Accepted Manuscript

Growth ability of gram negative bacteria in free-living amoebae

Zuhal Zeybek, Ali Rıza Binay

PII: S0014-4894(14)00147-7DOI: http://dx.doi.org/10.1016/j.exppara.2014.06.009Reference: YEXPR 6893

To appear in: Experimental Parasitology

Received Date: 22 December 2013Revised Date: 4 June 2014Accepted Date: 12 June 2014

Please cite this article as: Zeybek, Z., Binay, A.R., Growth ability of gram negative bacteria in free-living amoebae,Experimental Parasitology (2014), doi: http://dx.doi.org/10.1016/j.exppara.2014.06.009

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GROWTH ABILITY OF GRAM NEGATIVE BACTERIA IN FREE-LIVING AMOEBAE

Zuhal ZEYBEK*, Ali Rıza BİNAY

Istanbul University, Science Faculty, Biology Department, Fundamental and Industrial

Microbiology Division 34134, Vezneciler - Istanbul, Turkey

*Corresponding author

E-mail: zeybek@istanbul.edu.tr

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ABSTRACT

When bacteria and free-living amoebae (FLAs) live both in natural waters and man-

made aquatic systems, they constantly interact with each other. Some bacteria can

survive and grow within FLAs. Therefore, it has recently been thought that FLAs play

an important role in spreading pathogenic bacteria in aquatic systems. In this study we

investigated the intracellular growing ability of 7 different Gram-negative bacteria

(Pseudomonas fluorescens, Pseudomonas putida, Pasteurella pneumotropica,

Aeromonas salmonicida, L. pneumophila serogroup 1, L. pneumophila serogroup 3, L.

pneumophila serogroup 6) in four different FLA isolates (A1, A2, A3, A4). Among

these, four bacterial isolates (Pseudomonas fluorescens, Pseudomonas putida,

Pasteurella pneumotropica, Aeromonas salmonicida) and two free-living amoebae

isolates (A3, A4) were isolated from the tap water in our city (Istanbul). It was found

that 4 different Gram-negative bacteria could grow in A1, 2 different Gram-negative

bacteria could grow in A2, 4 different Gram-negative bacteria could grow in A3, 1

Gram-negative bacterium could grow in A4. In conclusion, we think that this ability of

growth could vary according to the characteristics of both bacteria and FLA isolates.

Also, other factors such as environmental temperature, bacterial concentration, and

extended incubation period may play a role in these interactions. This situation can be

clarified with future studies.

Key words: Gram-negative bacteria, free-living amoebae (FLAs), intracellular growth,

tap water, Istanbul.

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1. INTRODUCTION

Numerous microorganisms such as bacteria and free-living amoebae (FLAs) live

together both in nature and man-made aquatic systems (Bastian et al., 2009; Üstüntürk

et al., 2010; Burak and Zeybek, 2011; Üstüntürk and Zeybek, 2012; Türkmen, 2012).

When these microorganisms live in the same aquatic environment, they interact with

each other in different ways. Some bacteria are phagocytosed and used as food by FLA

while others can have suppressor/ lethal effects on these amoebal cells. Amoeba

Resistant Bacteria (ARB) entering to the amoebal cells due to unsuitable environmental

conditions (antibiotics, biocides, disinfectants etc.) grow through the mechanisms they

develop in order to survive to phagocytosis, and lyse their hosts and eventually spread

to the environment in large numbers (Tyndall and Domingue, 1982; Wadowsky et al.,

1988; Wang and Ahearn, 1997; Andra et al., 2003; Thomas et al., 2006; Thomas et al.,

2008). Free-living amoebae have gained significant attention for the role they play in

spreading pathogen bacteria in aquatic systems, in addition to their pathogenic activity

as described by many studies in the literature (Rowbotham, 1980; Brown and Barker,

1999; Molmeret et al., 2005; Thomas et al., 2006 ). Legionella pneumophila,

Mycobacterium spp., Francisella tularensis, Escherichia coli O157, Afipia felis,

Rickettsia pickettii, Pseudomonas spp., Burkholderia cepacia are listed among these

bacteria and Acanthamoeba, Hartmannella, Naegleria, Vhalkampfia are listed among

these free-living amoebae (Walochink et al., 1999; Landers et al., 2000; Winiecka-

Krusnell and Linder, 2001; Greub and Raoult, 2004; Molmeret et al., 2005; Thomas et

al., 2006; Decklerck et al., 2007). Research studies have also shown that nonpathogenic

bacteria gained pathogenic features after replication within free-living amoebae (Barker

et al., 1995; Brown and Barker, 1999; Winiecka-Krusnell and Linder, 2001).

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We detected, in our previous studies, Gram-negative rod-shaped bacteria and FLA are

abundant in home tap water samples (Burak and Zeybek, 2011; Üstüntürk and Zeybek,

2012), swimming pools (Türkmen, 2012) and dental unit water systems (Üstüntürk et

al., 2010) in Istanbul. We investigated whether Gram-negative rod-shaped bacteria

isolated from tap water in Istanbul can survive/grow in FLA or not in this study. This

study reports, for the first time, the observed interaction between Gram-negative

bacteria and FLA in our country.

2. MATERIALS AND METHODS

2.1 Bacteria:

L. pneumophila serogroup 1 (ATCC 33152), L. pneumophila serogroup 3 (ATCC

33155), L. pneumophila serogroup 6 (ATCC 33215), which are bacteria of Gram-

negative rod-shaped morphologies, were tested in the experiments. Also other

Gram-negative bacteria (Pseudomonas fluorescens, Pseudomonas putida,

Aeromonas salmonicida, Pasteurella pneumotropica strains) isolated from tap

waters in Istanbul were selected and used in this study.

2.2 Preparation of bacterial suspensions:

Each bacterial strain kept in -86 °C were thawed and L. pneumophlia bacteria were

plated on buffered charcoal yeast extract (BCYE) agar. Other Gram-negative rod-

shaped bacteria were plated on MacConkey agar. All petri dishes were incubated at

37 °C. The day when growth was observed, bacterial suspensions (1x108 CFU/mL)

were prepared for each bacteria seperately ( Feeley et al., 1979 ).

2.3 Free-Living Amoebae:

Acanthamoeba castellanii coded A1 (taken from Cumhuriyet University, Faculty of

Medicine), Acanthamoeba castellanii coded A2 (ATCC 50373), and 2 unnamed

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types of free-living amoeba cells (A3, A4) isolated from tap waters in Istanbul were

used in this study (Table 1). All cultures were kept on non-nutrient agar (NNA) at

+4 °C and subcultured to fresh NNA in a petri dish before use. For this purpose,

firstly a dense suspension was prepared from 18-24 hours culture of Escherichia

coli in the Page’s Amoeba Saline (PAS) solution and was inactivated at 121 °C in

an autoclave for 20 minutes. Then 200 µL of the suspension was spread on Petri

dishes with NNA. Finally, 1 cm2-pieces of each free-living amoeba cultures on

NNA were cut using a sterile lancet and were turned upside down and placed on the

surface of each fresh NNA. After making sure the pieces stuck to the agar, they

were incubated at 28 °C. All petri dishes were examined daily under light

microscope (x100) for the presence of trophozoites (HPA, 2005).

2.4 Preparation of Suspension for Free-Living Amoebae:

As explained above, the day when trophozoites were observed in petri dishes under

light microscope, 5 mL of PAS was added to petri dishes and amoeba cells were

gently harvested from the surface of agar. Then, they were centrifuged at 1000 ×g

(max. 3000 ×g) for 10 minutes. After the supernatant was discarded, the pellet

containing amoeba cells was resuspended by adding sterile PAS and suspensions

were prepared to a final concentration of 105 cells per mL.

2.5 Co-cultures of Bacteria and Free-Living Amoebae:

The co-cultures of bacteria and free-living amoebae assay were modified as based

on the method described by Moffat and Tompkins (1992). 1 mL portion of each

free-living amoeba suspension was distributed to each well of a 24-well tissue

culture dish and was incubated at 28 °C for an hour in order to allow the cells to

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adhere. Then, 100 µL of Gram-negative bacterial suspension (1x108 CFU/ml) was

added to the each well containing the amoeba cells, in a separate manner. Co-

cultures prepared in this way were incubated at 28 °C for an hour. Then, PAS inside

the wells was carefully aspirated and was replaced with PAS containing gentamicin

(100 µg/mL) to kill the extracellular bacteria and were incubated at 28 °C for an

hour. Then the antibiotic-containing PAS inside the wells was carefully removed

and they were washed 3 times with sterile PAS without antibiotics. (PAS was added

into the other wells, except the first three wells, and then was incubated for the

course of 24, 48 and 72 hours). After washing, 1000 µL portions of distilled water

were added to the first three wells and all content of wells were taken from the well

and were transferred to sterile test tubes separately. This time point was denoted as

zero (0.) hours. 500 µL portion of the liquid in test tubes was used as “lysis of

amoeba cells,” which will be explained later on. The remaining liquid was used for

the cultivation of Legionella bacteria and other Gram negative bacteria on BCYE

agar and MacConkey agar, respectively, to control if bacteria outside of amoeba

cells were inhibited by the antibiotic. All cultures were incubated at 37 °C for 3-14

days for Legionella bacteria and 24-48 hours for other Gram-negative rod-shaped

bacteria. Colonial growth on all agar media was examined at the end of the

incubation periods.

Other co-cultures in the 24-well tissue culture dishes incubated for 24-48-72 hours

were washed three times with PAS at the end of each period and all the process

explained above were repeated separately (Moffat and Tompkins, 1992; Bozue and

Johnson, 1996: Harb et al., 1998; Greub and Raoult, 2004). All experiments were

done in triplicate.

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2.6 Lysis of Amoeba Cells

For the lysis of amoeba cells the liquid in the test tube was placed for 15 minutes at

-80 °C, followed by 20 minutes at 37 °C, and 15 minutes at -80 °C. At the end of

the period, 10 µL portions were plated on BCYE agar and MacConkey agar for

Legionella bacteria and other Gram negative bacteria, respectively. All Petri dishes

were incubated at 37 °C for 3-14 days for Legionella bacteria and 24-48 hours for

other Gram negative rod-shaped bacteria and bacteria colonies were counted. These

counts were recorded as the zero hour bacteria counts after the lysis of amoeba

cells. The amoeba cells breaking process for the zero hour was repeated after the

24-48-72 hour incubations of co-cultures in other wells (Moffat and Tompkins

1992: Lück et al., 1998; Berk et al., 1998). Processes performed for every hour

were done in triplicate.

Control wells were set up in order to investigate whether or not the antibiotics used

have inhibiting effects on the bacteria and amoeba cells tested. For this purpose,

each of bacterial suspensions prepared in PAS were dispensed into 3 different wells

in order to control the bacterial survival in PAS. As seen in Section 2.4, each

amoebal cell suspansions prepared in PAS were pipetted into different three wells

in order to observe the viability of these cells during the experimental period (72

hours). Also, susceptibility of gentamycine of all tested amoeba were checked in

three different wells containing this antibiotic and each amoebal cells. Microplates

including all control wells were incubated during the experimental period. They

were examined microscopically at the end of every incubation period. Also

susceptibility of gentamycine of all tested bacteria was done according to disc

diffusion assay.

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RESULTS

4 free-living amoeba (FLA) isolates and 7 Gram-negative rod-shaped bacteria (GNCB)

isolates were used for the present study. These microorganisms are shown in the Table.

Figure 1,2,3 and 4 show only the results of intracellular growth of bacteria in tested

amoebae.

Table. Free living amoebae and Gram negative rod-shaped bacteria used in experiments.

It was found that Pseudomonas fluorescens replicated in amoebal cells coded A1 and

A3. Pseudomonas putida showed a fast burst of growth (48 hour) and then disappeared

(72 hour) in A1. Pasteurella pneumotropica replicated in amoebal cells coded A1, A2

and A3. Aeromonas salmonicida replicated in amoebal cells coded A1 only.

Furthermore, L. pneumophila serogroup 1 was observed only in A2, L. pneumophila

serogroup 6 only in A3 while L. pneumophila serogroup 3 was observed in A3 and A4

after 24 hours and then disappeared quickly after 48 hours (Figure 1, 2, 3, 4).

Four bacterial isolates (Pseudomonas fluorescens, Pseudomonas putida, Pasteurella

pneumoptropica, Aeromonas salmonicida) were observed in amoebal cells coded A1 in

various periods although P. fluorescens and P. putida disappeared at 72 hours (Figure

1). Two bacterial isolates (Pasteurella pneumotropica, L. pneumophila serogroup 1)

replicated in amoebal cells coded A2 up to 72 hours (Figure 2). Four bacterial isolates

(Pseudomonas fluorescens, Pasteurella pneumotropica, L. pneumophila serogroup 6, L.

pneumophila serogroup 3) were observed in amoebal cells coded A3, while the presence

of L. pneumophila serogroup 3 was not observed after 48 hour (Figure 3). One bacterial

isolate (L. pneumophila serogroup 3) was observed in amoebal cells coded A4 at 24

hour and then disappeared at 48th hour (Figure 4). Pasteurella pneumotropica,

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Aeromonas salmonicida, L. pneumophila serogroup1, L. pneumophila serogroup 6 were

detected in at least one of FLA isolates coded A1, A2, A3 even after 72 hours although

none of the bacteria tested were detected in the FLA coded A4 in this period.

Control wells containing the PAS suspensions of each free-living amoebal cells tested

were examined with invert microscope every day through the experiments and it was

observed that the cellular structures in these cells remained intact until the end of the

experiments. It was understood that each bacterial strains tested survived in PAS when

the growth was observed in the cultivations in suitable agar media in each experimental

hour. It was detected that each bacteria was inhibited by the test antibiotics with disk

diffusion method and that amoebal cell morphologies remained intact in the antibiotic

concentration during the invert microscope examinations performed every day.

Fig. 1. Growth of Pseudomonas fluorescens (G1), Pseudomonas putida (G2) Pasteurella pneumotropica

(G3) and Aeromonas salmonicida (G4) in A. castellanii (A1) cells

Fig. 2. Growth of Pasteurella pneumotropica (G3), L. pneumophila serogroup 1 (G5) in A. castellanii

(A2) cells

Fig. 3. Growth of Pseudomonas fluorescens (G1), Pasteurella pneumotropica (G3), L. pneumophila

serogroup 3 (ATCC 33155) (G6), L. pneumophila serogroup 6 (ATCC 33215) (G7) in A3 cells

Fig. 4. Growth of L. pneumophila serogroup 3 (ATCC 33155) in A4 cells

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DISCUSSION AND CONCLUSION

Studies performed in recent years on free-living amoebae have revealed their host role

for various bacteria in addition to infections induced by these amoebae. The first

research study that proved the relationship between some Legionella bacteria and free-

living amoebae like Acanthamoeba and Naegleria was conducted by Rowbotham

(1980). In later years, numerous studies have shown that the Legionella bacteria,

intracellular parasites, grew and survived inside protozoa (Wadowsky et al., 1988;

Moffat and Tompkins, 1992; Bozue and Johnson, 1996; Kwaik, 1996; Steinert et al.,

1997; Greub and Raoult, 2003). It was also shown that free-living amoebae were

infected by many amoeba-resistant bacteria (ARB) other than Legionella in water

environments like drinking water, tap water, swimming pools, and cooling towers

(Michel et al., 1998; Greub and Raoult, 2004; Molmeret et al., 2005; Hundt and

Ruffolo, 2005; Thomas et al., 2006; Pagnier et al., 2008). These findings confirm with

the findings from the present study that indicate Pseudomonas fluorescens,

Pseudomonas putida, Pasteurella pneumotropica, Aeromonas salmonicida, isolated

from the tap water used in the present study, can survive / grow inside tested free-living

amoebae. L. pneumophila serogroup 1 Philadelphia strain (ATCC 33152), used in the

present study, was able to grow inside A. castellanii (ATCC 50373), coded A2, for 72

hours, which is the maximum experimented time. On the other hand, the same bacteria

failed to grow inside A. castellanii, coded A1, and the unnamed free-living amoebae,

coded A3 and A4. In addition, it was detected that Legionella pneumophila serogroup 3

(ATCC 33155) strain grew inside the free-living amoebae coded A3 and A4 for up to 24

hours, while it did not grow in A. castellanii (A1, A2). It was found that Legionella

pneumophila serogroup 6 (ATCC 33215) did not grow in the free-living amoebae A.

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castellanii (A1, A2) and A4 but grew in the free-living amoeba coded A3. The literature

survey (Rowbothom, 1980; Bozue and Johnson, 1996; Steinert et al., 1997; Michel et

al., 1998) has yielded that the Legionella strains used in these studies were of different

strains of the same type as the strains used in the present study. These results indicate

that the growth ability of different L. pneumophila strains have varied in different free

living amoebal strains. All these data points out that the ability of growth of bacteria

inside free-living amoebae can vary from species to species.

It is thought that various properties of protozoan hosts and the mechanism of interaction

of protozoa and bacteria can be effective in the replication of bacteria in free-living

amoebae (Kwaik, 1996; Bozue and Johnson, 1996; Walochnik et al 1999; Molmeret et

al., 2005). It was detected in our study, out of the 7 bacteria tested, only 1 strain

(Legionella pneumophila serogroup 3) was able to grow inside free living amoeba

coded A3 and A4 in a short time. During the experiment, centrifuging and concentration

of coded A4 amoeba was fairly more difficult than amoebal cells coded A1, A2 and A3

and the problem was only overcome when the centrifuging speed and duration was

increased. It was found that more bacterial isolates grew inside amoebal cells coded A1,

A2 and A3. Therefore, it was ascertained that 4 different bacteria (P. fluorescens, P.

putida, P. pneumotropica, A. salmonicida) grew inside amoebal cell coded A1 (A.

castellanii), 2 different bacteria (P. pneumotropica, L. pneumophila serogroup 1) grew

inside amoebal cell coded A2 and 4 different bacteria (P. fluorescens, P.

pneumotropica, L. pneumophila serogroup 3, L. pneumophila serogroup 6) grew inside

amoebal cell coded A3. Similarly, Michel et al. (1998) reported that Naegleria sp. and

Hartmannella sp. could be infected successfully by Gram-negative bacteria. In contrast,

most strains of Willaertia sp., Vahlkampfia sp., Vannella sp., and Saccamoeba sp. were

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feeding on gram-negative bacteria instead of getting infected. Various researchers

indicated the presence of species Hartmannella, Naegleria, Vahlkampfia,

Paratetramitus, Adelphamoeba and Echinamoeba as well as Acanthamoeba in waters

(Thomas et al., 2008; Rohr et al., 1998; Muldrow et al., 1982; Thomas et al., 2006;

Declerck et al., 2007) and identified them with different techniques. Nowadays, the

identification of the amoebae has been done with different advanced molecular

techniques. So, we are planning to identify our unidentified free-living amoebal isolates

(A3, A4) used in the present study by these techniques in future studies.

We consider that capability of growth of bacteria in free-living amoebae depends on

environmental temperature. Walochnick et al. reported that temperature was apparently

of crucial importance for the interactions between these microorganisms (1999). Moffat

and Tompkins (1992) found that intracellular growth of virulent L. pneumophila and

other wild-type Legionella species was observed when the assay was performed at

37°C. At room temperature, none of the Legionella strains tested grew intracellularly,

while an avirulent L. pneumophila strain was unable to replicate in this assay at either

temperature. Consequently, once the bacteria entered the amoebal cell, only lowered

temperature could restrict replication. In our study, co-culture assay was tested at 28 °C.

We are planning to test the assay with same microorganisms at 30 °C and 37 °C in the

future studies.

Co-culture assay were done at maximum for 72 hours in our study. If the incubation

period is extended results may change. Because in a study it was shown that Naegleria

lovaniensis and Acanthamoeba royreba could use L.pneumophila as a sole food source.

On inoculation of L.pneumophila into axenic cultures of these amoebae, 99. 9% of the

L. pneumophila was destroyed within 24 h. After several weeks, however, some

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amoebal cultures became chronically infected and supported the growth of L.

pneumophila (Tyndall and Domingue, 1982).

Wang and Ahearn’s finding (1997) is also interesting on the survival and growth of A.

castellanii in the presence of bacteria. They reported that these amoebal cells were

markedly influenced by the species and densities of bacteria, especially Gram-negative

bacteria. Similarly, Weekers et al. (1993) found that amoebal growth, to some extent,

was detected in all test combinations, but E. coli K-12 proved to be a far better feed than

other tested bacteria. So, we think further studies are necessary for clarifying this

relationship using our isolates of free living amoebae and Gram-negative bacteria.

It is known that the free-living amoebae found in the water are resistant to the material

used in the disinfection of bacteria. Thanks to this resistance, they serve as an important

reservoir for various bacteria that cause diseases in humans. In addition, it was shown

that the bacteria become much more resistant to antibiotics once they take harbor inside

free living amoebae, they grow and lyse it. Thus, amoebae were described in the

different studies as “Trojan horses” of the microbial world, protecting bacteria from

unfavorable environmental conditions (Barker et al., 1995; Brown and Barker 1999;

Miltner and Bermudez, 2000). Therefore, we need new researches on the susceptibility

of each bacterial isolate (Pseudomonas fluorescens, Pseudomonas putida, Aeromonas

salmonicida, Pasteurella pneumotropica, Legionella pneumophila) against various

disinfectants (or antibiotics) prior to their intake to amoebal cells and following their

growth inside them.

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Isolation of Legionella bacteria from different samples such as water, sputum is

sometimes very difficult. Previous researches have shown the isolation of these bacteria

from clinical and environmental specimens via amoebae (Rowbotham, 1983; Sanden et

al., 1992). It was detected that Legionella, Pasteurella, Pseudomonas, Aeromonas

species, the tested bacteria in the present study are able to stay hidden for 0-72 hours

inside different amoebal cells. In the light of this information, a lack of growth observed

in microbiological analyses of any water sample for the culturing of these bacteria that

does not mean they are non-existent. In this case, it will be appropriate to examine the

same water examples for free-living amoebae and uncover any hidden bacteria inside

these amoebae with new methods. Moreover, free-living amoebae provide an alternative

research path to reveal the causes of diseases that cannot be detected. Additionally,

results obtained with these methods will also influence water system disinfection

processes. As is known, water disinfection is essential to remove the causes of

epidemics associated to water. However, especially the dose of the disinfectant used in

disinfection processes will not affect the bacteria hidden inside amoebal cells.

Moreover, it is possible that these bacteria will be more resistant when they lyse the

amoebae. As a result, it will be necessary to adjust/increase the dose of the disinfectant

used for the disinfection of water systems.

In conclusion, the free living amoebae that inhabit man-made aquatic systems play a

crucial role in the growth and transportation of Gram-negative rod-shaped bacteria in

the same environment. Survival of the Gram-negative rod-shaped bacteria inside free-

living amoebae depends on the species/types and concentration of microorganisms and

environmental conditions such as temperature. Because of the reservoir role of FLA for

some bacteria, water disinfection procedure should be checked in our city.

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Acknowledgment: This study has been supported by Istanbul University Scientific Research Projects Unit with no 3312 and BYP 29290, and was presented at 15th International Meeting on the Biology and Pathogenicity of Free Living Amoebae (FLAM 2013), Vienna, Austria.

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MICROORGANISMS

Code FLA Code Bacteria

A1 Acanthamoeba castellanii G1 Pseudomonas fluorescens

A2 Acanthamoeba castellanii (ATCC 50373) G2 Pseudomonas putida

A3 Unnamed isolate G3 Pasteurella pneumotropica

A4 Unnamed isolate G4 Aeromonas salmonicida

G5 L. pneumophila serogrup 1

(ATCC 33152)

G6 L. pneumophila serogrup 3

(ATCC 33155)

G7 L. pneumophila serogrup 6

(ATCC 33215)

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Highlights:

• The growth ability of different Gram negative rod bacteria in free living

amoebae

• The growth ability may vary according to the characteristics of microorganisms

• Three bacteria strains were detected within only one FLA strain after 72 hours