http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME09178

Microbes Environ. Vol. 25, No. 4, 266–274, 2010

Culturability and Survival of Marine, Freshwater and Clinical

Pseudomonas aeruginosa

NURUL H. KHAN1,2*, MAHBUBA AHSAN

1, WILLIAM D. TAYLOR2, and KAZUHIRO KOGURE

1

1Marine Microbiology Laboratory, Marine Ecosystem Dynamics, Ocean Research Institute, The University of Tokyo,

Tokyo 164–8639, Japan; and 2Department of Biology, Faculty of Science, University of Waterloo, 200 University

Avenue West, Waterloo, Ontario N2L 3G1, Canada

(Received November 14, 2009—Accepted July 3, 2010—Published online August 11, 2010)

Genetic typing of Pseudomonas aeruginosa isolated from the open ocean has revealed that marine strains formunique clusters. To clarify whether this genetic variation reflects differences in pattern of culturability and survival, amarine strain was compared with a freshwater strain and a clinical strain in microcosms with different levels of NaCl(0 to 7% [w/v]), pH (4.0 to 9.0) and temperature (−20, 0, 4, 25 and 37°C) in both artificial seawater (ASW) anddistilled water (DW). The viable but non-culturable (VBNC) state of P. aeruginosa was also monitored. The marinestrain 1200 grew better at high NaCl and pH, whereas the freshwater strain 1030 did better at 0 to 3% NaCl and a pHof less than 7.0. The clinical strain 1564 grew best at neutral pH and 0% NaCl. No significant differences wereobserved among the strains in culturability at different temperatures. Like other bacteria, P. aeruginosa enters a VBNCstate under stressful conditions. The marine P. aeruginosa isolate exhibits a unique pattern of culturability and survivalwhich demonstrates a physiological adaptation to the ocean environment.

Key words: Pseudomonas aeruginosa, marine, culturability, viable but non-culturable, freshwater

Pseudomonas aeruginosa is present in the open ocean and

marine strains with unique genotypes and forming distinct

clusters were recently confirmed by multilocus sequence

typing, pulsed field gel electrophoresis (PFGE) and phylog-

enetic analysis (22, 23). In addition, the geographic distribu-

tion of these marine strains is related to their phylogenetic

position (23).

The genus Pseudomonas is one of the most diverse and

ecologically significant groups of bacteria, playing important

roles in the carbon and nitrogen cycles in many natural envi-

ronments (47). P. aeruginosa is the quintessential oppor-

tunistic pathogen, causing a wide variety of infections in

compromised hosts (38, 49) and lethal when associated with

cystic fibrosis (8). P. aeruginosa also causes diseases in

plants (4) and animals (54). Numerous cases of folliculitis,

dermatitis, and ear and urinary tract infections due to P.

aeruginosa acquired by bathing in contaminated recreational

water have been reported (7, 16, 37, 45). This bacterium is

highly adaptable (48) and has diverse phenotypic character-

istics, being able to utilize a wide range of organic and inor-

ganic compounds (11, 53). As organic compounds generally

co-occur at low concentrations in the ocean, this physiologi-

cal versatility may aid its growth and survival in such envi-

ronments.

P. aeruginosa is also ubiquitous in freshwater environ-

ments including rivers (36), wastewater (57), bottled mineral

water (28), holy water (40), tap water (41), water-baths (32),

humidifiers (12), distilled water (6), etc. P. aeruginosa can

grow relatively fast in distilled water obtained in hospitals

and achieve high cell concentrations which remain stable for

long periods of time. Cells grown in distilled water react

quite differently to chemical and physical stress than cells

grown in standard laboratory culture media (6).

Although there is little information on the tolerance of

P. aeruginosa to NaCl, there are studies which demonstrate

the growth and survival of other pseudomonads at high

NaCl concentrations. Using media containing between 12

and 18% (w/v) NaCl, Hof (19) isolated a Pseudomonas-type

bacterium from beans preserved in brines with a salt concen-

tration of 6 to 29%. This organism, designated Pseudomonas

beijerinckii, grew in 3 to 18% salt but not 0.5% salt, showing

its obligate halophilic character. A Pseudomonas sp. growing

at 0.05% to 20% NaCl with an optimum at 5% and 30°C was

also found (46). A water sample from Lake Chaplin, Canada,

produced a bacteriophage that lysed a bacterium designated

Pseudomonas strain G3, which was able to grow at 1.5 to

over 18% NaCl (21). Salinity-dependent cadmium tolerance

was documented in Pseudomonas sp. strain 40. In 6% NaCl,

poor growth was obtained in the presence of 2 mg mL−1 of

CdCl2 although no growth was possible at 2.5 mg mL−1 (35).

It is now clear that the tolerance of P. aeruginosa to NaCl

depends on factors such as temperature and the culture

medium composition (51).

Marine environments are ones in which human pathogens

like P. aeruginosa are not expected to be present due to high

salinity and pH. Although P. aeruginosa has been isolated

from marine environments, its apparent distribution was

restricted to river outlets and shorelines (25, 30, 43, 50, 56).

Such isolates have been regarded as originating from fresh-

water or sewage (17, 25). Our detailed study on marine P.

aeruginosa demonstrated for the first time that this bacterium

is common in the open ocean (22, 23).

The goal of this study was to determine whether the

genetic distinctness of marine isolates of P. aeruginosa is

accompanied by physiological differences relative to fresh-* Corresponding author. E-mail: khanmnh@yahoo.com;

Tel: +1–519–888–4567; Fax: +1–519–746–0614.

Survival of marine Pseudomonas aeruginosa 267

water and clinical isolates. Therefore, we describe the cultur-

ability and survival of marine P. aeruginosa under stress due

to low nutrients, high NaCl, and different pH and tempera-

ture. Laboratory microcosms were prepared using distilled

water (DW) or artificial seawater (ASW) with various

amendments. In addition, the morphological characteristics

of P. aeruginosa cells in NaCl and temperature-stressed

conditions were observed. Specifically, we hypothesized

that a marine P. aeruginosa isolate would be culturable and

survive better at higher salinity and pH than P. aeruginosa

isolated from freshwater and clinical sources.

Materials and Methods

P. aeruginosa strains

Four strains of P. aeruginosa isolated from different geographi-cal locations and sources were used. Strain 1030, a river strain, wasisolated in 2003 from the uppermost reaches of the Arakawa River(22). Strains 100 and 1200 are marine strains, isolated from St. N7and S2 during the KH-05-01 and KT-03-07 cruises of R/V TanseiMaru, Ocean Research Institute, the University of Tokyo andJAMSTEC (22, 23). Human influence was considered minimal orabsent at these sampling sites. Strain 1564 was isolated from ablood sample of a hospital patient from Tokyo, Japan, in 2002 (22).These four strains have different serotypes, antibiotypes and PFGEpatterns (Table 1). Details of identification procedures for all thesestrains were provided previously (22). Strain 100 was used forcomparative analyses among the marine strains. As there were nosignificant differences among strains 100 and 1200 in the experi-ments on tolerance of NaCl, only strain 1200 was used for furthercomparative analyses.

Growth conditions

Bacterial strains maintained at −80°C in 40% (v/v) glycerol innutrient broth (Difco, Becton Dickinson, Sparks, MD, USA) werestreaked onto agar plates to obtain single colonies, and cultured onMueller Hinton agar (Difco) at 37°C under aerobic conditions. Twosubcultures were performed under similar conditions. Single colo-nies were selected and the inocula for the experiments wereprepared by growing the strains in 200 mL of M9 medium (22 mMKH2PO4, 42 mM Na2HPO4, 19 mM NH4Cl, 9 mM NaCl, 1 mMMgSO4 and 0.09 mM CaCl2, pH 6.8) (31) containing 5.5 mMglucose as a carbon source. The medium contained 93 mM Na+

which corresponds to c.a. 0.54% NaCl. The cells were incubated at35°C with shaking (150 rpm) as described by Mascher et al. (29)until mid-log phase (i.e., 108 CFU mL−1, corresponding to an OD600

of 0.12). The cells were washed three times in sterile distilled water(6,000×g for 10 min) prior to use in the experiments.

To quantify the effects of NaCl, pH and temperature, differentsets of microcosms (Table 2) were prepared with either DW orASW. ASW contained: NaCl, 23.4 g L−1 (400 mM); KCl, 0.8 g L−1;MgSO4·7H2O, 4.0 g L−1; CaCl2, 0.2 g L−1; KBr, 100 mg L−1;SrCl2·6H2O, 26 mg L−1; and H3BO3, 20 mg L−1 (26). The pH was7.5. When the pH was varied for DW there was no NaCl, and whenthe NaCl concentration was varied in DW the pH was unchanged,i.e., it remained at the original pH of DW which was around 5.0. Inthe case of ASW, when the pH was varied, the NaCl concentrationwas 3.0% and, when the NaCl concentration was varied, the pH was8.0. When temperature was varied, the pH was 5.0 and 8.0 andNaCl concentration was 0% and 3.0% for DW and ASW, respec-tively. The experiments with NaCl and pH were carried out at roomtemperature (25±1°C). For the −20°C experiment, the samples weretransferred to a refrigerator after inoculation. The cells were addedto each microcosm to obtain a final concentration of about 106 cellsmL−1. The flasks were not shaken.

Culturable cell counts

Culturable cells were counted by spread plating. Serially diluted

samples were spread in duplicate onto LB agar plates, which wereincubated at 35°C for 48 h. If no culturable cells were found oneither of the plate with 100 μL of sample, 1 mL of sample wascentrifuged, diluted with ASW and then plated. If no culturablecells were found in 1 mL of sample, they were considered non-culturable.

Viable and total cell counts

Viable and total cells were analyzed by using a LIVE/DEADBacLight bacterial viability kit (Invitrogen, Carlsbad, CA, USA). A1-mL aliquot of water with appropriate dilutions was mixed with 3μL of a mixture of 3.34 nM SYTO 9 green and 20 mM propidiumiodide (Molecular Probes, Eugene, OR, USA) dissolved in dimethylsulfoxide (DMSO) prepared according to the manufacturer’sinstructions, and incubated for 15 min in the dark at room tempera-ture (18). The mixture was filtered through 0.2-μm NucleporeTrack-Etch Membrane polycarbonate black filters (Whatman,Middlesex, UK). The filters were mounted with low-fluorescenceimmersion oil on glass microscope slides and observed by epifluo-rescence microscopy (BH2; Olympus, Tokyo, Japan). Cellsfluorescing green were considered viable and the numbers in 20microscopic fields were counted. The total cell count was the sumof the cells fluorescing green and red (the latter were considered tobe dead cells). When a sample of more than 1 mL was necessary,the water was filtered through 0.2-μm Nuclepore polycarbonateblack filters. The filters (still mounted in the filtration unit) werecovered with 3 μL of the stain mixture in 1 mL of sterile water.After 15 min in the dark at room temperature, the mixture wasfiltered and treated as described above.

Growth rate

As no organic nutrients were added to the ASW microcosms,the cells were generally not able to grow. However, the rate ofpopulation change (k) was defined as follows:

k=(lnZ−lnZ0)/(t−t0)

Here Z0 and Z are the number of bacterial cells at the beginning(t0) and end (t) of the experiment.

Slope

The rate of change in cell numbers varied with environmentalconditions. These relationships were indicated by the slope, deter-mined by least squares regression, of the rate of change as afunction of salinity, pH, or temperature.

Morphological observation using AFM

The morphological observations were conducted by atomicforce microscopy (SPM-9500 J2; Shimadzu, Kyoto, Japan) using

Table 1. Origin, source and date of isolation of the strains used

Strain number

Origin Source Date of isolation

1030 Freshwater Arakawa River, AK1 June 5, 2003

1200 Marine Open Ocean, S2 June 7, 2003

100 Marine Open Ocean, N7 2005

1564 Clinical, Human Blood, Tokyo, Japan 2002

Table 2. Microcosmic conditions

Factor examined Medium Conditions examined

NaCl DW 0, 3, 4, 5, 6 and 7% (w/v)

ASW 0, 3, 4, 5, 6 and 7% (w/v)

pH DW 5.0, 7.0 and 9.0

ASW 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0

Temperature DW −20, 0, 4, 25 and 37°C

ASW −20, 0, 4, 25 and 37°C

KHAN et al.268

the dynamic mode. Prior to observation, microcosm samples wereconcentrated on Isopore filters (0.2 μm pore size, Millipore,Bedford, MA, USA) and washed with 1 mL of DW that had beenpre-filtered through a polycarbonate filter (Nuclepore, 0.2 μm). Celllength and width were determined by the image analysis system(n=20) as was described previously (33). Cell volume (V) wascalculated from cell length (l) and width (w) according to theformula V=(π/4)w2(l−w/3) (3). The volume was determined in fL(10−15 L). To differentiate bacterial cells from non-living particles,cross sections of the particles and phase images were checked. Onlythose cells having a typical bacterial shape were counted.

Statistical analyses

Each point on the survival curves of bacterial numbers versustime represents a mean for duplicate flasks. The standard deviationvalues were generally less than 10% of the mean and thus were notpresented in the plots. Treatments were compared by conductingpaired t-tests using Systat 10.

Results

Change in cell numbers in DW with NaCl

When P. aeruginosa strains were incubated in DW with

different NaCl concentrations, culturable cell numbers

tended to decrease gradually. Marine strains 100 and 1200

survived better at 7% than the other two strains (Fig. 1,

paired t-tests, P<0.05). Culturable cells were detected even

after 192 h at 7% NaCl. The river strain 1030 and clinical

strain 1564 showed marked declines during the first 12 h,

followed by more gradual decreases thereafter. No culturable

cells were detected for river and clinical strains after 72 and

96 h, respectively (Fig. 1). There was virtually no difference

among the strains at up to 3% NaCl, thus the 1 and 2% NaCl

treatments were removed from the study (Table 3). The

differences became apparent at 4% NaCl, and were clear at

7% NaCl (Fig. 2). Culturable cell numbers relative to initial

numbers after 24 h were used to compare survival in elevated

NaCl concentrations (data not shown). Clear differences in

the numbers appeared at 4% NaCl or higher.

Change in cell numbers in ASW and NaCl

Fig. 2 shows the change in cell numbers of the marine

strain 1200 at various salinities in ASW. There were two

stages in the change of cell numbers; first until 48 h and

second after 48 h. In the first stage, the number increased

or decreased depending on the NaCl concentration. At 0%,

the numbers increased by nearly 50 times, whereas at other

concentrations they were rather variable. During the second

stage, a constant decrease was observed at 7% NaCl, which

is consistent with the result shown in Fig. 1. The changes in

other strains were much smaller until the end of the incuba-

tion period. Although the numbers at 5 and 6% NaCl were

consistently less than those at lower NaCl concentrations, all

values were within one order of magnitude after 320 h. On

the other hand, the river strain 1030 showed slight increases

Fig. 1. Culturable cell numbers of the four Pseudomonas aeruginosastrains in DW with 7% (w/v) NaCl. Symbols , , and repre-sent the marine (strain 1200), river (strain 1030) clinical (strain 1564)and marine (strain 100) strains of P. aeruginosa respectively. Valuesrepresent the mean of two determinations from duplicate experiments.Marine P. aeruginosa strains showed significant differences when com-pared with river (P<0.05) and clinical (P<0.05) strains. There was nodifference among the marine strains (P>0.05).

Fig. 2. Growth of the marine strain 1200 in artificial seawater (ASW)at 0 to 7% (w/v) NaCl. Symbols , , , , * and indicate 0%,3%, 4%, 5%, 6% and 7% NaCl. Values represent the mean of two deter-minations from two independent experiments.

Table 3. Percentage of culturable cell counts of Pseudomonas aeruginosa isolated from various sources at different NaCl concentrations, pH andtemperatures in DW or ASW after 24 h

StrainNaCl (%) pH Temperature (°C)

0 3 4 5 6 7 4.0 5.0 6.0 7.0 8.0 9.0 −20 0 4 25 37

Marine 1200 DW 26 18 36 45 64 74 ND 0.40 ND 7.3 ND 7.3 5.8 56 64 75 520

ASW 83 250 32 86 61 85 20 27 170 7600 9000 5400 80 100 300 2700 2900

River 1030 DW 35 34 5.7 32 2.0 10 ND 7.1 ND 8.5 ND 0.60 21 48 63 350 340

ASW 2700 910 540 200 105 22 3.1 140 53 1700 1400 1600 37 56 67 630 480

Clinical 1564 DW 24 42 8.5 8.7 5.2 0.40 ND 1.2 ND 41 ND 0.10 19 56 59 160 120

ASW 2700 380 90 52 30 40 0.90 24 32 4300 310 100 1.6 29 24 460 160

Survival of marine Pseudomonas aeruginosa 269

at 3 and 4% NaCl but the clinical strain 1564 did not show

an increase in cell number at NaCl concentrations of more

than 3% (data not shown). The percentage of culturable cells

was calculated after 24 h at 0% NaCl and it was found

that the river and clinical strains showed about 27 times

higher counts whereas the marine strain 1200 showed less

than the initial counts. When the culturable cell counts were

calculated after 24 h at 7% NaCl, they were 85.3, 21.8 and

41.4% (Table 3).

The slope was calculated for relative culturable numbers

(%) vs. NaCl concentration (%) (Fig. 3 and Table 4). This

value can be regarded as an index of the adaptability of

P. aeruginosa to NaCl. The slope varies depending on the

range of NaCl concentrations and period of incubation. In

order to see clear differences, the values obtained with up to

7% NaCl were used. As relative culturable numbers tend

to fluctuate in the early stages of incubation (Fig. 1 and 2),

the slopes at any one fixed time are not very reliable. There-

fore, all the values starting from 4 h up to 48 h are listed for

comparison in Table 4. In general, the slope decreases with

time, indicating that the cells lost their culturability with

increasing NaCl concentration and time. As the slope values

were higher in DW than ASW, the cells in DW are more

tolerant of higher NaCl concentrations than those in ASW.

Among the three strains, the marine strain 1200 had consist-

ently and significantly higher values than the other two

(P=0.038 and P=0.021, respectively). This indicates that the

marine strain is the most resistant to the elevated level of

NaCl under the present conditions.

Change in cell numbers in DW and ASW with different pH

In DW, the clinical strain 1564 and river strain 1030

showed optimum culturability at pH 7. For the latter strain, a

comparable number was detected at pH 5 as well. However,

for the marine strain 1200, survival was lowest at pH 7 and

there was no difference in culturability at pH 7 and 9 after 24 h

of incubation in DW (Table 3). The culturability of marine

strain 1200 at different pH was significantly different from

that of strain 1564 (P=0.01), but not strain 1030. In ASW,

survival was much higher than in DW. Although strains 1564

and 1030 both showed optimum survival at pH 7, they did

better at alkaline pH than at acidic pH (Table 3). As for strain

1200, its optimum pH was 8. The slopes (Fig. 4 and Table 4)

confirmed these tendencies. The values are generally much

higher in ASW than in DW, indicating the preference for

higher pH. The maximum cell count was observed after

either 48 or 72 h of incubation.

Table 4. Slope of survival of Pseudomonas aeruginosa isolated from various sources at different NaCl concentrations, pH and temperatures inDW or ASW

StrainsNaCl (0 to 7%) Time (h) pH (4 to 9) Time (h) Temperature (−20 to 37ºC) Time (h)

4 12 24 48 72 4 12 24 48 72 4 12 24 48 72

Marine 1200 DW −1.3 −2.2 7.5 −2.1 − 0.45 5.2 1.7 1.4 0.33 2.60 4.5 7.4 22 18

ASW 12 7.3 −7.1 −110 − 28 300 1300 9500 5000 3.8 25 59 54 78

River 1030 DW −12 −8.3 −4.1 −9.2 − −1.3 −4.6 −1.6 −1.7 −1.6 4.4 6.6 6.8 11 7.6

ASW −9.2 −37 −390 −450 − 23 66 380 2100 1800 1.2 6.2 11 10 16

Clinical 1564 DW −16 −12 −4.3 −15 − 0.84 −1.2 −0.29 0.12 0.010 −0.17 1.2 2.3 6.3 4.8

ASW −14 −73 −370 −340 − 17 22 19 19 430 0.25 3.2 5.7 8.3 5.4

Fig. 3. Slope of survival of three Pseudomonas aeruginosa strainscultured in DW with different NaCl concentrations (0 to 7% [w/v]) forup to 48 h. Linear regression lines are for each of the strains. Symbols

, and represent the marine (strain 1200), river (strain 1030) andclinical (strain 1564) strains of P. aeruginosa, respectively. Lines arerepresenting the data of marine (solid line), river (larger dotted line) andclinical (smaller dotted line) strains. Differences were significantbetween the marine and river (P=0.038) and marine and clinical(P=0.021) strains.

Fig. 4. Slope of survival of three Pseudomonas aeruginosa strainscultured in DW with different pH values (5.0, 7.0 and 9.0) for up to 72 h.Linear regression lines are shown for each of the strains. Symbols ,

, represent the marine (strain 1200), river (strain 1030) and clini-cal (strain 1564) strains of P. aeruginosa, respectively. Lines are repre-senting the data of marine (solid line), river (larger dotted line) and clin-ical (smaller dotted line) strains. Differences were significant betweenthe marine and river (P=0.008) and marine and clinical (P=0.032)strains.

KHAN et al.270

Change in cell numbers in DW and ASW at different

temperatures

Upon transfer to a freezer, DW froze within hours,

whereas ASW did not freeze until the end of the incubation

period (72 h). For all the strains, the percent culturability

after 24 h increased with the temperature (Table 3). For the

river strain 1030 and clinical strain 1564, the values were

less than 100 indicating that some cells lost culturability

during the 24 h. The cells increased several fold at 25 and

37°C. Although these two strains showed maximum growth

at 25°C, strain 1200 gave maximum counts at 37°C. Rather

large differences between the numbers in DW and ASW

were noticed for the marine strain. Except for one value,

the slopes were positive regardless of the incubation period,

indicating that all three strains preferred higher temperatures

(Table 4). The differences among the slope values of marine,

river and clinical P. aeruginosa were not significant (P>

0.05).

Viable but non-culturability (VBNC) at high NaCl

concentration

To understand the survival pattern of P. aeruginosa strains

and their entry into the VBNC state, marine, river and

clinical strains at 7% NaCl in DW were examined for up to

312 h (Fig. 5). The total counts remained essentially the

same during the first 48 h of incubation for all of the strains

(Fig. 5A, 5B and 5C). After 312 h, the count of viable cells in

the clinical strain was 70% (Fig. 5C) whereas in the river and

marine strains it varied from 87 to 90% (Fig. 5A and 5B).

There were no culturable cells of clinical and river strains

after 72 and 96 h, respectively, whereas the marine strain was

culturable for up to 192 h. The viable cell counts observed

using the BacLight kit declined rather steadily for the first 24

h, followed by a slower decrease in the marine and river

strains but a faster decrease in the clinical strain. A sharp

decline for all the strains was observed after 48 h and this

continued up to 144 h. The viable cell counts again became

steady and continued that way until the experiment was

discontinued. After 312 h of incubation, the count of viable

cells for the marine strain was highest (29%) whereas that

for the river and clinical strains varied between 11 and 1%.

There was a significant difference among the marine and

clinical strain (P>0.05), but not river and marine strains. The

viable count of the marine strain was significantly different

from that of the both river (P<0.01) and clinical (P<0.01)

strains.

Change in cell volume measured by AFM

Cell sizes were observed at each concentration of NaCl

after 22 days incubation (Fig. 6 and 7). Cell length increased

at 7% NaCl and at −20°C to more than that at 0% NaCl at

room temperature. There was no significant difference in cell

volume (fL) among the three strains except at 7% NaCl,

where the river strain decreased in volume in comparison to

both the marine and clinical strains (Fig. 6).

Discussion

The present study supports our hypothesis that P.

aeruginosa isolated from the open ocean (22, 23) possesses

physiological characteristics appropriate to that environ-

ment when compared to clinical or freshwater isolates. Al-

though it has not been possible to demonstrate a specific

Na+ requirement in most non-halophilic bacteria, this study

found that the marine isolate showed greater culturability

and survival at high NaCl concentrations, whereas river and

clinical strains showed reduced growth and survival in the

same environments.

Although 6 to 7% NaCl is not within the range of natural

Fig. 5. Viable but non-culturable, cuturable and total cell counts ofriver (A), marine (B) and clinical (C) Pseudomonas aeruginosa strainsin DW at 7% (w/v) NaCl. Symbols , and indicate the total, via-ble and culturable cell counts of P. aeruginosa respectively. Values arepercentage of average of 20 microscopic field counts.

Fig. 6. Volume of cells after 22 d incubation in DW with variousNaCl concentrations at room temperature. Symbols , , and rep-resent the river, marine and clinical Pseudomonas aeruginosa, respec-tively. Values are the average of 20 individual cell sizes in each group.

Survival of marine Pseudomonas aeruginosa 271

aquatic environments, except for the Dead Sea, Great Salt

Lake, or other brine environments, intertidal zones can

provide very high NaCl concentration when they are exposed

to evaporation. However, these experiments were designed

to impose stress on the P. aeruginosa cells to assess their

tolerance of NaCl. In our experiments, we did not notice any

significant differences in the growth of the P. aeruginosa

strains at 1 to 3% NaCl, which actually demonstrates their

ability to survive in marine environments.

It is possible that the freshwater and clinical isolates also

have a need for Na+. Phylogenetic analysis of bacterial

pathogens that use the Na+ cycle has demonstrated that

Pseudomonadaceae (including P. aeruginosa) fall in the

group Gammaproteobacteria (15, 55). Among other non-

halophiles, Na+ requirements have been detected only for

growth at the expense of certain specific carbon and energy

sources: e.g., Echerichia coli requires Na+ for growth at a

maximal rate with L-glutamate, and Enterobactor aerogenes

requires Na+ for growth on citrate (27). In both cases, the

quantitative requirements are small, and absolute growth

dependence on Na+ has not been demonstrated. In contrast,

bacteria of marine origin, most moderate halophiles (bacteria

which require NaCl for their growth and survival) and

extreme halophiles require Na+ for growth at concentrations

sufficiently high that their absolute dependence on Na+ can

be demonstrated experimentally without difficulty, even if

the basal medium employed has not been prepared from

specially purified (Na+ free) ingredients. In all of these

organisms, Na+ probably plays a number of different roles,

all indispensable to the maintenance of cellular function.

In marine bacteria, there is good evidence that it assures

the correct function of transport systems. In the extreme

halophiles, a high concentration of NaCl is essential in order

to maintain both the stability and catalytic activity of

enzymes (27).

There have been several studies on the survival of P.

aeruginosa in saline environments. Rawal and Nahata (39)

examined the survival and growth of aerobic and anaerobic

bacteria and yeast in intravenous fluids. Solutions were

experimentally contaminated with pathogenic strains of E.

coli, P. aeruginosa, Staphylococcus aureus, Bacteroides

fragilis and Candida albicans. Samples of these solutions

were tested for culturable bacteria daily for 3 d using a

membrane filtration method. Each organism showed a differ-

ent survival/growth pattern in various infusion fluids. In 5%

dextrose, C. albicans multiplied but only 2–3% of the initial

viable cells of E. coli, P. aeruginosa, and S. aureus were

detected after 3 d. In the present study, clinical P. aeruginosa

could not be cultured after 3 d whereas the marine strain

survived more than 6 d.

Boyle et al. (1) examined the survival of opportunistic

pathogens in distilled water using P. aeruginosa and S.

aureus as test organisms. Cultures were incubated at 10°C,

25°C, and 37°C. No viable S. aureus cells were detected after

the first week of incubation, whereas P. aeruginosa survived

up to 5 months at all three temperatures. We observed the

survival of P. aeruginosa in DW for 6 months even at 4°C

(unpublished observations).

No previous study has demonstrated the survival of P.

aeruginosa at 0 and −20°C. Bacteria in sea ice are generally

Fig. 7. Effect of low temperature and high NaCl concentration on thecell size of Pseudomonas aeruginosa. A, P. aeruginosa cells at 0%NaCl at room temperature, B, at −20°C with 0% NaCl after 22 d andC, at room temperature with 7% (w/v) NaCl after 22 d. The arrowsindicate P. aeruginosa cells.

KHAN et al.272

abundant compared to those in the underlying seawater, with

cell bio volumes 5 to 10 times larger and a high proportion

epiphytic or particle associated (5, 10). In the present study,

upon transfer to a freezer at −20°C, DW froze within hours,

whereas ASW did not freeze until the end of the incubation

period (72 h). All the strains survived at −20°C regardless of

their origin, indicating their ability to thrive in a frozen state.

Although the clinical and freshwater strains showed maxi-

mum growth at 25°C, the marine strain 1200 gave maximum

counts at 37°C. Rather large differences between the num-

bers in DW and ASW were noticed for strain 1200 at various

temperatures. Except for one value, the slopes were positive

regardless of the incubation period, indicating that all three

strains preferred higher temperatures.

In addition to DW we used ASW as a contrasting stress

medium that might highlight differences between marine and

freshwater strains. ASW contains not only NaCl but also K,

Ca, Mg and other elements. All of these elements have roles

in the metabolism of P. aeruginosa, and might have

enhanced its growth and survival. On the other hand, cells in

DW with high NaCl concentrations are under osmotic stress.

Another intention in choosing ASW as a medium was to

choose an osmotic environment similar to the open ocean. In

most of the experiments, the better survival and growth of

P. aeruginosa indicates the role of ASW or its components in

the physiological activities of P. aeruginosa. There is not

much literature available to interpret our data, but the present

study is a beginning for that kind of research. As a clinically-

significant organism, that is ubiquitous in many environ-

ments, P. aeruginosa deserves further attention from

researchers.

The slope of the rate of change of cell numbers with

environmental conditions was found to be higher in DW in

comparison to ASW in some cases for the river and clinical

P. aeruginosa strains. The marine strain had more potential

culturability than the river and clinical strains, thus it had a

higher slope than the others. With an increase in NaCl and

incubation time it was found that the slope for all strains

became higher in DW than in ASW. This reflects the increas-

ing stress due to NaCl at higher pH and with other ions in

ASW. On the other hand, the stress in DW is only due to

NaCl and low pH. At 0 and 4 h of incubation, the slope

values were higher for ASW than for DW indicating that

with the increased time, ASW imposed more stress on the

bacterial cells than DW. In all of the experimental circum-

stances, the marine P. aeruginosa had more culturability and

survival tolerance than the clinical and freshwater strains.

Bacteria in the open ocean are constantly exposed to

nutrient deprivation and a variety of other stresses (tempera-

ture, salinity, and solar illumination) that must be overcome

for survival. Various physiological changes, like size reduc-

tion, changes in protein synthesis, decreased metabolic

activity and cell wall modifications, aid in the survival of

bacteria in aquatic environments (34, 42). Various Gram-

negative bacteria are known to enter into a VBNC state,

when exposed to adverse environmental conditions (44). In

marine environments, many processes might play important

roles in VBNC strains of P. aeruginosa. This is a preliminary

study to see whether this bacterium is capable of entering a

VBNC state. There are many ways to induce VBNC state

but we used DW and high NaCl. These stresses, although

not present in the open ocean, are strong enough to cause

P. aeruginosa to enter into a VBNC state. The persistence

of P. aeruginosa in the ocean is partly determined by their

ability to endure the stress in that environment.

P. fluorescens cells can remain in a VBNC state in soil for

over a year (3), but this state can only be a significant means

of survival if they are able to again become metabolically

active. P. aeruginosa is a widely distributed organism, and

one of its main adaptive strategies appears to be recombina-

tion (24). This species has metabolic versatility, including

the ability to adapt to virtually all aquatic mesophilic habitats

(24). In the case of cystic fibrosis of the lung, P. aeruginosa

has a particularly complex challenge, requiring simultaneous

adaptation to dehydration, iron starvation, leukocyte influx,

antibacterial peptides, and frequently changing, aggressive,

and prolonged antibiotic therapy. In the case of marine envi-

ronments, perhaps, its metabolic versatility is not enough to

allow a rapid adaptation to this complex habitat. Further

studies are required to clarify this issue.

It is important to know how the morphology of P.

aeruginosa cells responds in stressful conditions. Usually

stressed bacterial cells shrunk or expand, depending on

the osmolarity of the medium, efflux of water, decrease or

increase of turgor pressure, etc. When a bacterium is

subjected to osmotic stress, such as being inoculated into a

hyperosmotic medium, it responds by rapidly accumulating

compatible solutes (2, 9). Staphylococcus aureus cells grown

in a defined medium under conditions of high ionic stress

(2.5 M NaCl) were significantly larger than cells grown

under unstressed conditions, even though the cells grew

much more slowly under stressed conditions (52). Similar

observations of larger cells in a complex medium containing

NaCl rather than in a medium alone have also been reported

by others (20). In the present study, while the P. aeruginosa

cells were exposed to high NaCl concentration, their size and

volume increased as in previous studies with other bacteria.

The increase could be necessary to meet the cells need in

unfavorable circumstances. The increased surface area could

allow the bacteria to absorb more extracellular materials. On

the other hand, the increase in P. aeruginosa cell volume at

low temperature is supported by previous studies where

frozen conditions increased cell size significantly (14). It has

also been demonstrated that some bacteria survive adverse

environmental conditions associated with the formation of

ice and adapt, albeit at a lower level of metabolic activity

(13). The increase in cell size at low temperature and high

NaCl could also be explained by an inability to undergo cell

division in these conditions. As P. aeruginosa is present in

the open ocean, it would not be surprising if it is associated

with sea ice where temperatures would be unfavorable for

normal growth. The ability to enter into a VBNC state and

increase in cell size and volume may indicate the survival

potential of P. aeruginosa in these aquatic environments.

In conclusion, P. aeruginosa can tolerate high NaCl

concentrations and pH levels, and has the capacity to grow

over a wide range of NaCl concentrations. Marine P.

aeruginosa can survive better at high NaCl concentrations

and high pH than clinical or river strains. The results of

this study strengthen our previous finding that P. aeruginosa

Survival of marine Pseudomonas aeruginosa 273

is present in the open ocean and that the marne strains are

unique in their culturability and survival compared to fresh-

water and clinical strains.

Acknowledgements

The study was supported by Grants-in aid for Creative BasicResearch #12NP0201 (DOBIS) and #14208063 from the Ministryof Education, Culture, Sports, Science and Technology (MEXT),Japan. We are grateful to Dr. Yoshikazu Ishii, Department ofMicrobiology and Infectious Diseases, Toho University School ofMedicine, Tokyo for providing clinical P. aeruginosa strains. Weremain indebted to Dr. Masahiko Nishimura and Dr. Minoru Wadaof the Ocean Research Institute of the University of Tokyo for theirtechnical support. Thanks to Ms. E. Ikemoto for her support in theAFM analysis. Special thanks to Prof. Rita R Colwell, University ofMaryland, USA for her valuable comments and suggestions.

References

1. Boyle, M., T. Ford, J.S. Maki, and R. Mitchell. 1991. Biofilms andthe survival of opportunistic pathogens in recycled water. WasteManagement Res. 9:465–470.

2. Bratbak, G. 1985. Bacterial bio volume and biomass estimations.Appl. Environ. Microbiol. 49:1488–1493.

3. Bunker, S.T., T.C. Bates, and J.D. Oliver. 2004. Effects oftemperature on detection of plasmid and chromosomally encodedgfp and lux-labeled Pseudomonas fluorescens in soil. Environ.Biosafety Res. 3:1–8.

4. Buysens, S., K. Heungens, J. Poppe, and M. Hofte. 1996.Involvement of pyochelin and pyoverdin in suppression of pythium-induced damping-off of tomato by Pseudomonas aeruginosa 7NSK2.Appl. Environ. Microbiol. 62:865–871.

5. Delille, D. 1992. Marine bacterioplankton at the Weddell Sea iceedge: distribution of psychrophilic and psychrotrophic populations.Polar Biol. 12:205–210.

6. Favero, M.S., L.A. Carson, W.W. Bond, and N.J. Peterson. 1971.Pseudomonas aeruginosa growth in distilled water from hospitals.Science. 173:836–838.

7. Feder, H.M., J.M. Grant-Kels, and R.C. Tilton. 1983. Pseudomonaswhirlpool dermatitis: report of an outbreak in two families. Clin.Pediatr. (Hagerstown) 22:638–642.

8. Finnan, S., J.P. Morrissey, F. O’Gara, and E.F. Boyd. 2004. Genomediversity of Pseudomonas aeruginosa isolates from cystic fibrosispatients and the hospital environment. J. Clin. Microbiol. 42:5783–5792.

9. Galinski, E.A. 1995. Osmoadaptation in bacteria. Adv. Microbiol.Physiol. 37:273–328.

10. Gleitz, M., S. Grossmann, R. Scharek, and V. Smetacek. 1996.Ecology of diatom and bacterial assemblages in water associatedwith melting summer sea ice in the Weddell Sea, Antarctica. Antarct.Sci. 8:135–146.

11. Green, S.K., M.N. Schroth, J.J. Cho, S.K. Kominos, and V.B.Vitanza-Jack. 1974. Agricultural plants and soil as a reservoir forPseudomonas aeruginosa. Appl. Microbiol. 28:987–991.

12. Grieble, H.G., F.R. Colton, T.J. Bird, A. Toigo, and L.G. Griffith.1970. Fine-particle humidifiers. Source of Pseudomonas aeruginosainfections in a respiratory-disease unit. N. Engl. J. Med. 282:531–535.

13. Grossman, S. 1994. Bacterial activities in sea Ice and open water ofthe Weddel Sea, Antarctica: A microautoradiographic study. Microb.Ecol. 28:1–18.

14. Haines, R.B. 1938. The effect of freezing on bacteria. Proc. R. Soc.Lond., B, Biol. Sci. 124:451–463.

15. Hase, C.C., N.D. Fedorova, M.Y. Galperin, and P.A. Dibrov. 2001.Sodium ion cycle in bacterial pathogens: Evidence from cross-genome comparisons. Microbiol. Mol. Biol. Rev. 65:353–370.

16. Havelaar, A.H., M. Bosman, and J. Borst. 1983. Otitis externa byPseudomonas aeruginosa associated with whirlpools. J. Hyg.90:489–498.

17. Hoadley, A.W. 1977. Potential health hazards associated withPseudomonas aeruginosa in water. Am. Soc. Test. Mater. Spec. Tech.Publ. 635:80–114.

18. Hoefel, D., P.T. Monis, W.L. Grooby, S. Andrews, and C.P. Saint.2005. Profiling bacterial survival through a water treatment processand subsequent distribution system. J. Appl. Microbiol. 99:175–186.

19. Hof, T. 1935. Investigations concerning bacterial life in strong brines.Rev. Trav. Bot. Neerl. 32:92–171.

20. Katinakis, P. 1989. The pattern of protein synthesis induced by heat-shock of the moderately halophilic bacterium Chromobacteriummarismortui: protective effect of high salt concentration against thethermal shock. Microbiologica 12:61–67.

21. Kanemasa, Y., K. Takai, T. Takatsu, H. Hayashi, and T. Katayama.1974. Ultrastructural alteration of the cell surface of Staphylococcusaureus cultured in a different salt condition. Acta Med. Okayama28:311–320.

22. Khan, N.H., Y. Ishii, N. Kimata-Kino, H. Esaki, M. Nishimura, andK. Kogure. 2007. Isolation of Pseudomonas aeruginosa from openocean and comparison with freshwater, clinical and animal isolates.Microb. Ecol. 53:173–186.

23. Khan, N.H., M. Ahsan, S. Yoshizawa, Y. Hosoya, A.Yokota, and K.Kogure. 2008. Multilocus sequence typing and phylogenetic analysesof marine Pseudomonas aeruginosa. Appl. Environ. Microbiol.74:6194–6205.

24. Kiewitz, C., and B. Tümmler. 2000. Sequence diversity ofPseudomonas aeruginosa: impact on population structure andgenome evolution. J. Bacteriol. 182:3125–3135.

25. Kimata, N., T. Nishino, S. Suzuki, and K. Kogure. 2004.Pseudomonas aeruginosa isolated from marine environments inTokyo Bay. Microb. Ecol. 47:41–47.

26. Kogure, K., U. Simidu, and N. Taga. 1979. A tentative directmicroscopic method for counting living marine bacteria. Can. J.Microbiol. 25:415–420.

27. Larsen, H. 1967. Biochemical aspects of extreme halophism. Adv.Microbiol. Physiol. 1:97.

28. Legnani, P., E. Leoni, S. Rapuano, D. Turin, and C. Valenti. 1999.Survival and growth of Pseudomonas aeruginosa in natural mineralwater: a 5-year study. Int. J. Food Microbiol. 53:153–158.

29. Mascher, F., C. Hase, Y. Moenne-Loccoz, and G. Defago. 2000.The viable-but-nonculturable state induced by abiotic stress in thebiocontrol agent Pseudomonas fluorescens CHA0 does not promotestrain persistence in soil. Appl. Environ. Microbiol. 66:1662–1667.

30. Mates, A. 1992. The significance of testing for Pseudomonasaeruginosa in recreational seawater beaches. Microbios. 71:89–93.

31. Miller, J.H. 1972. Experiments in molecular genetics: A LaboratoryManual. p. 431–432. Cold Spring Harbor Laboratory, Cold SpringHarbor, New York, USA.

32. Muyldermans, G., F. De Smet, D. Pierard, L. Steenssens, D. Stevens,A. Bougatef, and S. Lauwers. 1998. Neonatal infections withPseudomonas aeruginosa associated with a water-bath used to thawfresh frozen plasma. J. Hosp. Infect. 39:309–314.

33. Nishino, T., E. Ikemoto, and K. Kogure. 2004. Application of atomicforce microscopy to observation of marine bacteria. J. Oceanogra.60:219–225.

34. Nystrom, T., and S. Kjelleberg. 1989. Role of protein synthesis in thecell division and starvation induced resistance to autolysis of a marineVibrio during the initial phases of starvation. J. Gen. Microbiol.135:1599–1606.

35. Onishi, H., T. Kobayashi, N. Morita, and M. Baba. 1984. Effect ofsalt concentration on the cadmium tolerance of a moderatelyhalophilic cadmium tolerant Pseudomonas sp. Agric. Biol. Chem.48:2441–2448.

36. Pellett, S., D.V. Bigley, and D.J. Grimes. 1983. Distribution ofPseudomonas aeruginosa in a riverine ecosystem. Appl. Environ.Microbiol. 45:328–332.

37. Perrotta, D.M., R.E. Johns, Jr. R. Bradley, J. Jacobson, K. Miner,T. Sailer, and H.L. Gibbons. 1983. An outbreak of Pseudomonasfolliculitis associated with a waterslide. MMWR Morb. Mortal. Wkly.Rep. 32:425–427.

38. Poirel, L., E. Lebessi, M. Castro, C. Fèvre, M. Foustoukou, andP. Nordmann. 2004. Nosocomial outbreak of extended-spectrumß-lactamase SHV-5-producing isolates of Pseudomonas aeruginosain Athens, Greece. Antimicrob. Agents Chemother. 48:2277–2279.

39. Rawal, B.D., and M.C. Nahata. 1985. Variation in microbial survivaland growth in intravenous fluids. Chemotherapy. 31:318–323.

40. Rees, J.C., and K.D. Allen. 1996. Holy water—a risk factor forhospital-acquired infection. J. Hosp. Infect. 32:51–55.

KHAN et al.274

41. Reuter, S., A. Sigge, H. Wiedeck, and M. Trautmann. 2002. Analysisof transmission pathways of Pseudomonas aeruginosa betweenpatients and tap water outlets. Crit. Care Med. 30:2222–2228.

42. Rollins, D.M., and R.R. Colwell. 1986. Viable but nonculturablestage of Campylobacter jejuni and its role in survival in the naturalaquatic environment. Appl. Environ. Microbiol. 52:531–538.

43. Romaneko, L.A., N. Naoto, M. Uchino, N.I. Kalinovoskaya, andV.V. Mikhailov. 2008. Diversity and antagonistic activity of sea icebacteria isolated from the Sea of Japan. Microbes Environ. 23:209–214.

44. Roszak, D.B., and R.R. Colwell. 1987. Survival strategies of bacteriain the natural environment. Microbiol. Rev. 51:365–379.

45. Sausker, W.F., J.L. Aeling, J.E. Fitzpatrick, and F.N. Judson. 1978.Pseudomonas folliculitis acquired from a health spa whirlpool. J. Am.Med. Assoc. 239:2362–2365.

46. Simon, R.D., A. Abeliovich, and S. Belkin. 1994. A novel terrestrialhalophilic environment: the phylloplane of Atriplex halinus, a salt-excreting plant. FEMS Microbiol. Ecol. 14:99–110.

47. Spiers, A.J., A. Buckling, and P.B. Rainey. 2000. The causes ofPseudomonas diversity. Microbiology 146:2345–2350.

48. Stover, C.K., X.Q. Pham, A.L. Erwin, et al. 2000. Complete genomesequence of Pseudomonas aeruginosa PAO1, an opportunistic patho-gen. Nature 406:959–964.

49. Toyofuku, M., T. Nakajima-kambe, H. Uchiyama, and N. Nomura.2010. The effects of a cell-to-cell communication molecule,Pseudomonas quinolone signal (PQS), produced by P. aeruginosa onother bacterial species. Microbes Environ. 25:1–7.

50. Velammal, A., B. Aiyamperumal, V.K. Venugopalan, and S.Ajmalkhan. 1994. Distribution of Pseudomonas aeruginosa inPondicherry coastal environments. Ind. J. Mar. Sci. 23:239–241.

51. Ventosa, A., J.N. Nieto, and A. Oren. 1998. Biology of moderatelyhalophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62:504–544.

52. Vijaranakul, U., M.J. Nadakavukaren, B.L.M. de Jonge, B.J.Wilkinson, and R.K. Jayaswal. 1995. Increased cell size andshortened peptidoglycan interpeptide bridge of NaCl-stressedStaphylococcus aureus and their reversal by glycine betaine. J.Bacteriol. 177:5116–5121.

53. Williams, P.A., and M.J. Worsey. 1976. Ubiquity of plasmids incoding for toluene and xylene metabolism in soil bacteria: evidencefor the existence of new TOL plasmids. J. Bacteriol. 125:818–828.

54. Yeruham, I., D. Elad, Y. Avidar, T. Goshen, and E. Asis. 2004.Four-year survey of urinary tract infections in calves in Israel. Vet.Rec. 154:204–206.

55. Yoshie, S., T. Ogawa, H. Makino, H. Hirosawa, S. Tsuneda, and A.Hirata. 2006. Characteristics of bacteria showing high denitrificationactivity in saline wastewater. Lett. Appl. Microbiol. 42:277–283.

56. Yoshpe-Purer, Y., and S. Golderman. 1987. Occurrence ofStaphylococcus aureus and Pseudomonas aeruginosa in Israelicoastal water. Appl. Environ. Microbiol. 53:1138–1141.

57. Ziegert, E., and W. Stelzer. 1986. Comparative study of the detectionof Pseudomonas aeruginosa in water. Zentralbl. Mikrobiol. 141:121–128.