x10-6 4.1x10-6 5.0 x10-6 2.1x10-7 3.2x10-7 3.2x10-7 4.7x10-7
k2 5.6x10-5 8.1x10-5 8.0x10-5 1.1 x10-4 1.9x10-7 1.4x10-6 1.3x10-6 5.4x10-6
k3 4.1x10-5 6.8x10-5 6.8x10-5 1.0 x10-4 3.5x10-8 5.6x10-7 4.9x10-7 3.5x10-6
k4 5.5x10-5 8.1x10-5 7.9x10-5 1.1 x10-4 1.9x10-7 1.4x10-6 1.3x10-6 5.3x10-6
k5 2.4x10-8 1.4x10-6 1.1x10-6 1.0 x10-5 1.1x10-9 3.5x10-8 2.8x10-8 2.8x10-7
k6 6.2x10-6 7.9x10-6 7.8x10-6 9.5 x10-6 5.5x10-8 1.8x10-7 1.7x10-7 4.3x10-7
k7 2.2x10-6 2.9x10-6 2.9x10-6 3.7 x10-6 2.7x10-8 9.9x10-8 9.4x10-8 2.5x10-7
k9 5.4x10-6 2.0x10-5 2.8x10-5 8.3 x10-4 5.4x10-7 1.9x10-6 2.4x10-6 3.9x10-5
k10 2.2x10-6 2.9x10-6 2.9x10-6 3.7 x10-6 2.5x10-8 9.9x10-8 9.4x10-8 2.6x10-7

k11 9.0x10-7 1.2x10-6 1.2x10-6 1.7 x10-6 4.5x10-8 9.0x10-8 8.9x10-8 1.7x10-7

k12 1.7x10-6 1.1x10-5 1.5x10-5 1.0 x10-3 1.9x10-7 7.4x10-7 9.7x10-7 1.9x10-5

k13 5.6x10-7 7.0x10-7 7.1x10-7 8.9 x10-7 1.7x10-8 4.6x10-8 4.4x10-8 1.0x10-7
k14 5.2x10-7 6.7x10-7 6.7x10-7 8.8 x10-7 3.2x10-8 7.6x10-8 7.4x10-8 1.5x10-7
k15 3.1x10-7 3.9x10-7 3.9x10-7 5.0 x10-7 9.6x10-9 2.6x10-8 2.6x10-8 5.7x10-8
k15 1.2x10-7 1.6x10-7 1.6x10-7 2.1 x10-7 6.3x10-9 1.7x10-8 1.6x10-8 3.9x10-8
k16 2.3x10-7 4.2x10-6 5.1x10-6 4.4 x10-4 2.9x10-8 2.6x10-7 3.3x10-7 1.4x10-5

k17 4.9x10-8 7.9x10-8 7.6x10-8 1.1 x10-7 1.5x10-9 4.3x10-9 4.2x10-9 1.1x10-8

DISCUSSION

This study fit the multi-hit dose response equation to dose response data for P.

aeruginosa after neither the exponential or beta-Poisson model, models which represent the

majority of microbial dose response relationships, provided a statistically significant fit using

MLE methods. The optimized multi-hit model was further confirmed with a hierarchical

Bayesian analysis of additional dose response estimates used to benchmark the MLE analysis.

The multi-hit model has not been traditionally applied to microbial data but has been used in

toxic chemical risk assessment (Janardan 1986). The slope of the multi-hit model is greater than

that of the exponential at the median infectious dose and most microbial experimental data show

16
slopes equal to or less than the exponential model (Haas et al. 2014). One-hit models are based

on the hypothesis of independent action; a hypothesis that states that pathogenic individuals

behave independently of one another and each have an independent probability of causing

infection or death (Cornforth et al. 2015; Druett 1952). The statement of independent action

equates to a kmin value equal to 1. Single-hit models are not threshold models and exhibit low

dose linearity. The multi-hit model assumes a kmin >1, does not have low dose linearity, and is a

simple threshold model. Historically there has been stronger evidence for the biological

plausibility of the one-hit model than for the multi-hit (Haas et al. 2014).

Research into molecular mechanisms involved in infection have demonstrated that

several cooperative behaviors within pathogens help facilitate the evasion of host defenses and

initiate infection. There is concern that single-hit theory models cannot capture these behaviors

and host-pathogen interactions, making the models overly conservative (Coleman et al. 2017;

Coleman et al. 2018). It was demonstrated that the independent action theory failed in Bacillus

thuringiensis, an insect pathogen, because of the cooperative nature of the bacterial toxins. Cell

cooperation is necessary for B. thuringiensis to facilitate host invasion and septicaemic

proliferation (Cornforth et al. 2015). A study of Bacillus anthracis demonstrated that disruption

of the pathogen’s quorum sensing inhibited growth and virulence gene expression in vitro,

suggesting that a single spore would not result in growth, disease progression, or mortality

(Coleman et al. 2008; Jones et al. 2005). Coleman et al. (2008) presented evidence supporting

the concept of threshold regions for resistance to inhalation anthrax and suggested that in

response to the restrictions overestimated low dose risk place on risk management, it may be

necessary to analyze both threshold and non-threshold dose response models for B. anthracis.

Though the previous examples are spore-forming pathogens, limitations of single-hit theory

17
models have also been acknowledged for non-spore forming bacteria. Rahman et al. (2018)

developed a mechanistic dose response model for Listeria monocytogenes that quantifies the

interaction between the pathogen and the human gut environment with parameters such as

bacterial killing rate in the stomach, dispersal rate, and growth rate, and links infection to the

pathogen strain, the digestion process, and the host immune status. This model when compared

to the exponential model, predicted similar risks at high doses but predicted lower responses to

low doses and demonstrated that strain virulence and host immune status considerably affected

the response threshold of the model (Rahman et al. 2018).

These examples highlight a few demonstrated cases where the independent action

hypothesis fails and adds credence to the application of a simple threshold model to the P.

aeruginosa dose response data. Similar to the previously mentioned pathogens, there are

cooperative action indicators in the pathology of P. aeruginosa. P. aeruginosa rarely infects the

lungs of an immunocompetent host, despite its ubiquitous nature. Some of the bacterial factors

involved in the pathogenesis of P. aeruginosa lung infections include pili and flagella, a Type III

secretion system, and quorum sensing (Sadikot et al. 2005). Data suggests that P. aeruginosa

needs to express several virulence factors to initiate a pulmonary infection and the outcome of

the infection is dependent on the host response, which in the lung environment can be quite

robust (Tang et al. 1996; Sadikot et al. 2005). Quorum sensing is a form of cell-to-cell

communication that controls the expression of more than 300 genes in P. aeruginosa (Azam &

Khan 2017; Schuster et al. 2003; Schuster & Greenberg 2006). P. aeruginosa uses quorum

sensing to regulate the expression of extracellular virulence factors and to overcome innate

immune responses in the host (Pearson et al. 2000; Smith & Iglewski 2003; Kariminik et al.

2017). This communication also promotes biofilm formation which enables the pathogen to

18
evade host defenses and persist in the environment, as is common for P. aeruginosa (Shrout et

al. 2011; Sadikot et al. 2005). Targeting the quorum sensing system of P. aeruginosa is a

possible alternative to antibiotic treatment and previous studies demonstrated increased survival

of hosts when P. aeruginosa quorum sensing was targeted or quenched (Hraiech et al. 2014; Wu

et al. 2004; Pearson et al. 2000).

The reliance on cell-to-cell communication and the robustness of the immunocompetent

lung environment suggests that a single P. aeruginosa bacterium may not be enough to initiate

infection. A greater understanding of cooperative behavior in microorganisms indicates that dose

response modeling of microbes may not be limited to only the single-hit theory models

traditionally used. The multi-hit model attempts to capture some of the biological factors

involved in pathogen-host dynamics for the immunocompetent population with the kmin

parameter; for the immunocompetent population specifically, the results of the model fitting in

this study suggest that 11 P. aeruginosa bacteria are needed to overcome immune system

defenses and initiate a pulmonary infection. The LD50 of the simple threshold model fit in this

study for an inhalation exposure to P. aeruginosa is 2,588,047 CFU. The multi-hit model

indicates survivability below a certain point, and then a rapid increase in mortality with dose.

Compared to the exponential model, the multi-hit model estimates much lower probabilities of

response at low doses, as did the mechanistic model developed for L. monocytogenes (Rahman et

al. 2018).

Although more conservative estimates have often been preferred, it has more recently

been suggested that QMRA models often overestimate risk, likely due to overly conservative

dose response models (Coleman et al., 2017; Snary et al., 2016). However, it is important to note

with its application that the multi-hit model estimates de minimis risk at low doses. This model

19
attempts to describe the dose response relationship for the immunocompetent population only

and it is likely that the dose response curve is shaped differently for immunocompromised hosts.

As such, possible methods to utilize the same dose response model for more susceptible hosts,

such as using the 95th percentile parameter estimates, will likely greatly underestimate risk in the

case of P. aeruginosa.. Additionally, this model was fit to data of an intratracheal exposure, in

which the bacteria bypasses the additional defenses provided by the nasal passages (Ojielo et al.

2003). In a natural inhalation exposure, the additional nasal defense mechanisms may increase

the number of coliforms needed to initiate infection.

A major limitation of this study is that the MLE fitting was only done with a single dose

response data set. Fitting dose response models to several different data sets to evaluate

consistency across experiments or possible pooling would be much preferred. The absence of

additional data sets with more than three doses hindered the validation of the model and the

evaluation of its applicability. An attempt at validation was made, however, through the

hierarchical Bayesian analysis of single and double dose response estimates found in the

literature. A hierarchical Bayesian analysis was conducted for both the multi-hit and exponential

models in an effort to better understand if the multi-hit model was merely a feature of the Ojielo

et al. (2003) circumstances, or if it may be more consistently applicable for P. aeruginosa than

the more traditional models.

It is clear from Figures 5, 6, and 7 that there is over three orders of magnitude of

uncertainty between experiments for the P. aeruginosa dose response relationship for an

inhalation exposure. The ranges of σlnk for the hyperdistributions of both the multi-hit and

exponential k parameter suggest that it may be unlikely that a single set of dose response

parameters would be applicable across host species and pathogen strains for P. aeruginosa.

20
Although future dose response studies may narrow this uncertainty, the data suggests that the

dose response relationship may just be highly variable for P. aeruginosa. For this pathogen and

exposure route, it may be necessary to select a model based on the specific pathogen strain or to

apply the data-driven uncertainty factor produced in this analysis.

In addition to quantifying some of the uncertainty associated with this dose response

relationship, the benchmarking analysis also gave further credence to the multi-hit behavior of P.

aeruginosa. The 95% confidence interval for µlnk of the multi-hit model includes 17 of the

observed dose response data points, six more than the confidence interval for the exponential

µlnk. All of the data were captured by the 17 multi-hit curves in the analysis and the exponential

curves excluded three observations. Interestingly, one of the points excluded by the 17

exponential models fit in this analysis was the 1.0x108 CFU dose from the van Heeckern et al.

(2006) study. This study included a second observation of 100% mortality for a dose of 1.0x109

CFU (van Heeckern et al. 2006). Much like for the Ojielo et al. (2003) data, the exponential

model is not steep enough to be able to capture both observed responses. The steeper curve of the

multi-hit model is able to capture both points, as evidenced in Figure 5. The inclusion of a higher

percentage of the observed data points and this second instance of threshold-like behavior further

validates the use of the multi-hit model.

This study provides the best current estimate of the dose response relationship for P.

aeruginosa in an inhalation exposure. The multi-hit model in Table 4 provided a statistically

significant fit to the Ojielo et al. (2003) experiment and a more comprehensive look at dose

response estimates in the literature (Table 2) yielded a hyperdistribution with a similar median

value for k (3.11x10-6). Using the model in Figure 1 to describe all of the data in Tables 1 and 2

would provide an accurate or conservative estimate for 20 of the 26 dose response observations.

21
While the hierarchical Bayesian analysis may further validate the applicability of the multi-hit

model, it does suggest that an uncertainty factor or strain-specific model should be used in future

applications. It is important to note that this hierarchical analysis was only completed with a set

kmin of 11. If kmin were instead treated as a distribution, with less bacteria being needed to initiate

infections in some cases and more in others, it is possible that this would lead to more narrow

confidence bands of both the resulting kmin and k hyperdistributions. Further work is needed to

explore the validity of applying a distribution to the kmin value. In addition, more research into

the biological and physiological factors that contribute to the kmin parameter is needed to expand

the application of the multi-hit model to other populations at risk and additional strains of the

pathogen, which has specific relevance to P. aeruginosa as multi-drug resistant strains have been

categorized as a serious concern (CDC 2013).

CONCLUSIONS

The dose response model created in this study is the first dose response model for the

inhalation route of exposure for P. aeruginosa. The multi-hit model provided a significant fit to

the data and indicates that perhaps microbial dose response modeling should not be limited to

single-hit theory models. This was further suggested by the hierarchical Bayesian analysis of the

k parameter for both the multi-hit and exponential models. A dose response model is needed for

this route of exposure to facilitate the completion of quantitative microbial risk assessments

(QMRA) to address exposure scenarios of concern. Possible exposure scenarios may include

inhaling aerosols in a showering event, using a humidifier, or in pools and hot tubs. There were

limited primary data sets in the literature that could be modeled for this pathogen and exposure

route. This is a common type of uncertainty in microbial dose-response modeling as most data

sets available for modeling are small, dated and collected for other purposes. Because of this

22
limitation, the use of this model in future QMRAs should be coupled with an analysis of the

impact that using other dose response models may have on the risk estimates. If and when new

data sets become available, an uncertainty analysis using future models fit to new data sets,

modified versions of this model using the results of the benchmarking analysis, or surrogate

models from similar pathogens should be evaluated. This model and future QMRA models will

aid risk managers and decision makers in elucidating the potential risk posed by P. aeruginosa in

water.

Declarations of Interest: None.

Author Statement

Kara Dean: Formal Analysis, Writing-Original Draft. Jade Mitchell: Supervision, Project
Administration, Writing-Review and Editing.

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