On Feasibility of Fluorescence-based Bacteria Presence

Quantification: P.Aeruginosa

Alexander Caschera

and Gennadi Saiko

Ryerson University, Toronto, Canada

Swift Medical Inc., 1 Richmond St W, Toronto, Canada

Keywords: Bacteria, Bacterial Growth, Fluorophores, Fluorescence Imaging.

Abstract: Introduction: Wound healing typically occurs in the presence of bacteria at levels ranging from contamination

to colonization to infection. The role of bacteria in wound healing depends on multiple factors, including

bacterial concentration, species present, and host response. Thus, the determination of bacterial load is of

great importance. However, existing clinical bacteria load assessment methods (biopsy or swabbing combined

with culture methods) are slow, labor- and time-consuming. Pseudomonas aeruginosa is a known pathogen

implicated in numerous healthcare-associated infections and can express fluorescent metabolites during

proliferation. In particular, the siderophore pyoverdine produces a fluorescent emission between 450-520 nm

when excited at 400nm and can be measured quantitatively via fluorescence spectroscopy. The current project

aims to investigate the possibility of quantifying bacterial presence using fluorescence measurements.

Methods: Cultures of P.aeruginosa (PA01) were grown at various temperatures (ambient temperature, 30,

37-43°C), inoculum starting condition (5*10

-5*10

CFUmL

-1

), and initial nutrient’s concentration (0.6, 1.5,

3.0 gL

-1

) in Tryptic Soy Broth media. Media optical density (OD, as a proxy of bacterial concentration) and

fluorescence (ex. 400nm, em. 420- 520nm) were measured hourly for 10 hours. Results: Cultures remained

metabolically active in the whole temperature range, producing pyoverdine fluorescence (emission max at

455nm). We have correlated optical density with a fluorescent signal to establish a dependence between

fluorescence and growth stage. Noticeable pyoverdine accumulation started approximately 3 hours after the

beginning of the log growth phase and experienced saturation at the beginning of the stationary phase. Three

distinct regimes (a sigmoid curve) were observed: linear dependence of fluorescence on OD for low

concentrations, more rapid nonlinear dependence, and saturation when approaching the stationary phase.

Conclusions: The sigmoid dependence of bacterial fluorescence on their concentration persisted through

variations in temperature and inoculum starting condition; thus, it may have the potential for determining

culture growth phase progression. These results, combined with classical knowledge on disease progression,

could also lead to an advanced infection diagnosis before current pathogenesis observation techniques.

1 INTRODUCTION

Wound healing occurs in the presence of bacteria

(e.g., Staphylococcus, Streptococcus, Pseudomonas

species, and Coliform bacteria, including aerobic and

anaerobic types), at levels ranging from

contamination to critical colonization to infection.

The role of bacteria within wounds depends on

multiple factors, including bacterial concentration,

species present in the wound, and host response.

There are several distinct levels of bacteria

presence in the wound: contamination, colonization,

https://orcid.org/0000-0002-5697-7609

and infection. These levels delineate from the number

of microorganisms present per gram of tissue, which

can be highly variable and can range from less than 1

to 10

or 10

colony-forming units (CFU).

The increased bacterial burden may be confined

to the superficial wound bed or present deep within

the wound or even surrounding tissue.

Contamination and colonization by low microbial

concentrations are considered normal and are not

believed to inhibit healing. However, critical

colonization and infection are associated with a

significant delay in wound healing.

Caschera, A. and Saiko, G.

On Feasibility of Fluorescence-based Bacteria Presence Quantiﬁcation: P.Aeruginosa.

DOI: 10.5220/0010344001930200

In Proceedings of the 14th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2021) - Volume 2: BIOIMAGING, pages 193-200

ISBN: 978-989-758-490-9

193

With the ubiquity of microorganisms within the

natural environment, lacerations and skin tissue

lesions can lead to health-threatening issues if

infectious pathogens can take hold within a

compromising injury. With that in mind, the

importance of assessing the stage of infection and

identifying primary microorganisms involved can

assist with wound treatment. It can also help prevent

disease progression if pathogens are properly

characterized early on.

The gold standard collection method is to do a

tissue biopsy or needle aspirate of the wound's

leading edge after debridement. However, the

practical standard method for bacterial load

determination for skin infections is to culture (e.g.,

pour or spread plate) a microbiological swab of the

wound surface. Obviously, this method suffers from

multiple shortcomings. Among them, a) specimen

can be contaminated by normal skin or mucosa flora,

b) swabs frequently yield too small a specimen for

accurate microbiologic examination (Washington.

1996), and c) the duration of incubation for certain

cultures can be long. While most aerobic and

anaerobic bacteria will grow overnight, some

mycobacteria require as many as 6 to 8 weeks before

colonies are observed.

Other methods (e.g., Polymerase Chain Reaction

or PCR) have been proposed. However, they mostly

address the quantification step, while swabbing

remains the primary specimen collection technique.

Thus, other methods of bacterial load

quantification, preferably close to real-time, are

required. This could be accomplished by monitoring

the potential site of infection for unique metabolites

that can be quantified easily. Endogenous

fluorescence (FL) has great potential as a remote and

non-invasive modality. It can be performed remotely,

thus decreasing the risk of contamination.

Most clinically important strains (both gram-

positive and negative) clearly show a distinctive

double-peak of tryptophan fluorescence (emission

peaks at 340 nm, with two excitation maxima; at 230

nm and 280 nm) (Dartnell, 2013) with comparable

intensity between the strains studied. However, the

use of UVC light in vivo is quite problematic.

Fluorescence extending through the 400-500nm

emission range, from excitation of around 350 nm, is

also reported for many cells due to cellular

metabolites such as NAD(P)H. However, it is highly

variable, dependent on the microbial strain and

metabolic state, is not always detectable (Estes,

2003), and is much less intense than the tryptophan

signal (Dartnell, 2010).

In addition to that, it is known that most clinically

relevant bacteria (S. aureus, S. epidermidis, Candida,

S. marcescens, Viridans streptococci,

Corynebacterium diphtheriae, S. pyogenes,

Enterobacter, and Enterococcus) produces red FL

(from porphyrins (Kjeldstad, 1985)) when excited at

405nm. In contrast, P.aeruginosa produces a bluish-

green FL (from pyoverdin (Cody, 1987)) while

excited at 405nm. Considering the polymicrobial

nature of most chronic wounds, it is possible to use

this endogenous fluorescence to characterize bacterial

load.

Fluorescence has been previously used to

differentiate between different species. In particular,

the fluorescence of extracellular pyoverdines has

been used to distinguish between cultures of certain

strains of Pseudomonas (Shelly, 1980 a and b).

Leblanc et al. (Leblanc, 2002) successfully

distinguished between different species of bacteria

using principal component analysis (PCA) of the

autofluorescence of the aromatic amino acid,

nucleotide, and NADH components of the cell. Giana

et al. (Giana, 2003) successfully discriminated

between the clinically important Escherichia coli,

Enterococcus faecalis, and Staphylococcus aureus.

However, both porphyrins and pyoverdine are

mostly extracellular compounds produced by

bacteria. Moreover, their production and fluorescence

can be affected by numerous factors. Thus,

quantification of bacteria presence using porphyrin-

and pyoverdine-based fluorescence is not

straightforward.

The goal of the current project is to establish the

feasibility of fluorescence imaging of bacterial load

under clinical parameters and investigate the

possibility of quantifying bacterial presence using

fluorescence.

In this article, we will present the results of

bacteria fluorescence quantification on the cultures of

P.aeruginosa (PA01). The bacterium Pseudomonas

aeruginosa is an increasingly prevalent human

pathogen, responsible for 12% of hospital-acquired

urinary tract infections, 10% of bloodstream

infections, and 8% of surgical wound infections. In

the UK, 7.6% of acute hospital patients acquire

healthcare-associated infections, around a sixth of

which are caused by methicillin-resistant

Staphylococcus aureus and another sixth by

Clostridium difficile

(Smyth, 2008).

Pseudomonas aeruginosa is known to express

fluorescent metabolites during proliferation. These

metabolites include pyoverdine and pyocyanin and

are thought to play a role in the virulence of other

hemolytic pathogens as well. Of pyoverdine

BIOIMAGING 2021 - 8th International Conference on Bioimaging

194

specifically, 400 nm light is known to produce a

fluorescent emission between 450-500 nm. It can be

measured quantitatively based on concentration

within an appropriate growth media such as tryptic

soy broth. When correlated to optical density, this

fluorescent signature can be compared to the cell

quantity and growth stage. Additional factors, such as

temperature and initial starting concentration, also

play a role in cell growth and pyoverdine expression.

2 METHODS

2.1 Culture Preparation

Many of the materials and consumables used through

this study for the manipulation of microbial cultures,

including pipette tips, Petri dishes, and culture tubes,

were sourced from Sarstedt unless otherwise listed.

Tryptic Soy Broth (cat. 1054590500) was purchased

from Millipore-Sigma, and phosphate-buffered saline

tablets were purchased from Bio Basic (cat. PD0435)

and were used as indicated.

Bacterial strains used for this study were supplied

from ATCC and maintained at Ryerson University by

the Wolfaardt lab group. PAO1 and PA14 are

commonly used for studying the basic biology and

genetics of P. aeruginosa, and PA01 was chosen as

the representative strain for this study.

2.1.1 Inoculum Preparation

Several colonies of Pseudomonas aeruginosa (PA01)

were collected from a maintained 3 gL

-1

Tryptic Soy

Agar (TSA) culture plate using an inoculation loop

and deposited into a 50 mL conical tube containing

10 mL of 3 gL

-1

Tryptic Soy Broth (TSB) under

aseptic conditions. The tube was then sealed and

placed onto a shaking incubator set to 37 °C and left

to incubate to the late-exponential growth stage,

between 16-20 hours after initial cell deposition. The

following day, the overnight solution was rinsed by

centrifuging two 1 mL portions of overnight serum

collected into 2 mL microtubes, replacing the

supernatant solution with sterile phosphate-buffered

saline (PBS), and by resuspending the microbial

pellet within the solution. The process was repeated

twice to ensure proper rinsing of cells, and then both

rinsed aliquots were combined within a single 2 mL

microtube for sample inoculation.

Table 1: Inoculum Parameters.

Test microorganism

Pseudomonas aeruginosa

(PA01)

Growth media 3 gL

-1

Tryptic Soy Broth (TSB)

Incubation perio

16-20 hours (Overnight)

Incubation

tem

erature 37 °C

Rinse solution

1x Phosphate Buffered Saline

(PBS)

Centrifuge duration 4 min at 9000 x g

Expected inoculum

loa

-10

2.2 Culture Quantification

2.2.1 Microbial Reference Quantification

Following inoculum preparation, cells were

enumerated by serially diluting the prepared

inoculum (100 µL of the previous dilution was added

to 900 µL of PBS per step) and spot plating 0.1 µL of

the 10

, 10

, and 10

dilutions onto 3 g L

-1

TSA plates.

The plates were then left to incubate at ambient

temperature for 7 days prior to counting developed

colonies of PA01. The PA01 colonies grown during

spot plating are depicted in Fig 1. The Petri dish was

imaged by a camera with 425nm long pass filter and

405nm excitation. The bluish-green fluorescence of

pyoverdine is clearly visible.

Figure 1: The PA01 colonies grown during spot plating.

The colonies are imaged by a camera with 425nm long pass

filter and 405nm excitation. Two bright spots are caused by

specular reflection of the excitation light.

On Feasibility of Fluorescence-based Bacteria Presence Quantiﬁcation: P.Aeruginosa

195

Table 2: Microbial Quantification.

Quantification

Metho

Spot plate technique

Quantification

erio

Immediatel

after rinsin

cells

Maximum

dilution facto

Growth media 3 gL

-1

Tryptic Soy Agar (TSA)

Incubation

tem

erature 25 °C

(

ambient Tem

erature

)

Incubation

erio

7 days

2.2.2 Microbial Rapid Quantification

To allow for the rapid quantification of PA01

bacterial cell concentration within liquid media,

optical density (OD) measurements were calibrated to

colony-forming unit counts obtained from microbial

reference quantification. Since plate counting

requires multiple days and significant resources to

determine the bacterial concentration at any point in

time, rapid quantification of bacteria concentration

can be achieved by measuring the absorbance or

scattering of light. For this purpose, standard OD

measurements at 600nm were performed using

parafilm-sealed macro-cuvettes (cat. BR759035,

Millipore-Sigma) with a 10 mm light pathlength,

within a BioPhotometer (S/N 6131 21925, Eppendorf

AG). The concentration of bacterial cells suspended

within 3 mL of 3 g L

-1

TSB was then calibrated using

multiple concentration points between 10

-10

CFU

and graphed to produce a standard calibration curve.

For spectroscopic blanking purposes, a sterile TSB

control was maintained at 4 °C for each trial

performed.

2.2.3 OD-Correlated Fluorescence

Spectroscopy

Fluorescence spectroscopy was performed using an

LS 50 B Luminescence Spectrometer (S/N 50801,

Perkin-Elmer Ltd.) on macro-cuvettes containing 3

mL TSB inoculated with PA01, as defined by the

microbial quantification methods above.

Fluorescence emission scans were achieved using

400nm (2.5 nm slit width) as the excitation

wavelength, while emission was recorded in 420-

520nm (2.5 nm slit width) range with 0.5nm

increments for 1 minute. OD measurements at 600 nm

were taken prior to each fluorescence microscopy

scan. The typical raw fluorescence spectrum is

depicted in Fig 2.

Figure 2: An example of a raw fluorescence spectrum of the

PA01 sample.

A spectral integral was used over the whole 420-

520nm range to quantify the fluorescence signal:

𝑓

𝑙= 𝑆

(

𝜆

)

𝑑𝜆





(1)

Here, S(λ) is a fluorescence signal measured at a

particular wavelength λ. To eliminate background

fluorescence and dependence on various starting

conditions instead of absolute values, the normalized

fluorescence ration was used,

𝐹𝐿 =

𝑓

𝑙−

𝑓

𝑙



𝑓

𝑙



(2)

where fl

is the fluorescence spectral integral for a

sterile TSB control sample.

2.3 Experimental Protocol

Initial trials were performed by inoculating macro-

cuvettes containing 3 mL of 3 g L

-1

TSB with 30 µL

(1/100 dilution) PA01 inoculum, prepared and

quantified using the above methods. Samples were

then incubated at a predetermined temperature

(ambient temperature, 30°C, 37-43 °C) within a

standing incubator for 8-10 hours. Prior to incubation

and after each following 1 h interval, OD-correlated

fluorescence spectroscopy was performed on the

prepared samples to determine the concentration of

PA01 cells within the growth medium and the

quantity of fluorescence emitted by each sample.

To investigate the effect of the initial bacterial

concentration, another set of trials were performed at

37 °C, where the starting concentration of PA01 was

altered by inoculating the macro-cuvettes with 150

(1/20), 75 (1/40), and 37.5 µL (1/80) of quantified

PA01 inoculum.

BIOIMAGING 2021 - 8th International Conference on Bioimaging

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Finally, the impact of nutrient availability was

investigated by altering the initial concentration of

tryptic soy broth (0.6, 1.5, and 3.0 gL

-1

)) while

keeping the number of cells inoculated and

temperature the same (30 µL (1/100 dilution) PA01

inoculum and 37 °C, respectively).

3 RESULTS

Calibration procedure for PA01 shows the following

dependence between bacteria concentration N

(CFUmL

-1

) and optical density OD (R

=0.991):

𝑁=(5∗10



∗𝑂𝐷)

.

(3)

To estimate the carrying capacity K, we have

grown the culture in 3 g L

-1

TSB for 26 hours and

found that the optical density saturates at

approximately 0.462 value, which according to Eq.3,

corresponds to K=1.21*10

CFUmL

-1

We performed three types of experiments. All

results are presented in normalized fluorescence (FL)

vs. optical density (OD), a proxy for the bacterial

concentration.

3.1 Dependence on Temperature

Firstly, we studied growth and fluorescence at several

temperatures (ambient temperature, 30C. 37C, 38C,

39C, 40C, 41C, 42C, 43C), while maintaining

inoculation and nutrient's initial concentrations the

same (30 µL (1/100 dilution) PA01 inoculum and 3.0

-1

TSB, respectively). The results are depicted in

Fig 3.

Figure 3: Dependence of fluorescence (FL) on optical

density (OD) for various temperatures: ambient

temperature (blue rhombs ◊), 30C (red, squares □), 37C

(red, line), 38C (green, triangles Δ), 39C (green, line), 40C

(blue, star *), 41C (blue, |), 42C (purple, cross x), 43C

(brown, dot). All other parameters (inoculation and

nutrient’s concentration) were kept the same.

3.2 Dependence on Inoculums

Concentration

To investigate the effect of the initial bacterial

concentration, another set of trials were performed at

37 °C, where the starting concentration of PA01 was

altered by inoculating the macro-cuvettes with 150

(1/20), 75 (1/40), and 37.5 µL (1/80) of quantified

PA01 inoculum. The results are depicted in Fig 4. In

Fig 5, one can see the zoomed area with low OD.

Figure 4: Dependence of fluorescence (FL) on optical

density (OD) for various starting concentrations: original

stock (blue rhombs ◊), 1/20 (red, squares □), 1/40 (green,

triangles Δ), 1/60 (purple, cross x), 1/80 (blues, star *).

Figure 5: Dependence of fluorescence (FL) on optical

density (OD) for various starting concentrations: 1/20 (red,

squares □), 1/40 (green, triangles Δ), 1/60 (purple, cross

x), 1/80 (blues, star *).. Zoomed area with low OD.

On Feasibility of Fluorescence-based Bacteria Presence Quantiﬁcation: P.Aeruginosa

197

3.3 Dependence on Initial Nutrients

Concentration

Finally, the impact of the initial nutrient's

concentration was investigated by altering the initial

nutrient's concentration (0.6, 1.5, and 3.0 gL

-1

Tryptic

Soy Broth (TSB)) while keeping inoculation and

temperature the same (30 µL (1/100 dilution) PA01

inoculum and 37 °C, respectively). The results are

presented in Fig 4.

Figure 6: Dependence of fluorescence (FL) on optical

density (OD) for various starting nutrient's concentrations:

3gL

-1

(green, triangle, Δ), 1.5 gL

-1

(red, squares □), 0.6 gL

(blue, rhomb ◊)

4 DISCUSSION

Our data supports the view that P.aeruginosa is a

versatile and opportunistic microorganism. It remains

metabolically active even at temperatures

approaching 43 °C.

Our preliminary results support the nutrient-

dependent siderophore production model developed

in (Saiko, 2021). According to the developed

approach, siderophore production in a resource-

limiting environment has three distinct phases:

I. Slow siderophore production at low

bacteria concentrations where resources

are abundant (S> S

)

II. Rapid accumulation of siderophores

upon reaching a specific nutrient's

concentration S

. Linear dependence on

the bacteria concentration N.

III. Upon reaching resource limits, the

bacteria focus on growth solely, which

will result in saturation of compound

accumulation (while bacterial

concentration still growth)

The transition into rapid siderophore

accumulation regime (Phase I -> Phase II) occurs at

𝑁



=𝑁



+𝛾(𝑆



−𝑆



)

(4)

here N

and S

are the initial bacterial and resource

concentration, accordingly. S

is the resource

concentration below which bacteria start producing

siderophore rapidly. During that phase II, the

siderophore accumulation depends on the bacterial

concentration linearly:

𝐶=

𝜉

𝛾

(

𝑁−𝑁



)−𝜉(𝑆



−𝑆



)

(5)

Finally, when the bacteria population approaches

the carrying capacity, K (K=(N

+γS

)), the bacteria

divert all resources to replication only, thus reducing

siderophore production.

All these phases were observed in our

experiments.

We found that at the early stages of the growth,

where nutrients are abundant, the siderophore

production is relatively small.

We also found that the infliction point Nt

affected by the initial bacterial concentration N

(Fig

5) and the initial nutrient concentration S

(Fig 6).

There is a clear linear dependence of the infliction

point N

on the initial inoculums concentration N

The higher the initial concentration, the higher the

infliction point N

is (Fig 5). Also, we found a clear

linear dependence of the infliction point N

on the

initial nutrient's concentration S

. The higher the

initial concentration S

, the higher the infliction point

is (Fig 6).

In all our tests, upon reaching approximately K/2

bacteria concentration (OD=0.23), the siderophore

accumulation slope starts decreasing (see Fig 3 and

Fig 4). This finding agrees with the nutrient-

dependent siderophore production model (Saiko,

2021), and available experimental data from other

groups (Bren, 2013). Thus, our results support the

view that under starvation, bacteria will focus on

growth only (Bren, 2013) and stop diverting

resources to siderophore synthesis.

Despite promising results in culturing media, the

possibility of quantification of P.aeruginosa presence

based on pyoverdine fluorescence within wounds

requires further challenges to be solved. P.aeruginosa

is a particularly difficult model. Firstly, the

fluorescence of bacteria can be impacted by other

factors. Specifically, Pseudomonas fluorescence is

determined by two factors (Meyer, 1978): a) iron

bonded to pyoverdine quenches fluorescence, b)

pyoverdine production is affected by iron availability.

BIOIMAGING 2021 - 8th International Conference on Bioimaging

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Thus, P.aerugenosa fluorescence can be diminished

near blood vessels (due to fluorescence quenching

and/or decreased pyoverdine production).

Secondly, it is known (Smith, 2006) that P.

aeruginosa isolated from acute infections differ

substantially in phenotype from those isolated from

chronic infections. It was found (Morgan, 2019) that

P.aeruginosa isolated from chronic human wounds

were frequently defective in virulence functions and

biofilm formation. In addition to that, P.aeruginosa

has an extensive "quorum sensing" (QS) system with

three autoinducers. These QS sub-systems act

hierarchically and regulate cell survival, biofilm

formation, and virulence (Gellatly, 2013).

Thirdly, P.aeruginosa can sequester iron in ways

other than pyoverdine production. It can (i) produce

another siderophore (pyochelin); (ii) utilize a wide

range of siderophores synthesized by other organisms

(Cornelis, 2002); (iii) acquire Fe(II) through the Feo

system (Cartron, 2006). P. aeruginosa can also utilize

heme-iron by expressing two different heme-uptake

systems, namely phu and has (Ochsner, 2000).

Finally, a weak fluorescence signal from bacteria

in vivo can be masked by strong autofluorescence

from nearby tissues. Thus, the proper selection of the

excitation wavelength and emission filter may be

required. Therefore, quantification of P.aeruginosa

presence through pyoverdine fluorescence in vivo

seems quite challenging at this stage.

There are certain limitations regarding the

extrapolation of our results in vivo. They were

obtained in a resource-limiting environment, which

may or may not be the case in vivo. Thus, future

studies on animal models are required.

In future work, we plan to investigate porphyrins

production by another clinically relevant bacteria,

S.aureus.

5 CONCLUSIONS

We found that a fluorescent emissive signature

between 420-520 nm for PA01-produced pyoverdine

can be observed when excited with light at 400 nm in

a wide range of conditions.

Our temperature-dependence studies demonstrate

the production of fluorescent siderophores at

temperatures between ambient and 43 °C. Results

also point towards a local maximum in fluorescence

expression for P.aeruginosa around 40- 41 °C,

although further experimentation would be required

if this is to be determined.

We found that the sigmoid dependence of

bacterial fluorescence on their concentration

persisted through variations in temperature and

inoculum starting condition. This preliminary data

supports the hypothesis that siderophore production

in P.aeruginosa is governed by nutrient-dependent

mechanisms.

Starting nutrient concentration data also indicates

a positive relation between nutrient exhaustion and

fluorescent metabolite expression. This result agrees

with previous findings (Bren, 2013) and indicates that

siderophore production may become inhibited in

situations with high-nutrient concentrations.

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