Institutionen för medicinsk cellbiologi

Biomedicinska analytikerprogrammet

Examensarbete 15 hp

Optimization of a method for detection of Legionella pneumophila

in water samples

Charlotte Wilén

Examinator: Dick Wågsäter

Institutionen för medicinsk cellbiologi.

E-post: Dick.Wagsater@mcb.uu.se

2

Abstract

Legionella pneumophila is a bacterium which can be found in fresh water and causes

Legionnaires’ disease, which can be deadly for humans depending on the condition of the

infected individual. The bacterium is a gram-negative rod and can withstand severe conditions

such as high temperature. Therefore, various treatments including heat and acid treatment are

performed on the water to inhibit interfering microorganisms. However, to examine a larger

volume of water, the water needs to pass through a filter, which can be very time consuming,

and there are various variables that have a negative impact on the filtration speed. The aim of

this study was to examine these variables and find the fastest setup for detection of L.

pneumophila. To filtrate the water, a manifold with funnels, where you put the water, is used,

and the manifold is connected to a pump. Under the funnels, steel frits are placed, and the

filter is placed on the steel frits. To examine the fastest setup, different manifolds, pumps,

filters, and settings were investigated by timing the water running through in the different

settings. A new way of sterilization, that does not damage the steel frits was tested, and the

recovery of bacteria was examined on the filters with the top filtration speed. In conclusion,

the most efficient setup is the Cyclopore (GE Healthcare Life Sciences) filter, the pump from

KNF and the manifold MBS1 (Whatman), and the new way of sterilizing should be used to

reduce the damage of the steel frits.

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Populärvetenskaplig sammanfattning

Jämförande studie om vilka material och inställningar som får vattnet vid analys av

Legionella pneumophila att filtreras snabbast. L. pneumophila är en bakterie som lever i

sötvatten och orsakar infektioner som kan vara dödliga beroende tillståndet hos den

infekterade individen. Bakterien klarar av förhållanden som andra bakterier inte gör, till

exempel höga temperaturer, därför utsätts vattnet för värme- och syrabehandling för att

hämma bakgrundsflora. För att kunna undersöka en större volym vatten måste vattnet passera

genom ett filter, vilket kan vara väldigt tidskrävande. För att filtrera vattnet används ett

rörsystem där bägare som vattnet hälls i placeras. Under bägaren finns stålplattor där filtret

placeras. För att undersöka vilka material och inställningar som filtrerar vattnet snabbast,

noterades tiden det tog för vattnet att filtreras vid varje byte av material. Material som olika

pumpar, filter och rörsystem jämfördes. Även ett nytt sätt att sterilisera mellan proverna, som

inte skadade stålplattorna lika mycket, undersöktes. För de filter som var mest tidseffektiva

undersöktes även förmågan att fånga upp bakterier jämfört med det nuvarande använda filtret.

Den mest tidseffektiva utrustningen var filtret Cyclopore (GE Healthcare Life Sciences),

pump från KNF och rörsystemet MBS1 (Whatman). Det nya sättet att sterilisera borde även

användas för att minska slitage på stålplattorna.

Keywords

Filters, filtration speed, Legionnaires’ disease, manifold, water quality.

4

Introduction

Legionella is a bacillus that lives in fresh water and is most often found in large bodies of

water and at the surface of water from lakes and drinking water [1, 2]. It transmits to humans

through aerosols which for example occurs when taking a shower or flushing the toilet and

can be deadly depending on the condition of the infected. Around 6000 people get infected by

Legionella every year in Europe, and 9 % of them get so severe infections that they die from

them [3]. Since the effect of Legionella highly depends on the physical state of the person

who is infected, it often causes severe problems in hospitals. Cancer patients, transplant

patients and elderly people with many underlying diseases are groups that are extra

susceptible to the bacillus which makes it very important to have continuous monitoring to

make sure there is no presence of Legionella. Further risk factors are smoking and lung

diseases [2].

Legionella causes an infection called legionellosis which contains the syndromes Pontiac

fever and Legionnaires’ disease. The first known case of infection by Legionella was in 1968

in Pontiac, Michigan caused by defect air-conditioning system. At least 144 people who

visited or worked in the city’s health department caught Pontiac fever with symptoms that

reminiscent of influenza such as fever, headache, and myalgia. However, it took until 1976

before researchers could establish that Legionella caused the outbreak [4]. That year there was

another outbreak but this time among people who were at a Philadelphia convention of the

American Legion, 182 people who were at the convention caught a respiratory illness and 29

of them eventually died from the disease. It took the researchers half a year to establish that

the outbreak was caused by a bacterium, later known as Legionella and the disease caused by

the bacteria was named Legionnaires’ disease or legionellosis. Yet the cause of the outbreak

in 1976 never got confirmed [5].

5

When Legionella reaches the lungs, it attaches itself to the respiratory epithelial cells and

alveolar macrophages. Once the bacterium is inside the macrophages it starts to multiply,

which leads to recruitment of neutrophils, monocytes and bacterial enzymes which causes

alveolar inflammation. As mentioned, Pontiac fever manifests as an illness with resemblance

to influenza and shows no symptoms of pneumonia. Legionnaires’ disease on the other hand

is a more serious illness and causes severe pneumonia with symptoms such as coughing but

can also cause fever, muscle aches and headache. The preferred drug to treat symptomatic

Legionella is the antibiotic erythromycin and in severe cases rifampin can also be added. If

there is an ongoing outbreak prophylactic antibiotic can be used for immunosuppressed

patients to prevent infection. Pontiac fever, on the other hand, does not require antibiotic

treatment [1].

Legionella is considered a resistant bacterium because of its properties to withstand harsh

environment. It can survive high temperatures and can also use free-living amoebae as a host

which enhances Legionella’s pathogenicity even more by protecting them against

disinfectants and heat [3]. There are more than 66 species of Legionella but only about half of

them can infect humans and the species that causes approximately 90 % of the Legionnaires'

disease is called Legionella pneumophila. L. pneumophila can be divided into 15 different

serogroups, and serogroup 1 is the most common to cause severe infections to humans [6, 7].

ECCA Laboratory is a private laboratory founded in 1980 and is located in Flanders in the

northern part of Belgium. The samples for detection of Legionella are collected by the

customer or by ECCA’s staff and are often taken from cooling towers, swimming pools, and

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drinking water. This study summarizes various variables that make the method for detection

of Legionella slow and discusses the best possibilities to improve the filtration speed.

To concentrate a larger volume of water, the water is filtrated through a filter, and to achieve

this, various equipment is obligated. In routine, 500 or 250 mL of water needs to run through

the filter. The water can be dirty and contain a high concentration of bacteria making the rate

of filtration slower. The time it takes for the water to filter through can vary between minutes

to hours if the water is highly dirty. However, it is not only the interference of bacteria and

dirt that makes the rate of filtration slow, but there are also various other variables that have

an impact on the water filtration rate.

The manifold that is currently in use at ECCA has six positions to filtrate samples, and these

are connected to the same pump. The pump creates a vacuum which makes the water run

through the filter and is then transported to a waste container. A bar meter is connected to the

pump which is used to monitor the vacuum. It is important that the vacuum is consistent

during the whole time the water is being filtrated to maintain the speed of the filtration, since

without vacuum the water will not run through. The vacuum will decrease if there is a leak in

the system. On all positions on the manifold, steel frits are placed, and a filter is placed on

top, at the bottom of all steel frits there is a sealing layer to make sure no air enters. If this

layer is damaged, air can enter and decrease the vacuum. One reason the steel frits get

damaged is because of the way of sterilization.

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Method principle

The procedures are according to ISO 11731:2017 which describes methods for isolating and

counting Legionella. The filter that is currently used is made of polycarbonate and with a pore

size of 0.2 µm according to the standard.

Legionella is a gram-negative rod and needs special condition to grow in vitro. It is depended

on L-cysteine and iron and grows best in a temperature between 35-37℃. To discover the

bacteria in water samples, it is important to use cultivating plates that benefits the growth of

Legionella and inhibit other bacteria. To achieve this the water can be placed on three

different agars [3]. A GVPC agar which contains activated charcoal, that also benefits

Legionella, L-cysteine, glycine, and the selective antibiotics vancomycin, polymyxin and

cycloheximide 1. Secondly a BCYE+cys agar, that stands for Buffered Charcoal Yeast Extract

Agar + cys containing L-cysteine and activated charcoal and thirdly a BCYE+AB agar which

contains the same ingredients as the BCYE+cys but with the three antibiotics supplements

natamycin, cefazolin and polymyxin B. To inhibit background flora, acid and heat treatment

are also performed since Legionella can survive these conditions [8].

Different matrix

How the samples are prepared depends on how high the concentration of interfering

microorganisms is. The samples that are not believed to have a high concentration of

interfering microorganisms are analysed as “matrix A”, and samples that are believed to have

a high concentration of interfering microorganisms are analysed as “matrix B”. The samples

are prepared differently to achieve the best growth of Legionella and to inhibit the growth of

1 Standard operation procedure. Legionella in water. Prepared by Alexander De Meyer. 2021-

01-07

8

other bacteria. The water analysed as “matrix A” are added directly onto a BCYE+cys agar

and a BCYE+AB agar. To get bacteria from a larger volume water, 500 mL of the sample is

run through a filter and the filter is placed in a tube with PBS and vortexed. This is referred to

as the concentrated sample and is spread on a GVPC and BCYE+cys agar. The rest of the

concentrated sample is divided into heat treatment and acid treatment and are also placed on

GVPC and BCYE+AB plates. The heat and acid treatment are reducing the number of

interfering bacteria to be able to detect Legionella [9].

The water samples with a higher concentration of interfering microorganisms are analysed as

“matrix B”. Because of the high concentration some of the samples are diluted 10-1. The

procedure is similar to matrix A, but has a few modifications to optimize the growth of

Legionella and reduce the interfering microorganisms. The water is placed directly on an agar

and into acid and heat. These are placed on agar but also diluted 10-1 with PBS and placed on

agar. A volume of 250 mL of the sample is also concentrated through a filter and heat and

acid are performed the same way as matrix A. All agar plates used in matrix B are GVPC

plates and are incubated in 37℃ and are read after 4 and 10 days.

Detection of serogroup

When colonies that are believed to be Legionella occur, they are inoculated on another

BCYE+cys plate and a Columbia Agar with Sheep Blood (COS plate) and are incubated in

37℃ for 3-5 days. Since Legionella is dependent by L-cysteine and the COS plate does not

contain that, the colonies that grow on the BCYE+cys plate but not on the COS plate are

considered to be Legionella, and to find out the serotype of the colony, a Virapid Legionella

culture kit (Vircell) is used. The kit consists of a cassette with two membranes, one to confirm

the genus Legionella and one to confirm if it is L. pneumophilia serogroup 1-15 or a different

9

species. The kit is an immunoassay and when the sample is added to the application site,

bacterial components will attach to antibodies with colloidal gold and move along the

membrane. Complementary antibodies occur on the membrane and when the complexes reach

them a red line is created 2.

Aim

The method of Legionella demands a filter with a pore size of 0.2 µm. The small pore size

makes the water run through the filter very slowly, but there are several other components that

have an impact on the speed of the water running through the filter. The aim of this project

was to find the best setup to optimize ECCA Laboratory’s analysis for Legionella samples.

This is of big importance since a faster analysis would mean a big impact on the workload.

Materials and method

Study materials

The different equipment compared was borrowed from the different manufacturers. The

samples used for investigation of recovery of bacteria were collected between 22nd of

February and 25th of March 2021, and the samples that contained a high concentration of

interfering microorganisms (matrix B) were collected from different cooling towers while the

samples with a low concentration of interfering microorganisms (matrix A) were collected

from showers, water taps and drinking waters.

Ethics

Since the samples that contain the most bacteria are autoclaved, the risk of spreading the

bacteria is small. This study however includes an optimization of which settings that will

2 Virapid Legionella Culture (Vircell)

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make the analysis most time efficient which means a bigger load of samples can be analysed

and clients can get the result faster and remedy the concentration of bacteria. Since this study

does not include any biological material neither from humans nor animals an ethical review is

not required. Furthermore, there was no information that could reveal which company the

samples came from during this study.

Method

This report examines the best setup for Legionella analysis and to do this, various variables

have been investigated to find what is most time efficient. Before trying out different setups,

the wastewater was thrown in the sink, the pump was turned on and a vacuum was formed

which was seen on the bar manometer.

Equipment and settings

Different variables that could have had something to do with the speed the water runs through

the filter were tested and timed. Firstly, damaged, and non-damaged steel frits were

compared. The most damaged steel frit was placed on one position on the manifold, a funnel

was then placed on top and secured. A little bit of sterile water was filtered through, and the

funnel was removed, and a filter was placed on top of the frits, the funnel was placed on the

frits with the filter and secured. This procedure is always performed prior to pouring the water

sample in the funnel. A volume of 100 mL of tap water was poured into the funnel and the

time it took for the water to run through was noted. The vacuum at the start and at the end was

noted and the difference was calculated. This was performed five times. The same procedure

was then performed but with all six positions on the manifold. This was tested once. A non-

damaged steel frit was then tested the same way as the damaged one.

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Secondly, the impact a plastic ring had on the vacuum was investigated. The plastic ring was

put under the steel frits, and the funnel was prepared the same way as described, with a filter

on top of the frits. The damaged and the non-damaged steel frits were used and the difference

between using one and two plastic rings were investigated. Each variable was run and timed

five times each. The same procedure was also performed with a rubber ring instead of a

plastic ring.

Thirdly, the difference in time between having a plastic lid and tweezers and having no lid on

the funnel was investigated since it is common to put a lid on the funnel during the analysis.

The procedure was repeated five times with the non-damaged frits.

To investigate if there is any difference between using different filters, six different filters

including the filter that is currently used were compared. Each filter was tested five times and

the time it took for the water to run through was noted. The different filters can be seen in

table 1, where filter 1 is the filter currently used.

Table 1. The different filters used in the comparative study and data related to each filter.

Filter Manufacturer Name Pore

size Material Colour

1 GE Healthcare Life

Sciences

Nuclepore, Track-Etch

Membrane 0,2 µm Polycarbonate White

2 GE Healthcare Life

Sciences

Cyclopore, Track-Etch

Membrane 0,2 µm Polycarbonate White

3 Sartorius Polycarbonate Track-Etched

Filters 0,2 µm Polycarbonate White

4 Merck Millipore Isopore 0,2 µm Polycarbonate White

5 Merck Millipore Isopore 0,2 µm Polycarbonate Black

6 Merck Millipore Millipore Express 0,22

µm Polyethersulfone White

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Pumps

The pump that is currently used (pump 1) was compared with a different pump, see table 2.

Six positions and 200 mL of tap water were used. The test was run and tested five times on

the current pump, but the second pump stopped after two tries and therefore the test was only

run two times with the second pump.

Table 2. Data related to the two pumps tested.

Pump Manufacturer Type

1 KNF N035.1.2AN.18

2 Millipore EZ-Stream

Pump

Manifolds

Four different manifolds were also compared which can be seen in table 3. The test was run

five times when possible, and 200 mL of tap water were used on each manifold. The current

manifold (manifold 1) and manifold 4 were then tested again three times on each manifold.

Two positions were also added to the current manifold to investigate if there is a big

difference in speed between using six positions or eight positions.

Table 3. Data related to the four different manifolds that were tested.

Manifold Manufacturer Name

1 Whatman MBS1

2 Whatman AS600

3 Merck Millipore EZ-Fit

4 Sartorius Microsart Manifold

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Sterilization

To make the analysis even more time efficient and bring less damage to the steel frits, a new

way of sterilizing the steel frits between samples was tested. The current way is performed by

taking the frits out and placing them in 96% ethanol, cleaning the part of the manifold that is

under the frits, picking up the frits that are covered in ethanol and placing it in the fire before

putting the steel frits down on the manifold to cool off. The new way of sterilization

performed in this study is to spray 96% ethanol directly on the steel frits when it is still on the

manifold, and then use a lighter to put it on fire. In this way you never have to pick up the

steel frits which makes the procedure faster and most importantly, it does not damage the

sealing layer under the steel frits. To investigate if the new way of sterilization works as

effective as the current procedure, the two ways were compared by filtrating Pseudomonas

aeruginosa and thereafter sterilized water to see if any bacteria was still present after

sterilization. The frits were first sterilized the current way and 100 mL of sterile water was

then filtrated through a 0,45 µm filter (Whatman), the frits were then sterilized the same way

and 9 mL of P. aeruginosa, and PBS were filtered, the frits were then sterilized the same way

again before sterile water was filtrated. The three filtrations were then executed again the

same way but this time with the new way of sterilization between the filtrations. These

procedures were performed three times per way of sterilization, which makes a total of 18

plates. The filters were placed on CN plates and incubated in 37℃. They were supposed to

incubate for two days but were examined after one day since the result was already complete

but were also examined after two days. After an unexpected result, these procedures were

performed again the same way but this time on 22 plates and with 50 mL of sterile water.

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Recovery of bacteria

The currently used filter named Nuclepore (GE Healthcare Life Sciences, now called Cytiva)

was compared with Cyclopore (GE Healthcare Life Sciences) and Millipore Express (Merck

Millipore) to investigate the recovery of Legionella. In order to do so, 20 different water

samples were tested on all three filters, the same way as the analysis is normally performed on

ECCA. Ten samples were tested as matrix A and ten samples were tested as matrix B. One

filter was tested at the time, 100 mL of the sample was run through the filter, except for four

samples where there was less than 300 mL, 50 mL was used. The samples were finally spread

on cultivating plates after the different treatments, incubated in 37℃ and examined after 4

and 10 days. The Nuclepore and the Cyclopore filter were then compared again using 10

samples from matrix B and filtering 100 mL of the water.

Matrix A

After the sample had completely run through the filter, the filter was placed in a tube

containing 9 mL PBS, and vortexed for ten minutes. This procedure makes the concentrated

sample. A volume of 200 µL of the concentrated sample, containing possible bacteria was

spread on a GVPC and a BCYE+cys agar and 1 mL of the concentrated sample was then put

into 9 mL of Legionella Acid Buffer (HCl-KCl-Buffer; pH 2.2). After five minutes the tube

with the acid was vortexed and 200 µL were placed on a GVPC and BCYE+cys agar. The

volume left in the concentrated tube was transferred into a plastic tube and placed in a 50℃

water bath for 30 minutes. A volume of 200 µL of the concentrated and heated sample was

then spread on a GVPC and BCYE+cys agar. The same volume of the sample was also

directly put on a BCYE+cys and a BCYE+AB plate. These procedures were performed on all

three filters with the same 20 samples. The direct plating was only performed once since that

has nothing to do with the filters and would be the same procedure all three times.

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Matrix B

The samples were filtrated, and the filter was put in 9 mL of PBS and vortexed for 10

minutes, thereafter 200 µL from the concentrated sample was spread on a GVPC agar. A

volume of 1 mL of the concentrated sample was then added to 9 mL of Legionella Acid

Buffer (HCl-KCl-Buffer; pH 2.2), and after 5 minutes the acid sample was vortexed and

spread on another GVPC agar. What was left from the concentrated sample was added to a

plastic tube and heated for 30 minutes in the water bath, and thereafter 200 µL of the heated

sample was then spread on a GVPC agar. A volume of 3 mL of water directly from the

sample was also added a plastic tube and heated in the water bath for 30 minutes. Thereafter 1

mL of the heated sample was put in 9 mL of acid and 200 µL was spread on a GVPC agar

after 5 minutes. A volume of 200 µL was also spread on GVPC plates from the heated tube

and directly from the sample. All the direct samples were also spread on another GVPC plate

but after being diluted with PBS 10-1. The direct plating was only performed once since it had

nothing to do with the filters and would be the same procedure all three times.

Reading the plates

After four days the plates were examined for the first time. The colonies that were believed to

be Legionella were spread on a BCYE+cys agar and a COS-cys agar and incubated for three

days. If the colonies were growing on the BCYE+cys plate it was tested for serological

confirmation. If it grew on both plates, it was considered not to be Legionella and no further

tests were performed. If there was no growth on either, the plates were incubated again for

two more days.

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Serological confirmation

The serological confirmation was performed on a kit named Virapid Legionella Culture

(Vircell). Eight drops of the buffer were placed in an Eppendorf tube and the colonies

believed to be Legionella were picked up with an inoculation loop and mixed with the buffer.

Thereafter 90 µL of the solution were placed at the two application sites. The result was read

after 30 minutes.

Statistics

To present the spread of the results, the coefficient of variation was calculated on each

different setting. An unpaired two-tailed t-test with a significance level at 0,05 was used to

compare the different settings and investigate what variables that are statistically significant to

be true. This is presented by the p-value of all comparisons.

Results

The aim of this study was to optimize ECCA Laboratory’s analysis for detection of

Legionella. In order to do this, the setup that achieved the fastest filtration speed was

established. This includes trying out what equipment is optimal, a new way of sterilization

that will not damage the equipment and investigation if the faster filters pick up as much

bacteria as the one currently used. When nothing is mentioned about the state of the steel frits,

non-damaged steel frits are used.

Equipment and settings

Different variables were tested by using the different settings, and time the water filtration

five times. All settings described in table 4 were tested on one position on manifold 1, and

with filter 1, which is the current filter.

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Table 4. The filtration rate, loss of vacuum, average filtration time, coefficient of variation

and p-value for the different settings and comparisons.

Settings

Volume

water

filtrated

(mL)

Average,

loss of

vacuum

(bar)

Average,

time

(sec)

s/ml Standard

deviation CV% P-value

Damaged

steel frits 100 0,138 190,2 1,902 24,139 12,7 P=0,010146

Non-

damaged

steel frits

100 0 146,8 1,468 16,084 11,0

Damaged

steel frits,

with one

plastic ring

100 0,096 147 1,47 41,755 28,4 P=0,450184

Damaged

steel frits

with two

plastic

rings

100 0,072 162,6 1,626 13,686 8,4

Non-

damaged

steel frits,

with one

plastic ring

100 0,002 116,2 1,162 31,586 27,2 P=0,199517

Non-

damaged

steel frits,

with two

plastic

rings

100 0 136,8 1,368 9,338 6,8

Non-

damaged

steel frits,

with

plastic lid

100 0 140 1,4 6,633 4,7 P=0,028582

Non-

damaged

steel frits,

without

plastic lid

100 0,014 98,4 0,984 34,268 34,8

18

To investigate if any improvement was made when adding the plastic ring to the damaged

steel frits, a comparison between the “Damaged steel frits” and the “Damaged steel frits, with

one plastic ring” was made, resulting in a p-value at 0,080163.

Damaged frits were also compared with non-damaged frits on six positions once. The time it

took for the water to filter on the damaged frits were 59 minutes and 4 seconds with an

average loss of vacuum at 0,47 bar. When using non-damaged steel frits, it took 3 minutes and

35 seconds and a loss of vacuum at 0,15 bar.

A rubber ring was also tested instead of a plastic ring. This time, 200 mL, and a filter with a

pore size of 0,45 µm (Whatman) were used. Each setup was tested two times except non-

damaged steel frits with rubber ring, which was tested once, hence an t-test could not be

made. The t-test comparing damaged frits with and without rubber ring resulted in a p-value

at 0,203883. All results are shown in table 5.

Table 5. The filtration rate, loss of vacuum, average filtration time, coefficient of variation

and p-value for the effect of adding a rubber ring.

Settings

Volume

water

filtrated

(mL)

Average,

loss of

vacuum

(bar)

Average,

time (sec) s/ml

Standard

deviation CV% P-value

Non-

damaged

frits,

without

rubber ring

200 0 32,5 0,1625 3,536 10,9 -

Non-

damaged

frits, with

rubber ring

200 0 35 0,175 - -

Damaged

frits, 200 0,105 43 0,215 2,828 6,6

P=0,20388

3

19

without

rubber ring

Damaged

frits, with

rubber ring

200 0 35,5 0,1775 4,95 13,9

The filtration speed of the six different filters were compared by filtering 100 mL of water

through the different filters five times each, see table 6. Comparisons between filter 1, which

is currently used, and each filter were made and the p-value for each comparison when

calculated. The filters that were statistically significant faster than filter 1 were filter 2, 3 and

6.

Table 6. The result of the filtration speed of the six filters. The filtration rate, loss of vacuum,

average filtration time, coefficient of variation and p-value for the different comparisons are

presented in the table.

Filter

Volume

water

filtrated

(mL)

Average,

loss of

vacuum

(bar)

Average,

time (sec) s/ml

Standard

deviation CV% P-value

1 100 0,014 98,4 0,98 34,268 34,8 -

2 100 0 39,6 0,396 3,782 9,6 P=0,005136

3 100 0 59 0,59 4,416 7,5 P=0,034182

4 100 0 71,8 0,718 5,263 7,3 P=0,124575

5 100 0 99,2 0,992 12,617 12,7 P=0,962131

6 100 0 18,4 0,184 0,548 3,0 P=0,000803

Pumps

The currently used pump (KNF) was compared to a new one (Millipore) by letting 200 mL of

tap water run through filter 2 in all six positions five times with non-damaged steel frits. The

average time for pump 1 was 130 seconds, with no loss of vacuum, with a standard deviation

at 18,028 and a CV% at 13,9. On the first try of the second pump it took 316 seconds for the

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water to filter, but on the second try the pump stopped filtrating and the experiment was

aborted.

Manifolds

Four different manifolds were compared by using filter 2, the test was performed on one and

six positions five times and 200 mL of tap water was filtrated. Since the setting for manifold

1, which is the manifold currently used, was the same as when trying out pump 1, the results

for manifold 1 are taken from the previous test in the first try with six positions. Manifold 2

was leaking; therefore, the test could not be performed. The test of manifold 3 was aborted

after one try because of its many disadvantages and therefore the manifold would not be used

in routine. Since manifold 1 and 4 were the only ones that were tested, they are the only

manifold presented in the table. The results from the different manifolds can be seen in table

7, which shows that there is a statistically significant difference in time between manifold 1

and 4 using six positions.

Table 7. The filtration rate, loss of vacuum, average filtration time, coefficient of variation

and p-value for manifold 1 and 4 on one and six positions.

Settings

Volume

water

filtrated

(mL)

Average,

loss of

vacuum

(bar)

Average,

time (sec) s/ml

Standard

deviation CV% P-value

Manifold 1,

one position 200 0 78,8

0,39

4 8,556 10,9

P=0,32701

5

Manifold 4,

one position 200 0 74 0,37 5,701 7,7

Manifold 1,

six positions 200 0 130 0,65 18,028 13,9 P=0,00047

Manifold 4,

six positions 200 0 84 0,42 2 2,4

21

Manifold 1 and 4 was tested three more times with six positions but this time with filter 6,

which can be seen in table 8. This time no statistically significant difference could be

measured.

Table 8. The result from the comparison of manifold 1 and 4 using six positions.

Manifold

Volume

water

filtrated

(mL)

Average,

loss of

vacuum

(bar)

Average,

time

(sec)

s/ml Standard

deviation CV% P-value

1 200 0 39,333 0,197 2,082 5,3 P=0,851237

2 200 0 39 0,195 2 5,1

Two extra positions were also added to manifold 1. The experiment was performed three

times with 200 mL of tap water through filter 6. There was no loss of vacuum, the average

time it took for the water to run through filter 6 was 48,667 seconds with a standard derivation

of 1,155 and CV% at 2,4.

Sterilization

To investigate if the new way of sterilization was as efficient as the current way of

sterilization, P. aeruginosa was filtrated after the current way of sterilization and the new way

of sterilization. In between the sterilizations, sterile water was filtrated to investigate if the

sterilizations were effective. After incubation of the first 18 plates, every plate turned out as

expected expect one plate which showed growth of P. aeruginosa after sterilization with the

new way. When the test was performed again, all plates came out of incubation as expected.

Recovery of bacteria

The two filters that filtered the water the fastest were compared with the currently used filter.

Nuclepore (GE Healthcare Life Sciences), referred to as filter 1 is the filter that is currently

22

used and was compared with Cyclopore (GE Healthcare Life Sciences), referred to as filter 2,

and Millipore Express (Merck Millipore), referred to as filter 6. The recovery of L.

pneumophila was calculated to 64,86% on Millipore Express and 96,80% on Cyclopore

compared with the amount on Nuclepore. When comparing Nuclepore and Cyclopore again,

Cyclopore reached a higher amount of L. pneumophila than Nuclepore which was calculated

to 131,80%.

Discussion

When concentrating a sample for detection of L. pnemophila the sample needs to run through

a filter, this can be very time consuming depending on the concentration of interfering

microorganisms and on how high the presence of dirt is. However, there are several other

variables that make the water filter slowly. The aim of this study was to find the most

effective setup and optimize Laboratory ECCA’s analysis for detection of L. pnemophila.

The method used in this study was to change the setup and note the time it takes for a set

volume of water to filter through, and in most cases the same setting was tested five times.

This is an easy and simple method for investigating the fastest setup, but there are some

disadvantages with only doing five tries. In some cases, the CV% was as high as almost 35 %

which indicated a big spread in the results, the high CV% could have been avoided by more

attempts. More tries would also result in values closer to the mean and would therefor lead to

a smaller SD of mean [10]. However, almost 83 % of the different settings that were tested

had a CV% lower than 20 %.

To examine which settings that are statistically significant, an unpaired two-tailed t-test was

performed with a significance level at 0,05. There is a big difference between using damaged

23

steel frits and non-damaged steel frits and it is when using the damaged steel frits the loss of

vacuum is the highest. The loss of vacuum is reduced when adding a plastic ring under the

frits, but the time difference is not big enough to be statistically significant, neither when one

nor two plastic rings are added. There is also no statistic evidence that adding a rubber ring

under the frits reduces the filtration time. Adding a plastic lid on top of the funnel on the other

hand, seems to have a negative impact on the filtration speed.

The filters that are statistically significant to be faster than Nuclepore (GE Healthcare Life

Sciences), are Cyclopore (GE Healthcare Life Sciences), Polycarbonate Track-Etched Filters

(Sartorius) and Millipore Express (Merck Millipore). The two fastest filters where Cyclopore

and Millipore Express which was also compared with Nuclepore about the recovery of

bacteria. The Millipore Express had the fastest filtration speed by far, but the recovery of

bacteria was calculated to 64,86% compared with Nuclepore, while Cyclopore reached

96,80%, and later Cyclopore reached an even higher amount of recovery at 131,80% when

compared with the currently used filter.

Because the damage of the steel frits causes a large loss in vacuum and therefor a severe

reduction in filtration speed, a new way of sterilizing the steel frits was investigated where the

frits were not as damaged. This was made by letting P. aeruginosa run through a filter,

sterilizing, and then letting sterile water run through a filter. The current and the new way of

sterilizing was tested between the filtrations. On the first attempt one plate had bacterial

growth after being sterilized the new way, the experiments were repeated, and the result came

then out as expected. What caused the growth on the plate after sterilization is hard to tell, but

one possibility could be that there was not enough ethanol sprayed on the frits. The new way

24

of sterilizing seems to be as effective as the current way, but it is important to do the

sterilization properly in order to eliminate the risk of bacterial contamination.

When comparing the currently used pump (KNF) with a new pump (Millipore), the new pump

stopped filtrating after the first try. The reason for this was never established, but since it was

slower than the currently used one in the first try and because it stopped without a valid

reason, the first pump was concluded to be the most effective. When comparing the

manifolds, manifold 1 was leaking and the test of manifold 3 was aborted since it did not fit

the analysis, for example it had a funnel that could only fill 100 mL. The currently used one

(Whatman) and the manifold from Sartorius was the only ones that were compared, and the

results initially showed that the manifold from Whatman is not statistically significant to filter

the water faster on one position, but on six positions the time difference increases and the

manifold from Millipore, filters the water faster. When the two manifolds were compared

again on six positions the result showed no difference between the manifolds on six positions

either, this makes the result difficult to interpret, and further studies would be necessary to

find out which manifold that is better.

From a sustainability perspective this study has a small impact on the environment. The

concentrated and the acid sample are autoclaved before thrown out to reduce the number of

bacteria discarded, the untreated samples are discarded directly in the sink since the

concentration of bacteria is not as high. The purpose of this analysis is to discover Legionella

so that measures can be taken and hopefully eliminate the bacteria from the source that the

sample was collected from. The analysis is therefore important to maintain a low

concentration of Legionella to protect humans and nature.

25

There are not many previous studies in this research field which may be because this study

focuses on the equipment and settings on ECCA Laboratory in particular. However, there

have been studies made comparing different filters for detection of Legionella, that have

reached the conclusion that the filter made from polysulfone had a higher filtration speed, but

the filter made from polycarbonat had some better recovery of bacteria [11]. This study

confirms previous studies since the only filter made of different material than polycarbonate

was the Millipore express filter, made of polyethersulfone, which was the filter that filtrated

the water the fastest but had a low recovery of bacteria.

ECCA Laboratory is using a method containing conventional culture for detection of

Legionella. A different method that could be used is PCR which would mean no water needs

to be filtrated and the issues with slow filtration speed would be eliminated. No incubation

would have to be made which would result in a much quicker analysis with fewer steps.

However, though there are advantages with this method, it comes with disadvantages. When

using a method of conventional culture, the sensitivity can be improved by filtrating a larger

volume of water, which cannot be applied on PCR [12]. A low sensitivity results in a low

positive predictive value which means that the probability that a positive result truly is

positive gets lower. A positive result in PCR would therefore still need to be confirmed with a

conventional culture method [13]. Furthermore, PCR is significantly more expensive than

methods based on conventional culture [14].

To summarize, this study showed easy-to-interpret results, which can lead to a clear change in

the setup, but also results that were more difficult to interpret. What is clear is that damage

steel frits lead to loss in vacuum and has a very big impact on the filtration speed.

Unfortunately, putting rings of different material to seal the leak seem to have no effect. The

26

filter Cyclopore was the second to fastest filter with a high recovery of bacteria and is

therefore clearly the best filter used in this study. The results from the manifolds are unclear

since they show a statistically significant difference in some of the tests but not in all.

However, changing the manifold would mean much work and not only the manifolds but new

material as funnels would have to be purchased. Considering this, and the unclear result, it

would not be worth changing the manifold which makes the currently used one (Whatman)

the most efficient.

In conclusion, the setup that would lead to the fastest filtration speed for detection of L.

pneumophila in ECCA Laboratory is the filter Cyclopore, the manifold MBS1 (Whatman) and

the pump from KNF. The filtration should be made without plastic lids on top of the funnels

and the new way of sterilizing should be performed to lower the damage of the steel frits.

However, the dissemination of certain results was high in some cases and further studies

would be necessary to gain more reliable results.

Acknowledgements

Thanks to ECCA Laboratory for the opportunity to work with them and to Alexander De

Meyer for supervising me in this project.

27

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