Effect of Intracellular Resuscitation of Legionella pneumophila in

intracellular resuscitation in Acanthamoeba polyphage cells were investigated. Then, the survival profiles of L. pneumophila residing in the planktoni...
0 downloads 0 Views 137KB Size
Download PDF
Environ. Sci. Technol. 2006, 40, 7434-7439

Effect of Intracellular Resuscitation of Legionella pneumophila in Acanthamoeba polyphage Cells on the Antimicrobial Properties of Silver and Copper† MYOUNG GOO HWANG,* HIROYUKI KATAYAMA, AND SHINICHIRO OHGAKI Department of Urban Engineering, The University of Tokyo, 3-1-7 Hongo Bunkyo, Tokyo 133-8656, Japan

The property of Legionella pneumophila entering into a viable but noncultivable (VBNC) state under drinking water conditions (50 mL, pH 7.0, and 25 °C) and the intracellular resuscitation in Acanthamoeba polyphage cells were investigated. Then, the survival profiles of L. pneumophila residing in the planktonic phase and the endosymbiosis phase against antimicrobial silver and copper reagents were differentially compared with the case of Pseudomonas aeruginosa. The number of L. pneumophila in a cultivable state was rapidly reduced to below the detection limit (5.0 log reduction) within 30 days of incubation in synthetic drinking water, while the number of L. pneumophila in a viable state varied in only 0.1 log reduction during the same period, and the levels were sustained constantly for 190 days; in contrast, P. aeruginosa multiplied even in drinking water and continuously maintained its cultivability and viability for 190 days. Distinctively, the numbers of E. coli in both cultivable and viable states were simultaneously diminished as 3.0 log and 1.6 log reduction. The cultivability of L. pneumophila in the VBNC state was recovered and started to multiply after coincubation with A. polyphage in the same environment (initial population of inoculated amoeba was adjusted as 1.0 × 105 amoeba/ mL), and P. aeruginosa also multiplied in amoeba cells. Finally, the populations of L. pneumophila in the planktonic phase after 10 days coincubation were detected at 1.7 × 107CFU/mL, and this population was considered to have originated from the release of bacteria residing inside amoeba caused by the destruction of amoeba cells. Bacteria in the planktonic phase that were exposed to silver and copper were completely inactivated (more than 7 log reduction) within 30 min, while bacteria in the endosymbiosis phase showed much higher resistance against the exposure to the same concentrations of silver and copper. L. pneumophila and P. aeruginosa in A. polyphage cells survived to levels of 5.6 × 101 and 1.1 × 101 CFU/mL at the silver exposure (0.1 mgAg/L) and 7.3 × 103 and 6.1 × 104 CFU/ mL at the copper exposure (1.0 mgCu/L), respectively, after 7 days.

†

This article is part of the Emerging Contaminants Special Issue. * Corresponding author phone: +81-3-5841-6255; fax: +81-58418533; e-mail: [email protected]. 7434

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 23, 2006

Introduction The presence of pathogens in drinking water can be one of the causes of waterborne diseases. Water related pathogens are classified as three sorts, viruses, bacteria, and protozoa; and the route of infection can be differentiated as three routes, ingestion (intestines), inhalation (respiratory), and contact (skin) (2, 3). The source of the contamination in drinking water can be considerable from natural water, wastewater, water distribution networks, and storage tanks (1, 2). Of the pathogens, Gram-negative, rod-shaped bacteria, Legionella pneumophila and Pseudomonas aeruginosa, are water related pathogenic bacteria, and an infection route is not only drinking but also inhalation of aerosols or contact with water (3). L. pneumophila is generally detected as ubiquitous in freshwater environments and reported as the causative agent of Legionnaire’s disease (4-6). P. aeruginosa can infect the human lung and is responsible for the majority of morbidity and mortality in cystic fibrosis (7-10). P. aeruginosa and L. pneumophila are usually isolated from a natural water environment, but their physiological properties against starvation environments are very different. Bacteria released in an aqueous environment are often exposed to stresses due to limitations and changes in nutrient availability, temperature, salinity, oxygen, and pH (11, 12). In this case, bacteria often temporarily enter a noncultivable state generally referred to as viable but noncultivable (VBNC), in which they regulate cell metabolism to adapt to such stresses and then resuscitate when environmental conditions improve. L. pneumophila has been reported to be one type of bacteria that enters into the VBNC state to survive in a starvation environment, and many laboratory studies have demonstrated its resuscitation properties in vivo (5, 11, 13, 14). In contrast, P. aeruginosa is well-known to be capable of growth even at very low organic nutrient concentrations (15). In natural and artificial environments, free-living protozoa or amoebae, such as Hartmannella, Vahlkampfia, and Acanthamoeba spp., serve as host organisms in which bacteria multiply in organelle-studded phagosomes. In particular, Acanthamoeba polyphage, a causative agent of corneal ulcers (eye infection), has been well-established as the host for bacterial intracellular multiplication (16). The role of various bacterial species in promoting Acanthamoeba growth has been well documented by previous research (1, 4, 17). Several Gram-negative, water derived bacterial species such as Escherichia coli, Xanthomonas maltophilia, Flavobacterium breve, and Pseudomonas paucimobilis supported Acanthamoebae growth, while Legionella pneumophila and Pseudomonas aeruginosa inhibited the growth of Acanthamoeba spp (13, 18). Furthermore, there was recent evidence (18, 19) to suggest that intra-amoebal growth of bacteria significantly enhanced the resistance of these bacteria to disinfectants. Antimicrobial silver and/or copper reagents have been occasionally applied to the water distribution system for inactivation of pathogens (20). Silver compounds such as silver nitrate, silver sulfadiazine, silver acetate, and silver protein have antimicrobial properties and have long been used as microbiocidal agents (13, 21, 22). Mcdonnell and Russell (13) summarized that the mechanism of biocidal action of the silver ion was related to the interaction with thiol (sulfydryl, -SH) groups in enzymes and proteins. Other researchers verified the effects of silver as the release of K+ ions from microorganisms, hydrogen bond breaks, depression of nutrient uptake, inhibition of cell division, interference of proton transfer, and bonding to DNA, which resulted in increased stability of the double helix (18, 22, 23). The purpose 10.1021/es060412t CCC: $33.50

 2006 American Chemical Society Published on Web 08/26/2006

TABLE 1. Compositions of the Synthetic Drinking Water

a

compositions

amount (µg/L)

glucosea KNO3 KH2PO4 Na2SO4 CaCl22H2O MgCl26H2O FeCl36H2O KCl CoCl26H2O CuCl22H2O MnSO45H2O ZnCl2 (NH4)6Mo7O244H2O pHb

1.00 995.07 129.92 44.29 18.34 41.81 9.68 19.07 0.40 0.54 21.94 0.21 0.13 7.00

Unit: mgTOC/mL.

b

pH was adjusted by KOH or KCL.

of this study was to investigate the physiological properties of L. pneumophila: the entry into the VBNC state against a starvation environment and intracellular resuscitation from the inside of A. polyphage cells compared with the cases of P. aeruginosa and E. coli. In addition, susceptibilities of L. pneumophila and P. aeruginosa in different residences (planktonic phase and endosymbiosis phase) were evaluated following exposure to antimicrobial silver and copper reagents. Finally, the number of bacteria grown in an amoeba cell was calculated, and the correlation of this number with the number of bacteria in planktonic phase was estimated.

Materials and Methods Model Strains and Preparations. Four kinds of microorganism, Legionella pneumophila (ATCC 33152), Pseudomonas aeruginosa (ATCC 10145), Escherichia coli K12F+A/λ (IFO 3301), and Acanthamoeba polyphage (ATCC 30461) were used during this study. L. pneumophila was cultured for 4 days at 35 °C in buffered charcoal yeast extract R (BCYE R) medium (11.5 g of yeast extract, 1.5 g of activated charcoal, 6.0 g of N-(2-acetamido)-2-aminoethanesulfonic acid (C4H10N2O4S, ACES) buffer, 1.0 g of R-ketoglutarate, and 5.0 mL of Legionella agar enrichment (0.2 g of L-cysteine HCl, 0.125 g of ferric pyrophosphate) per liter of distilled water) (5). P. aeruginosa and E. coli were carefully incubated in Luria-Bertani (LB) broth medium for 24 h at 37 °C (5, 24), and A. polyphage was incubated in Peptone yeast-extract glucose (PYG) medium for 10 days at 25 °C (25). All of the model bacteria used in this study were collected in the exponential growth phase and adjusted to a density of 1 × 106cells/mL. Exponential growth phases were determined by consecutive monitoring of the variation of bacterial population using a flow cytometer (FCM) combined with fluorescent staining of bacterial nucleic acids with SYTO 9 and propidium iodide (PI). A. polyphage was adjusted to a population of 1 × 105 amoeba/mL by the hemocytometer counting method with a plankton counter (MPC-2000, Matsunami, Japan) using the following equation

Camoeba )

∑N n

n

×

amoeba 1mesh ×D × mesh 2.5 × 10-4 mL

where Camoeba is the population of A. polyphage in the sample (amoeba/mL), Nn is the counted number of A. polyphage in a mesh (0.5 mm × 0.5 mm) of amoeba counter observed by microscopic observation, n is the counting times, and D is the dilution rate of sample. Inoculation Processes. A synthetic drinking water (SDW, Table 1) (26) was used as the inoculation medium to prevent the occurrence of unknown disturbances on experiments, and three different experimental stages were successively

organized to observe the physiological properties of L. pneumophila, P. aeruginosa, and E. coli. First, to investigate the entry into the VBNC state, three model bacteria in an exponential growth phase were inoculated into 50 mL of SDW and incubated for 190 days at 25 °C without any injection of nutrients or minerals. Before inoculation into SDW, bacteria in abundant nutrients were centrifuged by a Kubota 5200 centrifuger (Kubota Co. Japan) at 1710×g for 15 min, and the supernatant was discarded to reduce the residual effect of culture media. Then, the sample was resuspended in SDW by vortexing, and this washing process was repeated three times. Finally, three model bacteria were individually inoculated to three glass vessels involving SDW (50 mL) with a density of 1.0 × 105 cells/mL. The second condition was the coincubations of A. polyphage with the model bacteria that were incubated for 190 days. Prior to inoculation of A. polyphage onto the sample, A. polyphage grown in PYG medium were washed three times with PY medium (PYG medium without glucose) and SDW to eliminate the remaining nutrients (centrifuging at 427×g for 30 min). To reduce the amoebal shock from environmental changes, washing was progressed subsequently with PY and SDW. Then, A. polyphage was inoculated into a sample at a density of 1.0 × 105amoeba/mL and coincubated for 7 days (10 days in the case of L. pneumophila) at 25 °C in order to observe the intracellular growth properties of model bacteria. In addition, the survivability of A. polyphage in the SDW without the presence of bacteria was simultaneously observed as a control test. The subsequent condition was the exposure of L. pneumophila and P. aeruginosa samples that were coincubated with amoeba cells to silver and copper reagents, successively. Silver originated from silver nitrate, copper originated from copper sulfate, and a combination of silver and copper reagents was injected into samples and finally adjusted to the concentration of 0.1 mgAg/L, 1.0 mgCu/L, and (0.1 mgAg + 1.0 mgCu)/L, respectively. Samples containing disinfectants were then incubated under the same conditions with the above two experimental conditions for 1 week to observe the survival profiles of the model bacteria. The concentrations of copper and silver reagents were determined with inductively coupled plasma mass spectrometry (ICP/MS, HP 4500 series, U.S.A.) after pretreatment with nitric acid (3%). Detection of Microorganisms. The cultivability of L. pneumophila, P. aeruginosa, and E. coli was determined periodically by counting the average number of colonies formed on BCYE and LB agar plates, respectively. The number of A. polyphage was determined directly by microscopic observation with a plankton counter. Bacterial viability is normally classified depending on the integrity of the cell membrane (27). The viability of model bacteria in this study was determined by using a flow cytometer (FCM, EPICS ALTRA flow cytometer, Beckman Coulter Inc., U.S.A.) after double staining of the nucleic acids of bacteria based on the permeability of molecular probes, SYTO 9 and PI. The applicability of FCM to the three model bacteria was previously verified with prepared samples of dead (pretreated with 70% isopropyl alcohol) and live (pure cultured) cells. FCM with Fluorescently Stained Cells. Nucleic acids of L. pneumophila, P. aeruginosa, and E. coli were stained with two kinds of fluorescent molecular probes, SYTO 9 and PI (LIVE/DEAD Baclight Bacterial Viability Kits, L-7012, Molecular Probes, Inc., U.S.A.) for detection of viability based on the permeability of the cell membrane and analyzed by FCM (7, 27). PI and SYTO 9 were simultaneously added to the samples at a concentration of 1.5 µg per mL sample, and samples were incubated at room temperature for 15 min in the dark. Flow count with a volume of 100 µL (assayed concentration was 1052 particles/µL, Beckman Coulter, U.S.A.) was added to count the bacterial number in samples. VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

7435

FIGURE 1. Variations in cultivability and viability of the model bacteria during the starvation period. Cultivability was detected by counting formed colonies, and viability was detected by FCM analysis following simultaneous fluorescent staining with PI and SYTO 9. Prepared samples were then analyzed with the FCM equipped with a 488 nm argon laser. The emitted fluorescence was split into three different channels: PMT1 for the flow counter, PMT2 with the wavelength range of 510-540 nm for SYTO9 (green fluorescence), and PMT4 (615-640 nm) for PI with red fluorescence. Determination of the Number of Bacteria in Two Different Phases. The numbers of bacteria in the planktonic state and in the endosymbiosis state were determined by application of filtration and homogenization based on the difference of size between bacteria and amoeba and the susceptibility to ultrasonic stress. The filtration with a pore size of 5 µm (Schleicher & Schuell Co., Germany) completely eliminated (over than 99.99%) amoeba cells, but three model bacteria were passed more than 99.5% through membrane pore. The number of bacteria in the planktonic phase was determined by counting the number of colonies formed on the surface of the agar plate after membrane filtration with a pore size of 5 µm. The number of bacteria in the endosymbiosis phase was determined from the resuspension of model bacteria residing inside amoeba cells into SDW by application of homogenization. The samples were homogenized at a power of 7 W for 1 min (11) at room temperature by using an Ultras homogenizer VP-60S (Taitec, Japan) to break up the amoeba cells. The amoeba cells were destroyed at 94% under the homogenizing condition, whereas almost 100% of the model bacteria cells were still alive. The homogenized samples were then cultured on agar plates, and the number of bacteria in the endosymbiosis phase was calculated by the subtraction of the number of colonies counted after filtration from the number of colonies counted after homogenization.

Results and Discussion Entry into the VBNC State of L. pneumophila. After inoculation onto SDW, rapid loss of L. pneumophila cultivability was evident, and no colony formation was detected

on the BCYE agar plate after a 30-day incubation (Figure 1). In contrast, P. aeruginosa multiplied from 5.5 × 104 to 1.7 × 106 CFU/mL in SDW within 1 day of incubation, and only 1 log reduction was detected at the observation of P. aeruginosa cultivability during the 190-day incubation period. In the case of E. coli, the number of cultivable bacteria decreased from 8.6 × 104 to 1.0 × 102 CFU/mL during the test period. The number of L. pneumophila in a viable state determined by FCM detection with SYTO 9 and PI showed only a 0.1 (11%) log reduction over the 190-day incubation. Results with P. aeruginosa were similar to those with L. pneumophila. The number of viable P. aeruginosa varied from 1.7 × 106 to 1.2 × 106 CFU/mL (0.2 log reduction, 19%) over the 190 days after multiplication. Remarkably, cultivability (2.9 log reduction) and viability (1.7 log reduction) decreased simultaneously in the case of E. coli during the experimental period (Table 2). Consequently, it could be determined from the results that P. aeruginosa cells did not lose their cultivability and could multiply its population in SDW: in contrast, L. pneumophila completely entered into the VBNC state after a 30-day incubation by reducing its cultivability and metabolic activities. This result was comparable with previous studies. Steinnert et al. (1) reported that L. pneumophila JR 32 entered into the VBNC state in sterilized tap water after 125 days. Another report (11) investigated that L. pneumophila serogroup 1 suzuki entered into the VBNC state after 60 days of incubation in hot spring water at 25 °C. Ohno et al. (11) and Kim et al. (12) demonstrated that the lack of minerals could result in the simultaneous loss of cultivability and viability of L. pneumophila under extreme starvation conditions such as in distilled water or seawater, but the loss of viability of L. pneumophila was not detected in this study. It is considerable that this maintenance of L. pneumophila viability is based on the presence of trace minerals in SDW. States and Conley (14) reported that certain minerals such as iron, zinc, and potassium were important factors in the survival of L. pneumophila in tap water. In other words, L. pneumophila could maintain its viability for a long time by reducing metabolic activities and loss of its cultivability as long as those minerals that are involved in starvation survival were present. Temperature is also a very important factor for the physiological properties of L. pneumophila against starving environments. Kolva et al. (28) demonstrated that the survivability and cultivability of L. pneumophila was enhanced at lower temperatures. Formation of Endosymbiosis and Bacterial Intracellular Growth. The change in the A. polyphage population at the coincubation with the different bacteria is shown in Figure 2. For the control test without any bacteria present, there was a 0.3 log reduction in A. polyphage in SDW. However, A. polyphage decreased 2.4 and 2.3 log when L. pneumophila and P. aeruginosa were coincubated, respectively. In contrast,

TABLE 2. Number of Bacteria, as Determined by Plate Culture Colony Counting (Cultivability) and FCM Analysis with Double Staining of Nucleic Acids (Viability), in the SDW Condition the number of bacteria (cellsa or CFU/mL)

L. pneumophila

P. aeruginosa

E. coli

time point (day)

cultivable

viable

cultivable

viable

cultivable

viable

0 14 30 190

4.4 × 104b 2.0 × 101 NDd ND

4.7 × 104c 6.3 × 104 4.3 × 104 4.2 × 104

5.0 × 104 1.3 × 106 9.5 × 105 1.3 × 105

6.1 × 104 1.7 × 106 1.6 × 106 1.2 × 106

8.6 × 104 2.4 × 104 3.4 × 103 1.0 × 102

8.7 × 104 4.6 × 104 8.9 × 103 1.7 × 104

a Detected by FCM (unit: cells/mL). were detected.

7436

9

b

Bacterial density detected by colony formation. c Bacterial density detected by flow cytometry.

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 23, 2006

d

No colonies

FIGURE 2. Variations in the population of A. polyphage corresponding to the presence of the model bacteria. The symbol b indicated the population of A. polyphage inoculated in SDW without bacterial existence (control test), O indicated the population of A. polyphage coincubated with L. pneumophila, 1 indicated amoeba coincubated with P. aeruginosa, and ∆ indicated amoeba coincubated with E. coli.

FIGURE 3. Intracellular resuscitation of L. pneumophila in the VBNC state after inoculation of A. polyphage. A. polyphage eventually grew from 8.9 × 104 to 2.6 × 107 amoeba/mL during a 7-day incubation in the presence of E. coli. The properties of in vivo resuscitation of L. pneumophila in the VBNC state and intracellular multiplication of P. aeruginosa cells by phagocytosis of amoeba cells was illustrated in Figure 3. After 2 days of coincubation with amoeba, L. pneumophila in the endosymbiosis phase exponentially multiplied, and L. pneumophila in the planktonic phase also started to multiply following the intracellular multiplication in amoeba cells. Finally, the population of L. pneumophila in two phases reached 2.6 × 107 CFU per mL of suspension after filtration with a membrane filter with a pore size of 5 µm (in planktonic phase) and 1.7 × 106 CFU per mL of suspension after destruction of amoeba cells (in endosymbiosis phase) after a 10-day incubation, respectively. The amount of P. aeruginosa in two different environments increased simultaneously to 1.5 × 107 CFU (planktonic phase) and 2.1 × 106 CFU/mL (endosymbiosis phase). In contrast,

the number of E. coli cells in both the planktonic state and within the amoeba gradually decreased to 1.6 × 100 and 2.1 × 101 CFU/mL (Table 3), respectively. From the results, an increase of the number of L. pneumophila in the planktonic phase was detected after an exponential increase of L. pneumophila in the endosymbiosis phase. Because L. pneumophila was not able to multiply in SDW conditions and entered into the VBNC state before inoculation of A. polyphage suspension, the increasing number in the planktonic phase seemed to originate from the released number of L. pneumophila that multiplied in amoeba cells. Interestingly, the populations of both L. pneumophila in the planktonic phase and the endosymbiosis phase did not change immediately but remained nearly constant during the 2- or 3-day incubation after inoculation of A. polyphage. In comparison with the case of L. pneumophila, the population of planktonic P. aeruginosa also did not change immediately but remained constant during the 2-day incubation after inoculation of A. polyphage, while the amount of P. aeruginosa in the endosymbiosis phase multiplied exponentially from no colony formation to 3.8 × 106 CFU/ mL of homogenized suspension during that period. This phenomenon was probably due to differences in the physiological status of the two model bacteria (29). Since L. pneumophila entered into the VBNC state due to the lack of nutrients in SDW, a certain time was required to recover its cultivability and metabolic activities for the intracellular multiplication of its cells in amoeba. Based on the number of bacteria in endosymbiosis phase and the number of amoeba cells detected, the number of L. pneumophila and P. aeruginosa cells that could reside inside of an A. polyphage cell was estimated. The maximum number of L. pneumophila in an amoeba cell was calculated to be 4.5 × 104 CFU/amoeba, and 4 × 104 CFU/amoeba was calculated in the case of P. aeruginosa. Barker et al. (16) demonstrated that the number of bacteria in the amoeba depends on the size of the amoebae vesicle or cyst. Berk and co-workers (30) calculated that each vesicle of A. polyphage could contain between 20 and 200 L. pneumophila cells, and using similar methods Donlan and Costerton (18) calculated from 365 to 1483 bacteria in a 5 µm diameter vesicle. Consequently, coincubation of A. polyphage with E. coli supported the growth of the amoeba and resulted in a decrease in the number of E. coli cells: in contrast, coincubation with L. pneumophila or P. aeruginosa proved lethal to A. polyphage, and the numbers of L. pneumophila or P. aeruginosa increased significantly. Additionally, the variation in the amount of planktonic bacteria after inoculation of A. polyphage depended on the ability of the organism to multiply within A. polyphage. In other words, certain pathogenic bacteria in the VBNC state cannot be detected by counting CFUs even though cells are present in samples, but they could recover their cultivability and multiply by intracellular resuscitation or growth within the other organism.

TABLE 3. Change in the Number of Bacteria, as Determined by the Plate Culture Colony Counting Method Following Filtration or Homogenizing, During the Coincubation with A. polyphage the number of bacteria (CFU/mL)

L. pneumophila time point (day) 0 3 7 10

planktonic

phasea

ND 1.5 × 101 1.3 × 106 2.6 × 107

in amoeba

P. aeruginosa phaseb

ND 1.3 × 103 7.6 × 106 1.7 × 107

E. coli

planktonic phase

in amoeba phase

planktonic phase

in amoeba phase

1.3 × 105 1.7 × 106 1.5 × 107 c

ND 3.8 × 106 2.1 × 107 c

1.0 × 102 7.3 × 101 1.6 × 100 c

ND 5.7 × 101 2.1 × 101 c

a The number of bacteria in planktonic phase determined by agar plate culture colony counting method after filtration (5 µm). b The number of bacteria in endosymbiosis phase determined by the agar plate culture colony counting method after homogenizing at the power of 7 W for 1 min. c Not tested.

VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

7437

FIGURE 4. Survival profiles of bacteria between the planktonic state and within the A. polyphage state against copper or silver exposure. The abbreviation LP means the number of L. pneumophila in the planktonic phase, LA means the number of L. pneumophila in endosymbiosis, PP means the number of P. aeruginosa in the planktonic phase, and PA means the number of P. aeruginosa in the endosymbiosis phase. Biocidal Profiles of Copper and Silver for Inactivation of Model Bacteria. L. pneumophila and P. aeruginosa that resuscitated and multiplied by phagocytosis of intracellular substances in amoeba were exposed to silver (0.1 mg Ag/L) and copper (1.0 mg Cu/L) reagents separately and in combination. Figure 4 shows the differences in the inactivation profiles of the model bacteria in the planktonic phase and the intracellular phase. All of the L. pneumophila and P. aeruginosa cells in the planktonic phase were completely inactivated after less than 0.5 h of exposure to silver or copper. However, the bacteria within A. polyphage did not lose as much activity or viability as those in the planktonic state even after exposure to copper for 7 days. For example, P. aeruginosa within A. polyphage cysts survived at a level of 6.1 × 104 CFU/mL (2.5 log reduction) after 7 days exposure to copper. A similar phenomenon was observed in the L. pneumophila test: 7.3 × 103 CFU/mL (3.4 log reduction) of L. pneumophila were detected from Acanthamoebae cells. One reason that bacteria grown in the amoeba cells are resistant to chemical disinfectants may be because amoeba cysts, similar to spores, are more impermeable to disinfectant molecules than vegetative cells (13). Barker et al. (16) demonstrated that 50-100% of L. pneumophila cells grown within A. polyphage could survive, but bacteria in the planktonic state lost 99.99% viability after 4 h exposure to polyhexamethylene biguanide (PHMB). Donlan and Costerton (18) summarized that 99% inactivation of planktonic L. pneumophila was accomplished following exposure to 0.5 mgCl/L for 1 min, but 10 mgCl/L for 2 h (pH 7.0, 25 °C) was required to inactivate 99% of L. pneumophila entrapped in Acanthamoeba castellani. Although the concentration of silver injected in SDW was 10 times less than the amount of copper reagent, the antimicrobial capacity of the silver reagent for inactivation of model bacteria within the amoeba was much higher. The silver reagent showed 100-fold higher antimicrobial capacity than copper after 7 days exposure in the case of L. pneumophila in the amoeba phase, and moreover, it showed almost 1000-fold higher antimicrobial activity in the case of P. aeruginosa (Figure 4). Antimicrobial capacity was increased when the two biocidal reagents were combined. No colonies were detected after 4 (P. aeruginosa) and 6 days (L. pneumophila) when both copper and silver were used simultaneously. 7438

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 23, 2006

According to this study, in vivo resuscitation and multiplication properties of L. pneumophila and P. aeruginosa that entered into the VBNC state to tolerate the starvation were observed, and the amoebal interruption to the susceptibility of those bacteria against exposure to antimicrobial silver and copper reagents was estimated in comparison with bacteria in planktonic phase. Based on the experiences of this study, investigation into the mechanism of amoebal interruption to the biocidal activities of disinfectant to inactivate pathogenic bacteria will be undertaken. Additionally in the focus of bacterial detection, the application of FCM to the simultaneous determination of bacterial viability and population in the planktonic phase was successfully performed throughout this study; however, it failed to detect the bacteria residing inside of amoeba (endosymbiosis phase). This was probably due to the interruption of the organic molecular substances released from the destruction of amoeba cells by homogenization. Further investigation into this phenomenon is required as new problems to detect bacteria in amoeba using FCM methods.

Acknowledgments We gratefully acknowledge the financial support provided by Grant-in-Aid for Young Scientists (A) 17686045, Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Literature Cited (1) Steinert. M.; Emo¨dy, L.; Amann, R.; Hacker, J. Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl. Environ. Microbiol. 1997, 63, 2047-2053. (2) Youwen, Y,; Jie, H.; Pei C. C.; Yan, J. Removal and inactivation of waterborne viruses using zerovalent iron. Environ. Sci. Technol. 2005, 39, 9263-9269. (3) Microbial aspects; World Health Organization (WHO) homepage. http://www.who.int/water_sanitation_health. (4) Paszko-kolva, C.; Yamamoto, H.; Shahamat, M.; Sawyer, T., K.; Morris, G.; Colwell, R., R. Isolation of amoebae and Psedomonas and Legionella spp. from eyewash stations. Appl. Environ. Microbiol. 1991, 57, 1630167. (5) Szeto, L.; Shuman, H. A. The Legionella pneumophila major secretory protein, a protease, in not required for intracellular growth or cell killing. Infect. Immun. 1990, 58, 2585-2592.

(6) Fry, N. K; Harrison, T. G. An evaluation of intergenic rRNA gene sequence length polymorphism analysis for the identification of Legionella species. J. Med. Microbiol. 1998, 47, 667-678. (7) Kievit, T. R. D.; Parkins, M. D.; Gillis, R. J.; Srinkumar, R.; Ceri, H.; Poole, K.; Iglewski, B. H.; Storey, D. G. Multidurg efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 2001, 45, 1761-1770. (8) Teitzel, G. M.; Parsek, R. M. Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol. 2003, 69, 2313-2320. (9) Head, N. E.; Yu, H. Cross-sectional analysis of clinical and environmental isolated of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity. Infect. Immun. 2004, 72, 133-144. (10) Govan, J. R.; Deretic, V. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev. 1996, 60, 539-574. (11) Ohno, A.; Kato, N.; Yamada, K.; Yamaguchi, K. Factors influencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water. Appl. Environ. Microbiol. 2003, 69, 25402547. (12) Kim, D. S.; Thomas, S.; Fogler, H. S. Effect of pH and trace minerals on long-term starvation of Leuconostoc mesenteroides. Appl. Environ. Microbiol. 2000, 66, 976-981. (13) Mcdonnell, G.; Russell, A. D. Antiseptics and disinfectants: activity, action, and resistance. Clin. Microbiol. Rev. 1999, 12, 147-179. (14) States, S. J.; Conley, L. F.; Ceraso, M.; Stephenson, T. E.; Wolford, R. S.; Wadowsdy, R. M.; McNamara, A. M.; Yee, R. B. Effects of metals on Legionella pneumophila growth in drinking water plumbing systems. Appl. Environ. Microbiol. 1985, 50, 11491154. (15) Oh, J. W. Evaluation of bacterial regrowth potential in drinking water system using cell cycle parameters. Ph.D. Dissertation, Department Urban Engineering, The University of Tokyo, Japan, 2004. (16) Barker, J.; Brown, M. R. W.; Collier, P. J.; Farrell, I.; Gilbert, P. Relationship between Legionella pneumophila and Acanthamoeba polyphage: physiological status and susceptibility to chemical inactivation. Appl. Environ. Microbiol. 1992, 58, 24202425. (17) Qureshi, M. N.; Perez, A. A., II; Madayag, R. M.; Bottone, E. J. Inhibition of Acanthamoeba species by Pseudomonas aeruginosa: Rationale of their selective exclusion in corneal ulcers and contact lens care system. J. Clin. Microbiol. 1993, 31, 19081910. (18) Donlan, R. M.; Costerton, J. W. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 2002,

15, 167-193. (19) Kilvington, S.; Price, J. Survival of Legionella pneumophila within cysts of Acanthamoeba polyphage following chlorine exposure. J. Appl. Bacterial. 1990, 68, 519-525. (20) Lin, Y. E.; Vidic, R. D.; Stout, J. E.; Yu, V. L. Legionella in water distribution systems. J. Am. Water Works Assoc. 1998, 90, 112121. (21) Brown, M. R. W.; Anderson, R. A. The bactericidal effect of silver ions on Pseudomonas aeruginosa. J. Pharm. Pharmacol. 1968, 20, 1-3. (22) Kim, J.; Cho, M.; Oh, B.; Choi, S.; Yoon, J. Control of bacterial growth in water using synthesized inorganic disinfectant. Chemosphere 2004, 55, 775-780. (23) Rusin, P.; Bright, K.; Gerba, C. Rapid reduction of Legionella pneumophila on stainless steel with zeolite coatings containing silver and zinc ions. Lett. Appl. Microbiol. 2003, 36, 69-72. (24) Hoefel, D.; Grooby, W. L.; Monis, P. T.; Andrews, S.; Saint, C. P. A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester and indicators of bacterial activity. J. Microbiol. Methods 2003, 52, 379-388. (25) Kahane, S. B.; Dvoskin, M. M.; Friedman, M. G. Infection of Acanthamoeba polyphage with Simkania negevensis and S. negenensis Survival within amoebal cysts. Appl. Environ. Microbiol. 2001, 6, 4789-4795. (26) Sathasivan A.; Ohgaki, S.; Yamamoto, K.; Kaniko, N. Role of inorganic phosphorus in controlling regrowth in waterdistribution system. Water Sci. Technol. 1997, 35, 37-44. (27) Amor, K. B.; Breeuwer, P.; Verbaarschot, P.; Rombouts, F. M.; Akkermans, W. A.; De Vos, M.; Abee, T. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress. Appl. Environ. Microbiol. 2002, 68, 5209-5216. (28) Paszko-Kolva, C.; Shahamat, M.; Colwell, R. R. Effect of temperature on survival of Legionella pneumophila in the aquatic environment. Microb. Releases 1993, 2, 73-79. (29) Rowbotham, T. J. Current views on the relationships between amoebae, Legionellae and man. Isr. J. Med. Sci. 1986, 22, 678689. (30) Berk, S.; G.; Ting, R. S.; Turner, G. W.; Ashburn, R. J. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl. Environ. Microbiol. 1998, 64, 279-286.

Received for review February 20, 2006. Revised manuscript received July 24, 2006. Accepted July 28, 2006. ES060412T

VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

7439