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Eradication of Acinetobacter baumannii by photosensitized agents in vitro
Journal
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of
Photochemistry Photobiology
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and
B:Biology
E L S E V I ER
Journal of Photochemistry and I)hou>hioh)gy B: Biology 42 ( 1998 ) 21 I 218
Eradication of Acinetobacter baumannii by photosensitized agents in vitro Yeshayahu Nitzan *, Anat Balzam-Sudakevitz, Helena Ashkenazi Health Science~ Research Center, l)eparlme,t o/L!'/~" Sciences, Bar-l/an Univerxio', Ramat-Gan, 52900, Israel Received 28 Atlgttsl
1997: accepted 13 February 1998
Abstract
The photodynamic effects of photosensitizers on Acinetohacter baumam#i were studied. These Gram negative bacteria have recently been implicated in various infections, mainly acquired in hospitals. They have outstanding characteristics of multidrug high resistance to antimicrnbial agents. The best photodynamic effect was obtained when A. bammmnii cultures were treated with light activated deuteroporphyrin (Dp) at a concentration of 34 Ixmoles I ~and polymyxin nonapeptide (PMNP) at a concentration of 200 txmoles 1 ~. At these concentrations the culture in brain heart infusion (BH1) broth was tbund to be sterile after l h of treatment. Some inhibition was also obtained under the same conditions with Cd-texapfiyrin (Cd-Tx) in the presence of PMNP. Treatment with various other photosensitizers in the presence of PMNP exhibited only marginal antibacterial activity. The cationic photosensitizer tetra-methylpyridyl porphme (TMPyP) did not exhibit any photodynamic efl;ect on A. baumam#i when illuminated during its growth in BHI broth. Bacteria grown in nutrient broth or suspended in saline and treated by TMPyP resulted in a significant photoinactivalion by the sensitizer alone even in the absence of PMNP. h was found that a high concentration of the proteins present m BHI or in serum prevent TMPyP fiom acting as a photosensitizer against A. haumannii. Bovine serum albumin at the same high protein concemration prevents Dp ( in the presence of PMNP ) to act as a photosensitizer. The anionic photosensitizer tetra-sulfouatophenyl porphine I TPPS,) did not show any photodynamic effect in high or low. protein media. In this study it was found that despite the high resistance of the Acinetohacter haumamfii to antibiotics, these bacteria can be significantly photoinactivated by treatment with either Dp + PMNP or TMPvP in low protein content environments. When the protein concentration is high, photoinactivation efficiency depends on the type o f protein present in the medium. ~E:,1098 Elsevier Science S.A. All rights reserved. Kevwords: Actinetol+acwr: Photosensitization: Cationic porphyrins: Dcuteroporphyrin: Polymyxin nonapeptide; (]ran>negative bacteria
1. Introduction A c i n e t o b a c t e r b a u m a n n i i is one of the species in the genus A c i n e t o b a c t e r , which is an aerobic Gram negative rod related
to the Pseudomonas group [ I ]. In recent years,Acinetobacter b a u m a m f f i has emerged as an important nosocomial pathogen in intensive care units [ 2 - 7 ] . This microorganism is capable of causing life-threatening infections such as bacteremia, pneumonia, meningitis, endocarditis, skin and wound infections 18-1 I ]. A c i n e t o b a c t e r microorganisms are very often multidrug-resismnt to unrelated antibiotics and are a significant problem in hospitals [ 12,13] due to the difficulty to eradicate them [ 14-16]. They produce potent fl laclamases, aminoglycoside modifying enzymes and chloramphenicol acetyltransferase as one of the mechanisms of resistance [ 17 ]. ~: (7orresponding author. Tel.: +972 3 531 8592: Fax: + 972 3 535 1824: e-mail: n itzay (a' mail.biu.ac.il
101 I - 1344/98/$19.00 (~) 1998 Elsevier Science S.A. All righls reserved. PIISIOI 1-1344(981(t(1073-6
To overcome the problem of multidrug resistance of bacteria, the photodynamic treatment by photosensitizers was established for photoinactivation of bacteria. Gram-positive bacteria such as S t a p h y l o c o c c u s a u r e u s are susceptible to porphyrin-induced antibacterial activity [18,19]. The efficient and non-recovering antimicrobial killing effects due to irradiation are independent on the antibiotic susceptibility spectrum of the treated pathogen I 18,20-22 ]. Gram-negative bacteria exhibited induced damage with light, only when using the small non-toxic peptide derived from polymyxin B nonapeptide (PMNP), which stimulated the translocation of porphyrins through the membranes of Gram-negative bacteria and enabled photodynamic damage [ 231. Anaerobic bacteria have also been shown to be sensitive to several of the above photosensitizers [ 24]. Porphyrin binding to the cytoplasmic membrane is a prerequisite for photosensitization of Gram-positive and Gramnegative bacteria [23,25] regardless of whether they are aerobic or anaerobic 124]. Recently, cationic porphyrins
212
E Nil=an el a[. /,lourmd ot I'hoto¢'/wmi~lr 3 emd t'hotohiolok'y B: Biology 42 (199,~) 211 21,~
have been shown to photoinduce the direct inactivation of Gram-negative bacteria without the presence of an additional permeabilization agent [ 26]. The same phenomenon has also been shown for cationic water soluble zinc phthalocyauines [ 27 ]. In the present study, we have examined pholoinactiration of the opportunistic multidrug resistant Acinetohacter baumannii by various hydrophobic photosensitizers, as well as by two differently charged hydrophilic sensitizers: the anionic 5,10,15,20-tetra (4-sulfonatophenyl) porphine (TPPSa) and the cationic 5. I 0,15,20-tetra( 4-mefllylpy ri dy I ) porphine (TMPyP). In the present work, we demonstrate the photoinactivation of the multidrug resistant bacterium A. haumannii by various photosensitizers in the presence of PMNP. In addition, we also attempted to establish the relationships of cationic photosensitizers in different physiological environments. These environments contain different contents or concentrations of proteins.
2. Materials and methods
2.1. Bacterial strain Acinetobacter baumannii biotype 9, recovered from clinical material submitted to the clinical bacteriology laboratory at the Meir Hospital, Kfar Saba, Israel. was used in this study. This strain was multi-resistant to the lbllowing antibiotics: ampicillin, mezlocillin, piperacillin, cefoxitin, ceftriaxone, aztreonam, tetracycline, chloramphenicol, gentamycin, tobrarnycin, amikacin, ciprofloxacin and norfloxacm, ll was found to be sensitive to colistin, imipenem, and polymyxin B.
function of time. Viable bacteria were monitored by counting the number of colony forming units on agar plates.
2.3. Growth inhibition cahtdalions The rate of growth inhibition of Acinetobacter baumannii by the various photosensitizers was calculated by measuring the optical density at 660 nm at the time when the photosensitizer was added (OD,) and 3 h later (OD,). In the control sample the second measurement was also perlormed after 3 h ( ()D~.i. Percent inhibition of the culture was calculated by the following equation: I
OD~-OD"tXIO0
~i;i inhibition of the culture
o & . - OD,, I
0I),, was always 0.3 and is indicated by an arrow in the relevant figures. The survival fraction N/N. was determined [ 28,29 ] by counting the colony-rotating units per ml, before starting the treatment by' the sensitizer under illumination (N,,) and after treatment for the indicated times under illumination (N). Bacterial cultures grown under the same conditions and light exposures but without any photosensitizer served as controls.
2.4. Preparation of PMNP PMNP was prepared by, cleavage of polymyxin B sulfate with Ficin as described previously [ 30 ]. The purity of each batch of PMNP was tested on cellulose-coated aluminum foil thin layer chromatography (TLC) plates ( Merck, Darmstadt, Germany ). Polymyxin B sulfate and licin ( Ec 3.4.22.3 ) from lig trees were obtained from Sigma ( St. Louis, MO).
2.5. Photosensili=er sohttions 2.2. Bacterial growth and photosensitization pro~'edure Cultures of A. baumamfii which were grown on brainheart agar (Difco, Detroit, MI ) for 18 h were transferred into brain heart infusion medium (Difco) and adjusted to pH 6.5. In other experiments overnight cultures were grown on nutrient agar (Difco) and were transferred into nutrient broth (Difco) at pH 6.5. In some indicated experiments the bacteria were transferred into 0. l M PBS pH + 6.5 supplemented with 15 mg/ml Bovine serum albumin (BSA) (Sigma, USA) or 15 mg/ml fetal calf serum (Biological Industries. Beit-Haereek, Israeli. The cultures were transferred into the broth media to a final volume of 25 ml at an initial optical density of 0. I at 660 nm and growth continued at 37 °C with aeration. Photosensitizers were always added when the culture reached an optical density of 0.3 at 660 nm. PMNP was added in the indicated experiments prior to the addition of the photosensitizer. Cultures were irradiated at 140 W m -' using two unfiltered tungsten lamps placed 30 cm above both sides of the flasks. Bacterial growth was determined with a Novaspec, Biochrom LKB (Cambridge, UK) spectrophotomctcr by monitoring the increase in optical density at 660 nm as a
Stock solutions of deuteroporphyrin (Dp), hematoporphyrin, hematoporphyrin derivative and protoporphyrin were prepared by dissolving 5 mg of the photosensitizer in 200 ~l of 1 N NaOH with vigorous agitation. A sterile NaCI solution (0.85~/~) was added to achieve a final concentration of 2.5 mg/ml. The sensitizers merocyanine 540, chloro-aluminium phthalocyanine, tetra-sulfonatophenyl porphine (TPPS,) and tetra-methyl pyridyl porphine (TMPyP) were dissolved to a concentration of 2.5 rag/ml in sterile distilled water. Zntetrabenzoporphyrin, Mg-tetrabenzoporphyrin and Cd-texaphyrin (Cd-Tx) were dissolved in dimethyl formmnidc to a tinal concentration of 2.5 mg/ml. All stock solutions were stored in the dark at 4 ~(" for a maximum of I week. Protoporphyrin, Zn-tetrabenzoporphyrin Dp, TPPSa and TMPyP were obtained from Porphyrin Products ( Logan, UT). Hcmatoporphyrin and merocyanine 540 were purchased Irom Sigma (St. Louis, MO). Chloro-aluminium phthalocyanine was obtained fi-om Eastman-Kodak USA. Cd-Tx was synthesized by Dr Sessler as described previously 131 ]. Mg-tetrabenzoporphyrin was prepared by Professor Ehrenberg according to published reactions [ 32,33 ].
Y. Nit=an el al. / Jottrmtl o/ Pholochemistrv .rid PI olobiolo~,,v B." Biology 42 (1998) 211 218
213
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Photosensitization ofA. baumannii treated by various porphyrins ( natural, semi-synthetic or synthetic ) in the presence of PMNP resuhed in bacterial growth inhibition effects ( Fig, I ). These inhibitory effects were 88-90~;¢, respectively, when the bacteria were treated by Cd-Tx or by Dp in the presence of PMNP (200 ixmoles 1 I ). Only marginal inhibition was exhibited by all the other photosensitizers (3-23~A inhibition). Inhibition of 6()°/~ was exhibited by Mg-tetrabenzoporphyrin in the presence of PMNP. The amount t)f PMNP present in the treatment was found to be important in the photosensitization procedure (Fig. 2 ). When the amount of PMNP reached 200 ixmoles I ~ or more, the growth inhibition rate of the photosensitizer Dp increase to 87 88c~. Increasing the amount of PMNP to 300 p~moles I I have increased the growth inhibition of A. baumwmii upon photosensitization with Dp to 80% and only marginally affected the growth of the bacteria upon photosensitization with the other sensitizers (results not shown). In parallel, treatment of the bacteria with Dp or Cd-Tx it] the presence of PMNP had a dramatic effect on the viability of the bacteria { Fig. 3 ). Viability decreases upon treatment with either 17 ~ m o l e s I Dp or Cd-Tx, in the presence of the appropriate concentration (I
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Fig. 2. Growth inhibition of Acinetoha(Wr bamnannii by Dp and wtrious concentrations of PMNP under illumination. Bacterial cells were grown in BHI and treatment was begun by addinf the indicated concentrations of PMNP and Dp { 17 ixlnoles I i). Optical densilies were monitored at 661) nnl lk)l-3 h. Treatment was begun at 0 . 3 0 D ( see [lrrow ). I, Untreated control: 2,50p+lnolesl ~ P M N P + D p ; 3 , 1 0 0 1 ~ m o l e s l ~ P M N P + b p ; 4 . 1 5 0 p . m n l e s l r P M N P + D p : 5, 200 #moles l f P M N P + D p : 6, p.molesl P M N P + I)p: 7. 300 ixmoles I i PMNP F I)p.
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Fig. I. G r o w t h inhibition ol'Aeineu~bacter baumannii by various photosensitizers in the presence ol PMNP under illumination. Photosensitizers with PMNP ( 200 #moles 1 ' ) were added to bacterial cultures grown m BHI. Optical densities were monitored for 3 h at 660 nm. Treatlnent was begun at 0 . 3 0 D I see arrow). 1. Untreated control: 2. polymyxm nnnapeptide (PMNP) alone: 3. deuteroporphyrin ( 17 #.molesl I I + P M N P : 4 . hema toporphyrin derivative ( 14 p.moles I ') + PMNP; 5. protoporphyrin 118 p.lnoles I t ) + PMNP; 6, hemalop,arphyrin ( 17 lamoles } i ) + PMNP: 7. Znqetrabenzoporphyrin ( 18 ~nmles I ~ ) + PMNP; 8, Mg tetrabenzupur phyrin ( 19 #_moles I ~) + PMNP: 9, Cd-texaphyrin ( 17 Fmoles I ) PMNP: 10. chloro+alummimn phthalocyanine i l 8 g.molesl ~ ) + P M N P : ll.merocyaImle540(ISgmolc~l i)+PMNP.
Fig. 3. Photndynamic effects on the ,Aability o f Acinetobacter baun+annii. Acinelobacter cuhures grown in BHI were incubated under illumination at the indicated concentrations of the photosensitizer in the presence of PMNP 200 p.molesl r. Dp (17 lamole,,,1 ~ ) + P M N P ( O ) : Dp 134 txmolesl i ) + P M N P ( C ) ) ; o r C d _ T x ( l T t x m u l e ~ l t)+PMNP(•).Thecon. trol culture ( A ) was not treated at all. Counts of the viable bacteria were monitored as a fimclion of illumination time.
of PMNP, and both demonstrated inactivation curves with double slopes. One slope shows a fast killing rate in the first 90 rain of illumination. The survival fraction calculated for 90 min was N I N , = 2 X 10 5 and 2 . 5 × 10 4, respectively. for the above two sensitizers. Viability decreased further at a slower rate of killing as shown by the second slope R~r each
214
Y. Nitzan et al. I Journal of Photochemistt3' aml Pkombioh~,w B." Biology 42 (1998) 211 218
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3 4 2 Fig. 4. Growth inhibition of Acinetobacter bamnannii by TMPyP. TPPS4 and Dp + PMNP grown in various media with illumination. Bacterial cells were grown to the beginning of the log phase and treatment was begun by adding the indicaled photosensitizer. Bacteria were grown at 37 ~'(' for an additional 3 h. Optical densities were monitored fi)r 3 h at 660 nm. Treatment always started at 0.30D I see arrow ). 1, Untreated comml; 2, TMPyP ( 8 ixmolesl ~);3, TPPS4(8,txmolesl '):4, Dp(171.zmolesl ~)+PMNP ( 200 gmoles 1 t ). Bacteria grown in BHI are shown in the uppcr panel and those grown in NB are shown in lhe lower panch photosensitizer. Only by treating the bacteria with 34 # ` m o les 1 t Dp and P M N P was a sharp killing rate o t ' 6 orders of magnitudes obtained within 30 rain of illumination. Using this protocol, no viable bacteria were found in the culture m e d i u m after 35 min from the beginning o f the photosensitization treatment. T M P y P , a cationic porphine photosensitizer, did not demonstrate any effect on A. baumannii upon photosensitization when grown and photosensitized under the same conditions. These growth conditions included growth in BHI broth which is a protein-rich medium. This cationic porphine ( T M P y P ) demonstrated a growth inhibition when the medium in which the bacteria were grown was nutrient broth ( N B ) (Fig. 4). Furthermore, growth inhibition was obtained by T M P y P alone, in the absence o f the P M N P as a m e m b r a n e disorganizing agent. The difference between BHI broth and NB is that BHI contains 14.75 m g / m l protein while NB contains only 1.95 m g / m l protein. This is a 7-fold difference in the protein concentration, which may evidently cause a big difference in the behavior of the photosensitizer in these two media. When e x a m i n i n g the viable counts o f A . baumamtii treated in BH1 broth, only marginal effects were observed even with 32 t x m o l e s l ' T M P y P (Fig. 5). H o w e v e r , in NB, when the bacteria are treated with only 8 Ixmoles I ~ o f T M P y P a double slope of inactivation can be seen: N / N , , = 1 × 10
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Fig. 5. Viability of Acitwtobacter haumannii treated by TMPyP in NB or
BHI. Acinelobacter cultures were incubated under illumination at concentrationsof8#molesl 'TMPyPinNB(•):32txmolesl 'TMPyPinNB ( O ): 32 bunoles 1 t TMPyP in BHI ( • ). The control cullnres in NB ( D ) and ill BHI ( • ) were not treated. Colony forming units were monitored as a function of illumination time. within 30 min and in the other slope N / N , , = 2 × 10- 2 during 3 h was observed. With 32 #`moles I ~T M P y P , a single sk)pe and a survival rate of N/N,, = 5 × l0 ~' was obtained within 30 rain and sterilization of the culture could be obtained 5 min later (Fig. 5 ). The next step was to determine which of the f o l l o w i n g factors has the greatest effect on the photosensitizer's ability to photoinactivate the A. haumannii microorganisms: (a) protein concentration alone is the factor that affects the photoinactivation ability of the cationic photosensitizer; (b) the protein composition is also an important factor, in an attempt to solve this problem, we e x a m i n e d the photoinactivation ability of Dp + P M N P and of T M P y P in media containing 15 m g / m l serum proteins and in media containing an equal concentration of BSA. Pertorming photoinactivation experiments in serum at a protein concentration of 15 m g / m l demonstrated (Fig. 6) that 34 i.tmoles 1 J Dp with P M N P acts much better than 32 #.moles 1 ' T M P y P . The killing rate with Dp + P M N P was 7 orders of magnitudes and the survival fraction was calculated as N / N o = 10 7 in one slope within 3 h. As for the T M P y P , the same slope was obtained for only the first hour, as the survival fraction was N / N . - 5 × 10 -'. After 1 h of treatment, the killing rate was drastically reduced. With B S A (15 m g / m l ) , the opposite effect was observed. The photoinactivation rate by T M P y P (32 ixmolcs I ~) was
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Fig. 7. Viability ofAcinelobacter bamnamtii treated by TMPyP or Dp in thc
presence of PMNP in serum ( 15 mg/ml protein). Acinetohacwr cultures were incubaled under illumination at concentrations o1=8txmolcs I ~TMPyP (~); 32 ixmolesl t TMPyP ( i ) : 17 o.moles I ~ Dp+PMNP (C~}: 34 bunolesl ~ Dp+PMNP (•);Thecontrolculture in serum ( • ) was not treated. Cohuay forming units were counted as a function oI illuufination time.
presence of PMNP iu BSA ( 15 rag/iiil ). Acinembacwrcultures were incubated under illumination at concentrations o18 ~moles I ~TMPyP ([~): 32 p.molesl ITMPyP ( I ) ; 17 bu'nolesl ~ Dp+PMNP (()): 341xmoles I ~l)p + PMNP ( • ), The control culture with BSA ( • ) was not treated Colony forming units were counted as a function of illumination time.
better than the inactivation rate by 34 ixmoles I ~ Dp with P M N P (Fig. 7). The killing rate was 7 orders of magnitude and the survival rate of T M P y P was calculated to be 10 7 in 1 h. Although the same survival rate was achieved lot Dp and PMNP, it was achieved only after 3 h. Apparently, not only the protein concentration but also the type of protein in the m e d i u m affects the photosensitization ability of the various photosensitizers against Acmetobacter.
4. Discussion The Gram negative bacterium Acinetobacter baumatmii is of special interest for photodynamic therapy since it is resistant to many kinds of antimicrobial agents. The ability of photosensitizers to inactivate this microorganism and thus overcome its multidrug resistance may offer an alternative method to the use of antibiotics. However, photoinactivation of A. baumannii appears to be limited to selected photosensitizers such as deuteropolphyrin and Cd-texaphyrin, both require the presence of the m e m b r a n e idsorganizing peptide P M N P 123]. Other photosensitizers, such as hematoporphyrin derivative, hematoporphyrin or protoporphyrin, which
are effective against Gram positive bacteria [ 18,29 ] or some anaerobic Gram negative bacteria [241, are not effective against this particular microorganism. Furthermore, the concentration of P M N P required for optimal disorganization of the bacterial m e m b r a n e s is different from those used with other species of G r a m negative bacteria. For example 500-700 Ixmoles I ~ P M N P are required for the photoinactivation of E. coil by Dp or hydrophobic porphines [ 23,28 I, whereas only 50 Ixmoles I ~ P M N P can cause the disorganization of m e m b r a n e s of Pseudomonas aeruginosa and enable its photoinactivation by Dp. The differences between the Gram negative bacteria mentioned here and the amounts of P M N P needed for disorganization of their barriers may be a reflection of the hydrophobic state of the bacterial barriers. The barriers of A. baumannii are similar to those of other Gram negative bacteria, and include the outer and inner membranes. In addition, some strains of A. baumamfii produce a slime capsule on the surface of the bacteria rendering a surface more hydrophilic. In strains isolated lyon] catheters or tracheal devices, their hydrophobicity was found to be higher than in strains isolated in other sites [ 34 ]. It is possible that in other strains of the bacteria a higher or lower a m o u n t of P M N P will be required for their photoinactivation, in the present study, it
216
Y. Nitzan et al./ Journal o/Pholochemist;y and Pholohiology B: Biology 42 (1998) 211 21,~
was found that 200 Ixg/ml PMNP is needed fl)r the photoinactivation of Acinetobacter baumannii by Dp or Cd-texaphyrin. With this amount of PMNP and Mg-tetrabenzoporphyrin as photosensitizer, we observed a 6()~ growth inhibition and this increased to 80~J when the amnount of the disorganizer was increased to 300 txmoles I ~. This might indicate that for certain photosensitizers, a higher degree of barrier disorganization is necessary in order to penetrate into the bacteria. The amounts of PMNP used in these experiments were not toxic to A. baumannii. It was flmnd previously ] 301 that PMNP is not toxic by itself on enteric bacteria up to an amount of 800 i~moles I ~. In the present work, we used amounts of PMNP of up to 300 ixmoles I ~ with no toxic effects on A. baumam~ii bacteria. It is possible that even higher amounts of PMNP are not toxic to this bacteria. It is now well recognized that in Gram negative bacteria, which possess two membrane layers, photosensitization is possible only if the photosensitizers penetrate through the outer membrane as well as through the cytoplasmic inner membrane [ 2326,27,35,361. Furthermore, although binding of the photosensitizer to the bacterial membranes is a prerequisite for the photosensitization process, it is not by itself sufficient for the photoinactivation of the bacteria. This statement was made in a previous study [ 25 ] where E. colt spheroplasts with exposed inner membranes were capable of binding porphyrins lo the same extent as Gram-positive bacteria, and, in spite of this fact, the E. colt spheroplasts were not killed by the porphyrin and light but needed also the assitance of a membrane disorganizing agent. In the method of photoinactivation of Gram-negative bacteria by Dp and PMNP, we are dealing with two molecules which, as we have shown previously [ 23 ], probably tend to bind by electrostatic forces through the two carboxyl groups of Dp and two out of the four positively charged groups of the PMNP molecule. It is postulated that this complexed or combined molecule ( D p - P M N P ) , with 2 residual positive charges, acts as a detergent having both a hydrophobic and a hydrophilic nature. Such a complex will penetrate and will concomitantly disorganize the lipid layers of both the outer and the inner membranes of Gram negative bacteria. Use of a porphine ( T M P y P ) , or phthalocyanine which is positively charged, a very efficient photoinactivation of wuious Gram negative bacteria was demonstrated 126,27,371. This photoinactivation was a direct inactivation of the Gram negative bacterial membranes without any need of the disorganizing peptide or any other disorganizing means [ 26,27,37 ]. The advantage of these cationic photosensilizers is that their action is due to one stable molecule having the same properties as the Dp-PMNP complex. However, in a previous study where we were using TMPyP, we failed to efficiently photoinactivate the Gram negative bacterium Escherichia coli ] 35]. Our experimental data with Acinetobacter baumannii treated with the cationic photosensitizer TMPyP in the growth medium has also failed to show photoinactivation of the bacterial culture. In the same
medium the complex Dp-PMNP caused efficient photoinactivation ofA. baumannii. From the experimental protocols of other groups that have worked with TMPyP or Zn-phthalocyanine [27,36], it is obvious that they photoinactivate the bacteria in PBS with or without washing the bacteria from the growth medium. In the present work, reducing the protein content 7 fold, which meant exchanging the medium from BHI to NB, resulted in a dramatic decrease in the viability of A. haumannii cultures when photoinactivated by TMPyP in the low protein medium. Protein concentration alone does not seem to be the major factor lk)r the inability of the cationic porphyrin to cause photokilling of bacteria in BHI medium. From the results obtained with different contents of proteins in high concentrations, it could be shown that the complex Dp-PMNP and the cationic TMPyP each interact differently with different proteins. This is probably due to the differences in their net charge and the hydrophobic state of the porphyrinic moiety. The nature of the different proteins in the environment is apparently another important factor affecting the photoactivation ability of photosensitizers. It is obvious that BSA, which is known to bind to porphyfins 138 l, will bind differently than serum or BHI, each of which contains many different proteins. Furthermore, a protein of the cationic porphyrins are probably captured by proreins in the medium and are thus unavailable for penetration through the bacterial membranes and cannot induce photoinactivation. More PMNP than Dp is always required depending on the bacteria, since PMNP binds to the proteins, saturating them and leaves some of the complex molecules to interact with the bacteria cell membranes. Even the cationic TMPyP, when acting on bacteria in media containing proteins, needs to be treated by a concentration of 32 txmoles ml ~, while the concentration of the same photosensilizer can be 8 txmoles 1 ' or less in low protein media. Other positively charged sensitizers have also been shown to be effective photosensitizers for inactivation of Propionibacterium aches [ 37 ], Porpllyromonas gingivalis and other anaerobic bacteria 139,401. In these studies, the effect of methylene blue or loluidine blue was also demonstrated only when the bacteria or sensitizer were in aqueous solution, but not in bacteriological culture media. TPPS4, the anionic photosensitizer, does not demonstrate any activity against the tested Gram positive or Gram negative bacteria [ 26,35,36 ]. This is also true for its photoactivity on Acitlelobacter I)aumannii. Failure of TPPS4 to photodamage Gram positive bacteria is conceivably a result of its inability to cross the bacterial cell wall. which contains a high amount of negatively charged leichoic acids. Gram negative are not photodamaged by TPPS4 as a result that they have two membrane layers which are composed of phospholipids. The inability of TPPS4 to photomactivate bacteria is not dependenl on the protein content or concentration in the environment, but rather on its inability to reach and cross the inner membrane as a result of electrostatic repulsion. Our resistance on performing the photosensitization experiments in bacteriological culture media is for a specific rea-
Y. \~ilcan et al, /,hmrnal ql Photochemi.stt 3 and Plunohiot¢*ex B." Biolo~ 3 42 (19989 .II "~ =1,~ ~ '
s o n . If w e w a n t to a p p l y t h e p h o t o s e n s i t i z e r s to p h o t o d y n a m i c therapy of bacterial infections, we must remember microenvironment
thal the
o f t h e b a c t e r i a in t h e t i s s u e s o r b o d y f l u i d s
[5]
c o n t a i n s v a r i o u s c o n c e n t r a t i o n s o f p r o t e i n s . T h e a b i l i t y o! Dp-PMNP
or TMPyP
to a c t in v i v o m a y b e i m p a i r e d u n d e r
[ 61
physiological conditions and protein content. Photodynamic therapy of k. baumannii
b y c a t i o n i c p h o t o s e n s i t i z e r inl'ec-
t i o n s in h u m a n s m a y be o f m e d i c a l r e l e w m c e to t h e s k i n and
[7 ]
w o u n d i n f e c t i o n s c a u s e d by t h i s m i c r o o r g a n i s m . In t h i s s t u d y , we have again clearly shown that the multidrug resistance of a microorganism
d o e s n o t p r o t e c t it f r o m p h o t o i n a c t i v a t i o n .
[8 ]
Further studies on the binding and uptake of the photosensit i z e r s a n d t h e e f f e c t s o f i l l u m i n a t i o n b y v a r i o u s l i g h t s in different wavelengths
[ 91
h a v e b e e n c a r r i e d o u t to e x t e n d t h e 101
p r e s e n t s t u d y o f p h o t o s e n s i t i z a t i o n o f A. b a u m a m f i i .
1I I
5. Abbreviations 12 I BHI
brain heart infusion
BSA
bovine serum albumin
Cd-Tx
Cd-texaphyrin
Dp
deuteroporphyrin
NB
nutrient broth
131
14[
PBS
phosphate buffered saline
PMNP
polymyxin nonapeptide
TMPyP
5, 1 0 , 1 5 , 2 0 - t e t r a ( 4 - N m e t h y l p y r i d y l ) p o r p h i n e
TPPS4
5, 1 0 , 1 5 , 2 0 - t e t r a ( 4 - s u l p h o n a t o p h e n y l )
151 porphine 16 ]
Acknowledgements
17]
T h e a u t h o r s w o u l d like to t h a n k M r s R. D r o r f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . T h i s w o r k w a s s u p p o r t e d in part by a
grant f r o m t h e H e a l t h S c i e n c e s R e s e a r c h C e n t e r F u n d s ( t o Y.N.).
181
19]
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K Nitza;t et a[. /,Iotowot O/Pito/oc'/tenti~trv am/I'hotohiolog, 3 B. Biology 42 (199~') 211 218 { occtt.s aureus and Escherichia c o i l Photochen/. Photobiol. 41 ( 1985 ) 429-435. M, Merchal, G. Bermlini, P. Giacolnini. A. Vilhmueva. (1. ,hu-i, Meso>ubstitutcd cationic porphyrins as eflicient photoscnsitizers of Grain posilive and Gram-lmgative bacteria. J. Photochem. Pholobiol. B: Biol. 32 ( 1 9 % ) 153-157. A. Minnock, D.I. Vcrnon, J. Scholield. J. Griffiths, J.H. Parish, S.B. Brown, Photoillactivatitm of bacteria. [ ]se of ~Lcationic v~atcr soluble zinc phlhalocyanine l,a photoinactJvale both (~Ianl-negati~c and (]rain-positive bacteria, .I. Photoehem. Phot,.:,biol. B: Biu]. 72 (1996) 159 164. Y. Nitzun, H. l.adam S. (]ozhansky. Z. Malik. (_'haracteri/alima c~l heroin anlibacterial action on Slalgo'lococcu~ rmre~v. |:IiMS Micrubiol. 1.ett. 48 ( 1987} 157-163. Y. Nitzan, B. Shainberg. Z. Malik. T h c m e c b a n i s m o f p h o t o d y n a l n i C inactivation of ~'Id/)hy/ocot'clt,~ ~ItIIt,H~ by deutel'OpOl'ph}lill, ('LILT, Microbiol. 19 (1989) 265-269. 1',,I.Vaara. T. Vaara. PolycatiOl> sensitize enteric bacteria to anlibioties. Antilnicroh. Agents('hemothcr. 24(19g3; 1(17 113. A. Harrilnan. B.G. Maiya. T. Murai. G. Hemmi. J.[,. Ses,,ler. "LIE. Mallouk, Melallotexaphyrins: A new talni] 3 ol photoscnsilizer~, lot c flicient gcneralion (if"singlet oxygen. ,I. ('heln. Soc. (~heln. ( "ollUllUll. 5 { 1t)89) 314-316. R.P. Ban'et. R.P. I,instead, F.G. Rundall. G.A.D. Tuey. Phthalocyanine', and related con)potuld~.. XlX. Tctrabcnzop(uphy, rin. Ietl-:.lbcno-
1331 1341
135 ]
136l
1371
138 ] ] 3 tj I
[ 40l
inonazaporphyrm and lheir metallic derivatives. J. Chem. Soc. 92 ( 1940 ) 1079-1092, P. l_indslead. Discoveries ;unong Ctuljugated na:.icrocyelJc con]poullds. J. Chem. Soc. 1(15 (1953/ 2873 2884. E. Belgognc-Berezm, K..1. 'Fowner, Acilwlot)¢tcter spp. as nosocomial pathogens: Microbiological. clinical and epidemioh~gieal leatures, Clin. Microbio]. Rev. 9 q 19961 148-165. Y. Nilzan, R. Dl+or, H. [,adam Z. Malik, S. Kimcl, V. ( ;ottfl'ied. Stl+UC ture-activily relatitmship el porphyrins for pholmnactivation of bac teria, Photocheln. Pholobiol. t,2 (1995) 342 347. m. Merchat, J.D. Spikes. (Z Bcrtolini, O. Jori, Studies or[ file mech ~lnisn/o1 baclerJtl ph,.)l,,tsen,,itiz:.ltJoll by meso .-,ubstitllled cationic porphyrins, J, Photochem. Photobiol. B: Biol. 35 (1996) 149 157. K. Konig. H. Meyer, Photodynamically induced inacti',ation of Pro i~ioml~acleritmt acm'~ using the photosensilizer methylene blur and red ]ighl. Dermalol. Munalsschr. 178 (1992) 297-300. M. R~)lenbcrg. R. Margalit, l)cuteroporph',rm albumin binding equi lihrium. Biochem. ,1. 220 {I t)g5) 197-203 M. Wib, on. J. Dobson, W. tiar\ey, Sensitization of oral bacteria iu killing b)hive-power laser radiation. (Turf. Microbild. 25 ( 19921 77 81. M. Bhaui, A. MacRohert. 5;. Megjhi, B. Henderson. M. Wilson, Effect el doshnetliC and ph) siologiual factors on the lethal pholosensilizalion (if Pot7gzyr~mlmlo~ vi,~'iv, div in vitro, I'hotochcm. I~hotohiol. 65 (1997) 1026 103l.
i
of
Photochemistry Photobiology
"
and
B:Biology
E L S E V I ER
Journal of Photochemistry and I)hou>hioh)gy B: Biology 42 ( 1998 ) 21 I 218
Eradication of Acinetobacter baumannii by photosensitized agents in vitro Yeshayahu Nitzan *, Anat Balzam-Sudakevitz, Helena Ashkenazi Health Science~ Research Center, l)eparlme,t o/L!'/~" Sciences, Bar-l/an Univerxio', Ramat-Gan, 52900, Israel Received 28 Atlgttsl
1997: accepted 13 February 1998
Abstract
The photodynamic effects of photosensitizers on Acinetohacter baumam#i were studied. These Gram negative bacteria have recently been implicated in various infections, mainly acquired in hospitals. They have outstanding characteristics of multidrug high resistance to antimicrnbial agents. The best photodynamic effect was obtained when A. bammmnii cultures were treated with light activated deuteroporphyrin (Dp) at a concentration of 34 Ixmoles I ~and polymyxin nonapeptide (PMNP) at a concentration of 200 txmoles 1 ~. At these concentrations the culture in brain heart infusion (BH1) broth was tbund to be sterile after l h of treatment. Some inhibition was also obtained under the same conditions with Cd-texapfiyrin (Cd-Tx) in the presence of PMNP. Treatment with various other photosensitizers in the presence of PMNP exhibited only marginal antibacterial activity. The cationic photosensitizer tetra-methylpyridyl porphme (TMPyP) did not exhibit any photodynamic efl;ect on A. baumam#i when illuminated during its growth in BHI broth. Bacteria grown in nutrient broth or suspended in saline and treated by TMPyP resulted in a significant photoinactivalion by the sensitizer alone even in the absence of PMNP. h was found that a high concentration of the proteins present m BHI or in serum prevent TMPyP fiom acting as a photosensitizer against A. haumannii. Bovine serum albumin at the same high protein concemration prevents Dp ( in the presence of PMNP ) to act as a photosensitizer. The anionic photosensitizer tetra-sulfouatophenyl porphine I TPPS,) did not show any photodynamic effect in high or low. protein media. In this study it was found that despite the high resistance of the Acinetohacter haumamfii to antibiotics, these bacteria can be significantly photoinactivated by treatment with either Dp + PMNP or TMPvP in low protein content environments. When the protein concentration is high, photoinactivation efficiency depends on the type o f protein present in the medium. ~E:,1098 Elsevier Science S.A. All rights reserved. Kevwords: Actinetol+acwr: Photosensitization: Cationic porphyrins: Dcuteroporphyrin: Polymyxin nonapeptide; (]ran>negative bacteria
1. Introduction A c i n e t o b a c t e r b a u m a n n i i is one of the species in the genus A c i n e t o b a c t e r , which is an aerobic Gram negative rod related
to the Pseudomonas group [ I ]. In recent years,Acinetobacter b a u m a m f f i has emerged as an important nosocomial pathogen in intensive care units [ 2 - 7 ] . This microorganism is capable of causing life-threatening infections such as bacteremia, pneumonia, meningitis, endocarditis, skin and wound infections 18-1 I ]. A c i n e t o b a c t e r microorganisms are very often multidrug-resismnt to unrelated antibiotics and are a significant problem in hospitals [ 12,13] due to the difficulty to eradicate them [ 14-16]. They produce potent fl laclamases, aminoglycoside modifying enzymes and chloramphenicol acetyltransferase as one of the mechanisms of resistance [ 17 ]. ~: (7orresponding author. Tel.: +972 3 531 8592: Fax: + 972 3 535 1824: e-mail: n itzay (a' mail.biu.ac.il
101 I - 1344/98/$19.00 (~) 1998 Elsevier Science S.A. All righls reserved. PIISIOI 1-1344(981(t(1073-6
To overcome the problem of multidrug resistance of bacteria, the photodynamic treatment by photosensitizers was established for photoinactivation of bacteria. Gram-positive bacteria such as S t a p h y l o c o c c u s a u r e u s are susceptible to porphyrin-induced antibacterial activity [18,19]. The efficient and non-recovering antimicrobial killing effects due to irradiation are independent on the antibiotic susceptibility spectrum of the treated pathogen I 18,20-22 ]. Gram-negative bacteria exhibited induced damage with light, only when using the small non-toxic peptide derived from polymyxin B nonapeptide (PMNP), which stimulated the translocation of porphyrins through the membranes of Gram-negative bacteria and enabled photodynamic damage [ 231. Anaerobic bacteria have also been shown to be sensitive to several of the above photosensitizers [ 24]. Porphyrin binding to the cytoplasmic membrane is a prerequisite for photosensitization of Gram-positive and Gramnegative bacteria [23,25] regardless of whether they are aerobic or anaerobic 124]. Recently, cationic porphyrins
212
E Nil=an el a[. /,lourmd ot I'hoto¢'/wmi~lr 3 emd t'hotohiolok'y B: Biology 42 (199,~) 211 21,~
have been shown to photoinduce the direct inactivation of Gram-negative bacteria without the presence of an additional permeabilization agent [ 26]. The same phenomenon has also been shown for cationic water soluble zinc phthalocyauines [ 27 ]. In the present study, we have examined pholoinactiration of the opportunistic multidrug resistant Acinetohacter baumannii by various hydrophobic photosensitizers, as well as by two differently charged hydrophilic sensitizers: the anionic 5,10,15,20-tetra (4-sulfonatophenyl) porphine (TPPSa) and the cationic 5. I 0,15,20-tetra( 4-mefllylpy ri dy I ) porphine (TMPyP). In the present work, we demonstrate the photoinactivation of the multidrug resistant bacterium A. haumannii by various photosensitizers in the presence of PMNP. In addition, we also attempted to establish the relationships of cationic photosensitizers in different physiological environments. These environments contain different contents or concentrations of proteins.
2. Materials and methods
2.1. Bacterial strain Acinetobacter baumannii biotype 9, recovered from clinical material submitted to the clinical bacteriology laboratory at the Meir Hospital, Kfar Saba, Israel. was used in this study. This strain was multi-resistant to the lbllowing antibiotics: ampicillin, mezlocillin, piperacillin, cefoxitin, ceftriaxone, aztreonam, tetracycline, chloramphenicol, gentamycin, tobrarnycin, amikacin, ciprofloxacin and norfloxacm, ll was found to be sensitive to colistin, imipenem, and polymyxin B.
function of time. Viable bacteria were monitored by counting the number of colony forming units on agar plates.
2.3. Growth inhibition cahtdalions The rate of growth inhibition of Acinetobacter baumannii by the various photosensitizers was calculated by measuring the optical density at 660 nm at the time when the photosensitizer was added (OD,) and 3 h later (OD,). In the control sample the second measurement was also perlormed after 3 h ( ()D~.i. Percent inhibition of the culture was calculated by the following equation: I
OD~-OD"tXIO0
~i;i inhibition of the culture
o & . - OD,, I
0I),, was always 0.3 and is indicated by an arrow in the relevant figures. The survival fraction N/N. was determined [ 28,29 ] by counting the colony-rotating units per ml, before starting the treatment by' the sensitizer under illumination (N,,) and after treatment for the indicated times under illumination (N). Bacterial cultures grown under the same conditions and light exposures but without any photosensitizer served as controls.
2.4. Preparation of PMNP PMNP was prepared by, cleavage of polymyxin B sulfate with Ficin as described previously [ 30 ]. The purity of each batch of PMNP was tested on cellulose-coated aluminum foil thin layer chromatography (TLC) plates ( Merck, Darmstadt, Germany ). Polymyxin B sulfate and licin ( Ec 3.4.22.3 ) from lig trees were obtained from Sigma ( St. Louis, MO).
2.5. Photosensili=er sohttions 2.2. Bacterial growth and photosensitization pro~'edure Cultures of A. baumamfii which were grown on brainheart agar (Difco, Detroit, MI ) for 18 h were transferred into brain heart infusion medium (Difco) and adjusted to pH 6.5. In other experiments overnight cultures were grown on nutrient agar (Difco) and were transferred into nutrient broth (Difco) at pH 6.5. In some indicated experiments the bacteria were transferred into 0. l M PBS pH + 6.5 supplemented with 15 mg/ml Bovine serum albumin (BSA) (Sigma, USA) or 15 mg/ml fetal calf serum (Biological Industries. Beit-Haereek, Israeli. The cultures were transferred into the broth media to a final volume of 25 ml at an initial optical density of 0. I at 660 nm and growth continued at 37 °C with aeration. Photosensitizers were always added when the culture reached an optical density of 0.3 at 660 nm. PMNP was added in the indicated experiments prior to the addition of the photosensitizer. Cultures were irradiated at 140 W m -' using two unfiltered tungsten lamps placed 30 cm above both sides of the flasks. Bacterial growth was determined with a Novaspec, Biochrom LKB (Cambridge, UK) spectrophotomctcr by monitoring the increase in optical density at 660 nm as a
Stock solutions of deuteroporphyrin (Dp), hematoporphyrin, hematoporphyrin derivative and protoporphyrin were prepared by dissolving 5 mg of the photosensitizer in 200 ~l of 1 N NaOH with vigorous agitation. A sterile NaCI solution (0.85~/~) was added to achieve a final concentration of 2.5 mg/ml. The sensitizers merocyanine 540, chloro-aluminium phthalocyanine, tetra-sulfonatophenyl porphine (TPPS,) and tetra-methyl pyridyl porphine (TMPyP) were dissolved to a concentration of 2.5 rag/ml in sterile distilled water. Zntetrabenzoporphyrin, Mg-tetrabenzoporphyrin and Cd-texaphyrin (Cd-Tx) were dissolved in dimethyl formmnidc to a tinal concentration of 2.5 mg/ml. All stock solutions were stored in the dark at 4 ~(" for a maximum of I week. Protoporphyrin, Zn-tetrabenzoporphyrin Dp, TPPSa and TMPyP were obtained from Porphyrin Products ( Logan, UT). Hcmatoporphyrin and merocyanine 540 were purchased Irom Sigma (St. Louis, MO). Chloro-aluminium phthalocyanine was obtained fi-om Eastman-Kodak USA. Cd-Tx was synthesized by Dr Sessler as described previously 131 ]. Mg-tetrabenzoporphyrin was prepared by Professor Ehrenberg according to published reactions [ 32,33 ].
Y. Nit=an el al. / Jottrmtl o/ Pholochemistrv .rid PI olobiolo~,,v B." Biology 42 (1998) 211 218
213
2.6. Data calculation
0
1.4 + L\x\~
E
Each experimental treatment with the various sensitizers and in the mentioned media conditions was repeated five times and the results presented are the means of all these experiments. The difference of error fell within IOC~ and the curves were drawn as the tendency of the line.
20
=r
~o
_o~o g-
08
"~) 0 4
3. Results
02 ~
Photosensitization ofA. baumannii treated by various porphyrins ( natural, semi-synthetic or synthetic ) in the presence of PMNP resuhed in bacterial growth inhibition effects ( Fig, I ). These inhibitory effects were 88-90~;¢, respectively, when the bacteria were treated by Cd-Tx or by Dp in the presence of PMNP (200 ixmoles 1 I ). Only marginal inhibition was exhibited by all the other photosensitizers (3-23~A inhibition). Inhibition of 6()°/~ was exhibited by Mg-tetrabenzoporphyrin in the presence of PMNP. The amount t)f PMNP present in the treatment was found to be important in the photosensitization procedure (Fig. 2 ). When the amount of PMNP reached 200 ixmoles I ~ or more, the growth inhibition rate of the photosensitizer Dp increase to 87 88c~. Increasing the amount of PMNP to 300 p~moles I I have increased the growth inhibition of A. baumwmii upon photosensitization with Dp to 80% and only marginally affected the growth of the bacteria upon photosensitization with the other sensitizers (results not shown). In parallel, treatment of the bacteria with Dp or Cd-Tx it] the presence of PMNP had a dramatic effect on the viability of the bacteria { Fig. 3 ). Viability decreases upon treatment with either 17 ~ m o l e s I Dp or Cd-Tx, in the presence of the appropriate concentration (I
E
~4
1
(5 ~0 >. o cm (18
4
5
6
7
107
106 :D E~
E
105
LL 104
k._
+
lO a
,+
101[
" i
I
I
0.5
I()
1.5
I
20
I
I
I
2.5
3.0
3.5
Time (hr)
04 02~ --
3
10 z
06 0
2
108
O -6 £.3 16
1
Fig. 2. Growth inhibition of Acinetoha(Wr bamnannii by Dp and wtrious concentrations of PMNP under illumination. Bacterial cells were grown in BHI and treatment was begun by addinf the indicated concentrations of PMNP and Dp { 17 ixlnoles I i). Optical densilies were monitored at 661) nnl lk)l-3 h. Treatment was begun at 0 . 3 0 D ( see [lrrow ). I, Untreated control: 2,50p+lnolesl ~ P M N P + D p ; 3 , 1 0 0 1 ~ m o l e s l ~ P M N P + b p ; 4 . 1 5 0 p . m n l e s l r P M N P + D p : 5, 200 #moles l f P M N P + D p : 6, p.molesl P M N P + I)p: 7. 300 ixmoles I i PMNP F I)p.
1
__
2
E_ 3
4
5
6
7
8
9
1()
11
Fig. I. G r o w t h inhibition ol'Aeineu~bacter baumannii by various photosensitizers in the presence ol PMNP under illumination. Photosensitizers with PMNP ( 200 #moles 1 ' ) were added to bacterial cultures grown m BHI. Optical densities were monitored for 3 h at 660 nm. Treatlnent was begun at 0 . 3 0 D I see arrow). 1. Untreated control: 2. polymyxm nnnapeptide (PMNP) alone: 3. deuteroporphyrin ( 17 #.molesl I I + P M N P : 4 . hema toporphyrin derivative ( 14 p.moles I ') + PMNP; 5. protoporphyrin 118 p.lnoles I t ) + PMNP; 6, hemalop,arphyrin ( 17 lamoles } i ) + PMNP: 7. Znqetrabenzoporphyrin ( 18 ~nmles I ~ ) + PMNP; 8, Mg tetrabenzupur phyrin ( 19 #_moles I ~) + PMNP: 9, Cd-texaphyrin ( 17 Fmoles I ) PMNP: 10. chloro+alummimn phthalocyanine i l 8 g.molesl ~ ) + P M N P : ll.merocyaImle540(ISgmolc~l i)+PMNP.
Fig. 3. Photndynamic effects on the ,Aability o f Acinetobacter baun+annii. Acinelobacter cuhures grown in BHI were incubated under illumination at the indicated concentrations of the photosensitizer in the presence of PMNP 200 p.molesl r. Dp (17 lamole,,,1 ~ ) + P M N P ( O ) : Dp 134 txmolesl i ) + P M N P ( C ) ) ; o r C d _ T x ( l T t x m u l e ~ l t)+PMNP(•).Thecon. trol culture ( A ) was not treated at all. Counts of the viable bacteria were monitored as a fimclion of illumination time.
of PMNP, and both demonstrated inactivation curves with double slopes. One slope shows a fast killing rate in the first 90 rain of illumination. The survival fraction calculated for 90 min was N I N , = 2 X 10 5 and 2 . 5 × 10 4, respectively. for the above two sensitizers. Viability decreased further at a slower rate of killing as shown by the second slope R~r each
214
Y. Nitzan et al. I Journal of Photochemistt3' aml Pkombioh~,w B." Biology 42 (1998) 211 218
1.6 1.4
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._
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3 4 2 Fig. 4. Growth inhibition of Acinetobacter bamnannii by TMPyP. TPPS4 and Dp + PMNP grown in various media with illumination. Bacterial cells were grown to the beginning of the log phase and treatment was begun by adding the indicaled photosensitizer. Bacteria were grown at 37 ~'(' for an additional 3 h. Optical densities were monitored fi)r 3 h at 660 nm. Treatment always started at 0.30D I see arrow ). 1, Untreated comml; 2, TMPyP ( 8 ixmolesl ~);3, TPPS4(8,txmolesl '):4, Dp(171.zmolesl ~)+PMNP ( 200 gmoles 1 t ). Bacteria grown in BHI are shown in the uppcr panel and those grown in NB are shown in lhe lower panch photosensitizer. Only by treating the bacteria with 34 # ` m o les 1 t Dp and P M N P was a sharp killing rate o t ' 6 orders of magnitudes obtained within 30 rain of illumination. Using this protocol, no viable bacteria were found in the culture m e d i u m after 35 min from the beginning o f the photosensitization treatment. T M P y P , a cationic porphine photosensitizer, did not demonstrate any effect on A. baumannii upon photosensitization when grown and photosensitized under the same conditions. These growth conditions included growth in BHI broth which is a protein-rich medium. This cationic porphine ( T M P y P ) demonstrated a growth inhibition when the medium in which the bacteria were grown was nutrient broth ( N B ) (Fig. 4). Furthermore, growth inhibition was obtained by T M P y P alone, in the absence o f the P M N P as a m e m b r a n e disorganizing agent. The difference between BHI broth and NB is that BHI contains 14.75 m g / m l protein while NB contains only 1.95 m g / m l protein. This is a 7-fold difference in the protein concentration, which may evidently cause a big difference in the behavior of the photosensitizer in these two media. When e x a m i n i n g the viable counts o f A . baumamtii treated in BH1 broth, only marginal effects were observed even with 32 t x m o l e s l ' T M P y P (Fig. 5). H o w e v e r , in NB, when the bacteria are treated with only 8 Ixmoles I ~ o f T M P y P a double slope of inactivation can be seen: N / N , , = 1 × 10
,o.f t 10zl1
-.
I 0 1 ~ 0.5
~0
~5
2.0
Time
(hr)
2.5
30
3.5
Fig. 5. Viability of Acitwtobacter haumannii treated by TMPyP in NB or
BHI. Acinelobacter cultures were incubated under illumination at concentrationsof8#molesl 'TMPyPinNB(•):32txmolesl 'TMPyPinNB ( O ): 32 bunoles 1 t TMPyP in BHI ( • ). The control cullnres in NB ( D ) and ill BHI ( • ) were not treated. Colony forming units were monitored as a function of illumination time. within 30 min and in the other slope N / N , , = 2 × 10- 2 during 3 h was observed. With 32 #`moles I ~T M P y P , a single sk)pe and a survival rate of N/N,, = 5 × l0 ~' was obtained within 30 rain and sterilization of the culture could be obtained 5 min later (Fig. 5 ). The next step was to determine which of the f o l l o w i n g factors has the greatest effect on the photosensitizer's ability to photoinactivate the A. haumannii microorganisms: (a) protein concentration alone is the factor that affects the photoinactivation ability of the cationic photosensitizer; (b) the protein composition is also an important factor, in an attempt to solve this problem, we e x a m i n e d the photoinactivation ability of Dp + P M N P and of T M P y P in media containing 15 m g / m l serum proteins and in media containing an equal concentration of BSA. Pertorming photoinactivation experiments in serum at a protein concentration of 15 m g / m l demonstrated (Fig. 6) that 34 i.tmoles 1 J Dp with P M N P acts much better than 32 #.moles 1 ' T M P y P . The killing rate with Dp + P M N P was 7 orders of magnitudes and the survival fraction was calculated as N / N o = 10 7 in one slope within 3 h. As for the T M P y P , the same slope was obtained for only the first hour, as the survival fraction was N / N . - 5 × 10 -'. After 1 h of treatment, the killing rate was drastically reduced. With B S A (15 m g / m l ) , the opposite effect was observed. The photoinactivation rate by T M P y P (32 ixmolcs I ~) was
Y. Nitza, el a l . / . h m r , al o/ Photochemi~tr~ and Photol)iology B: Biolot,,y 42 (1998) 211 21,'~'
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Fig. 6. Viability ol'AcinembacWr haummmii treated by TMPyP or Dp in the
Fig. 7. Viability ofAcinelobacter bamnamtii treated by TMPyP or Dp in thc
presence of PMNP in serum ( 15 mg/ml protein). Acinetohacwr cultures were incubaled under illumination at concentrations o1=8txmolcs I ~TMPyP (~); 32 ixmolesl t TMPyP ( i ) : 17 o.moles I ~ Dp+PMNP (C~}: 34 bunolesl ~ Dp+PMNP (•);Thecontrolculture in serum ( • ) was not treated. Cohuay forming units were counted as a function oI illuufination time.
presence of PMNP iu BSA ( 15 rag/iiil ). Acinembacwrcultures were incubated under illumination at concentrations o18 ~moles I ~TMPyP ([~): 32 p.molesl ITMPyP ( I ) ; 17 bu'nolesl ~ Dp+PMNP (()): 341xmoles I ~l)p + PMNP ( • ), The control culture with BSA ( • ) was not treated Colony forming units were counted as a function of illumination time.
better than the inactivation rate by 34 ixmoles I ~ Dp with P M N P (Fig. 7). The killing rate was 7 orders of magnitude and the survival rate of T M P y P was calculated to be 10 7 in 1 h. Although the same survival rate was achieved lot Dp and PMNP, it was achieved only after 3 h. Apparently, not only the protein concentration but also the type of protein in the m e d i u m affects the photosensitization ability of the various photosensitizers against Acmetobacter.
4. Discussion The Gram negative bacterium Acinetobacter baumatmii is of special interest for photodynamic therapy since it is resistant to many kinds of antimicrobial agents. The ability of photosensitizers to inactivate this microorganism and thus overcome its multidrug resistance may offer an alternative method to the use of antibiotics. However, photoinactivation of A. baumannii appears to be limited to selected photosensitizers such as deuteropolphyrin and Cd-texaphyrin, both require the presence of the m e m b r a n e idsorganizing peptide P M N P 123]. Other photosensitizers, such as hematoporphyrin derivative, hematoporphyrin or protoporphyrin, which
are effective against Gram positive bacteria [ 18,29 ] or some anaerobic Gram negative bacteria [241, are not effective against this particular microorganism. Furthermore, the concentration of P M N P required for optimal disorganization of the bacterial m e m b r a n e s is different from those used with other species of G r a m negative bacteria. For example 500-700 Ixmoles I ~ P M N P are required for the photoinactivation of E. coil by Dp or hydrophobic porphines [ 23,28 I, whereas only 50 Ixmoles I ~ P M N P can cause the disorganization of m e m b r a n e s of Pseudomonas aeruginosa and enable its photoinactivation by Dp. The differences between the Gram negative bacteria mentioned here and the amounts of P M N P needed for disorganization of their barriers may be a reflection of the hydrophobic state of the bacterial barriers. The barriers of A. baumannii are similar to those of other Gram negative bacteria, and include the outer and inner membranes. In addition, some strains of A. baumamfii produce a slime capsule on the surface of the bacteria rendering a surface more hydrophilic. In strains isolated lyon] catheters or tracheal devices, their hydrophobicity was found to be higher than in strains isolated in other sites [ 34 ]. It is possible that in other strains of the bacteria a higher or lower a m o u n t of P M N P will be required for their photoinactivation, in the present study, it
216
Y. Nitzan et al./ Journal o/Pholochemist;y and Pholohiology B: Biology 42 (1998) 211 21,~
was found that 200 Ixg/ml PMNP is needed fl)r the photoinactivation of Acinetobacter baumannii by Dp or Cd-texaphyrin. With this amount of PMNP and Mg-tetrabenzoporphyrin as photosensitizer, we observed a 6()~ growth inhibition and this increased to 80~J when the amnount of the disorganizer was increased to 300 txmoles I ~. This might indicate that for certain photosensitizers, a higher degree of barrier disorganization is necessary in order to penetrate into the bacteria. The amounts of PMNP used in these experiments were not toxic to A. baumannii. It was flmnd previously ] 301 that PMNP is not toxic by itself on enteric bacteria up to an amount of 800 i~moles I ~. In the present work, we used amounts of PMNP of up to 300 ixmoles I ~ with no toxic effects on A. baumam~ii bacteria. It is possible that even higher amounts of PMNP are not toxic to this bacteria. It is now well recognized that in Gram negative bacteria, which possess two membrane layers, photosensitization is possible only if the photosensitizers penetrate through the outer membrane as well as through the cytoplasmic inner membrane [ 2326,27,35,361. Furthermore, although binding of the photosensitizer to the bacterial membranes is a prerequisite for the photosensitization process, it is not by itself sufficient for the photoinactivation of the bacteria. This statement was made in a previous study [ 25 ] where E. colt spheroplasts with exposed inner membranes were capable of binding porphyrins lo the same extent as Gram-positive bacteria, and, in spite of this fact, the E. colt spheroplasts were not killed by the porphyrin and light but needed also the assitance of a membrane disorganizing agent. In the method of photoinactivation of Gram-negative bacteria by Dp and PMNP, we are dealing with two molecules which, as we have shown previously [ 23 ], probably tend to bind by electrostatic forces through the two carboxyl groups of Dp and two out of the four positively charged groups of the PMNP molecule. It is postulated that this complexed or combined molecule ( D p - P M N P ) , with 2 residual positive charges, acts as a detergent having both a hydrophobic and a hydrophilic nature. Such a complex will penetrate and will concomitantly disorganize the lipid layers of both the outer and the inner membranes of Gram negative bacteria. Use of a porphine ( T M P y P ) , or phthalocyanine which is positively charged, a very efficient photoinactivation of wuious Gram negative bacteria was demonstrated 126,27,371. This photoinactivation was a direct inactivation of the Gram negative bacterial membranes without any need of the disorganizing peptide or any other disorganizing means [ 26,27,37 ]. The advantage of these cationic photosensilizers is that their action is due to one stable molecule having the same properties as the Dp-PMNP complex. However, in a previous study where we were using TMPyP, we failed to efficiently photoinactivate the Gram negative bacterium Escherichia coli ] 35]. Our experimental data with Acinetobacter baumannii treated with the cationic photosensitizer TMPyP in the growth medium has also failed to show photoinactivation of the bacterial culture. In the same
medium the complex Dp-PMNP caused efficient photoinactivation ofA. baumannii. From the experimental protocols of other groups that have worked with TMPyP or Zn-phthalocyanine [27,36], it is obvious that they photoinactivate the bacteria in PBS with or without washing the bacteria from the growth medium. In the present work, reducing the protein content 7 fold, which meant exchanging the medium from BHI to NB, resulted in a dramatic decrease in the viability of A. haumannii cultures when photoinactivated by TMPyP in the low protein medium. Protein concentration alone does not seem to be the major factor lk)r the inability of the cationic porphyrin to cause photokilling of bacteria in BHI medium. From the results obtained with different contents of proteins in high concentrations, it could be shown that the complex Dp-PMNP and the cationic TMPyP each interact differently with different proteins. This is probably due to the differences in their net charge and the hydrophobic state of the porphyrinic moiety. The nature of the different proteins in the environment is apparently another important factor affecting the photoactivation ability of photosensitizers. It is obvious that BSA, which is known to bind to porphyfins 138 l, will bind differently than serum or BHI, each of which contains many different proteins. Furthermore, a protein of the cationic porphyrins are probably captured by proreins in the medium and are thus unavailable for penetration through the bacterial membranes and cannot induce photoinactivation. More PMNP than Dp is always required depending on the bacteria, since PMNP binds to the proteins, saturating them and leaves some of the complex molecules to interact with the bacteria cell membranes. Even the cationic TMPyP, when acting on bacteria in media containing proteins, needs to be treated by a concentration of 32 txmoles ml ~, while the concentration of the same photosensilizer can be 8 txmoles 1 ' or less in low protein media. Other positively charged sensitizers have also been shown to be effective photosensitizers for inactivation of Propionibacterium aches [ 37 ], Porpllyromonas gingivalis and other anaerobic bacteria 139,401. In these studies, the effect of methylene blue or loluidine blue was also demonstrated only when the bacteria or sensitizer were in aqueous solution, but not in bacteriological culture media. TPPS4, the anionic photosensitizer, does not demonstrate any activity against the tested Gram positive or Gram negative bacteria [ 26,35,36 ]. This is also true for its photoactivity on Acitlelobacter I)aumannii. Failure of TPPS4 to photodamage Gram positive bacteria is conceivably a result of its inability to cross the bacterial cell wall. which contains a high amount of negatively charged leichoic acids. Gram negative are not photodamaged by TPPS4 as a result that they have two membrane layers which are composed of phospholipids. The inability of TPPS4 to photomactivate bacteria is not dependenl on the protein content or concentration in the environment, but rather on its inability to reach and cross the inner membrane as a result of electrostatic repulsion. Our resistance on performing the photosensitization experiments in bacteriological culture media is for a specific rea-
Y. \~ilcan et al, /,hmrnal ql Photochemi.stt 3 and Plunohiot¢*ex B." Biolo~ 3 42 (19989 .II "~ =1,~ ~ '
s o n . If w e w a n t to a p p l y t h e p h o t o s e n s i t i z e r s to p h o t o d y n a m i c therapy of bacterial infections, we must remember microenvironment
thal the
o f t h e b a c t e r i a in t h e t i s s u e s o r b o d y f l u i d s
[5]
c o n t a i n s v a r i o u s c o n c e n t r a t i o n s o f p r o t e i n s . T h e a b i l i t y o! Dp-PMNP
or TMPyP
to a c t in v i v o m a y b e i m p a i r e d u n d e r
[ 61
physiological conditions and protein content. Photodynamic therapy of k. baumannii
b y c a t i o n i c p h o t o s e n s i t i z e r inl'ec-
t i o n s in h u m a n s m a y be o f m e d i c a l r e l e w m c e to t h e s k i n and
[7 ]
w o u n d i n f e c t i o n s c a u s e d by t h i s m i c r o o r g a n i s m . In t h i s s t u d y , we have again clearly shown that the multidrug resistance of a microorganism
d o e s n o t p r o t e c t it f r o m p h o t o i n a c t i v a t i o n .
[8 ]
Further studies on the binding and uptake of the photosensit i z e r s a n d t h e e f f e c t s o f i l l u m i n a t i o n b y v a r i o u s l i g h t s in different wavelengths
[ 91
h a v e b e e n c a r r i e d o u t to e x t e n d t h e 101
p r e s e n t s t u d y o f p h o t o s e n s i t i z a t i o n o f A. b a u m a m f i i .
1I I
5. Abbreviations 12 I BHI
brain heart infusion
BSA
bovine serum albumin
Cd-Tx
Cd-texaphyrin
Dp
deuteroporphyrin
NB
nutrient broth
131
14[
PBS
phosphate buffered saline
PMNP
polymyxin nonapeptide
TMPyP
5, 1 0 , 1 5 , 2 0 - t e t r a ( 4 - N m e t h y l p y r i d y l ) p o r p h i n e
TPPS4
5, 1 0 , 1 5 , 2 0 - t e t r a ( 4 - s u l p h o n a t o p h e n y l )
151 porphine 16 ]
Acknowledgements
17]
T h e a u t h o r s w o u l d like to t h a n k M r s R. D r o r f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . T h i s w o r k w a s s u p p o r t e d in part by a
grant f r o m t h e H e a l t h S c i e n c e s R e s e a r c h C e n t e r F u n d s ( t o Y.N.).
181
19]
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[32[
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1331 1341
135 ]
136l
1371
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