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Phage-antibiotic synergism: a possible approach to combatting Pseudomonas aeruginosa
Research in Microbiology 164 (2013) 55e60 www.elsevier.com/locate/resmic
Phage-antibiotic synergism: a possible approach to combatting Pseudomonas aeruginosa Petar Knezevic a,*, Sanja Curcin b, Verica Aleksic a, Milivoje Petrusic a, Ljiljana Vlaski c a
Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 2, Novi Sad, Vojvodina, Serbia b Molecular Plant Physiology, Centre for Organismal Studies (COS) Heidelberg, INF 360 D-69120 Heidelberg, Germany c University ENT Clinic, Clinical Center of Vojvodina, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3, Novi Sad, Vojvodina, Serbia Received 19 March 2012; accepted 24 August 2012 Available online 7 September 2012
Abstract Pseudomonas aeruginosa is a highly resistant opportunistic pathogen and an important etiological agent of various types of infections. During the last decade, P. aeruginosa phages have been extensively examined as alternative antimicrobial agents. The aim of the study was to determine antimicrobial effectiveness of combining subinhibitory concentrations of gentamicin, ceftriaxone, ciprofloxacin or polymyxin B with P. aeruginosa-specific bacteriophages belonging to families Podoviridae and Siphoviridae. The time-kill curve method showed that a combination of bacteriophages and subinhibitory concentrations of ceftriaxone generally reduced bacterial growth, and synergism was proven for a Siphoviridae phage s-1 after 300 min of incubation. The detected alteration in morphology after ceftriaxone application, resulting in cell elongation, along with its specific mode of action, seemed to be a necessary but was not a sufficient reason for phage-antibiotic synergism. The phenomenon offers an opportunity for future development of treatment strategies for potentially lethal infections caused by P. aeruginosa. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Bacteriophages; Antibiotics; Synergism; Pseudomonas aeruginosa
1. Introduction Pseudomonas aeruginosa, one of the most common opportunistic pathogens in humans, is an important etiological agent of various types of infections and one of the main nosocomial pathogens. It stands out among Gram-negative rods as extremely resistant to a number of antimicrobials due to its intrinsic and acquired mechanisms of resistance, including an attached mode of growth in the form of a biofilm (Hoiby et al., 2001; Livermore, 2004). The resistance phenomenon in P. aeruginosa is a global health concern that is rapidly increasing in magnitude. Some groups of antibiotics can be successfully applied to infections caused by
* Corresponding author. E-mail addresses: [email protected] (P. Knezevic), sanja. [email protected] (S. Curcin), [email protected] (V. Aleksic), [email protected] (M. Petrusic), [email protected] (Lj. Vlaski).
this bacterium: some b lactams, aminoglycosides, fluoroquinolones and polymyxins (Giamarellou and Antoniadou, 2001). The prospects of novel anti-Pseudomonas agents are poor, since most do not represent new classes of antimicrobials, but rather, are derivatives of existing antibiotic molecules (Nordmann et al., 2007). In addition, almost all currently used anti-Pseudomonas agents are toxic, teratogenic or cause reactions of hypersensitivity (Adam, 1989; McKinnon and Davis, 2004; Russell, 1998; Tam et al., 2005). Thus, alternative treatments of potentially lethal P. aeruginosa infections must be sought. One solution involves a combination of two or more conventional antimicrobial agents (reviewed in Wroblewska, 2006). These treatments prolong the emergence of antibiotic-resistant strains and effectively kill bacteria, which is not the case when antimicrobials are used separately. During the last decade, bacteriophages have also been extensively examined as potential antimicrobial agents for therapy, including various P. aeruginosa-specific phages (Heo et al.,
0923-2508/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.resmic.2012.08.008
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P. Knezevic et al. / Research in Microbiology 164 (2013) 55e60
2009; McVay et al., 2007: 20, Wang et al., 2006; Watanabe et al., 2007). However, few studies have dealt with combining phages and conventional antibiotics. Hagens et al. (2006) noted that filamentous phages could increase P. aeruginosa sensitivity to several antibiotics (gentamicin, tetracycline, chloramphenicol and carbenicillin), while Comeau et al. (2007) reported that among tetracyclines, gentamicin, quinolones and b-lactams (aztreonam and several cephalosporins), only the latter two groups gave phage-antibiotic synergy against Escherichia coli. Existing data are scarce and inconsistent, and the activity of some important groups of antibiotics such as polymyxins has not yet been examined in combination with phages. Furthermore, the phenomenon has not been confirmed for P. aeruginosa-specific phages of the order Caudovirales. The aim of the present study was to examine the efficacy of subinhibitory concentrations of conventional anti-Pseudomonas agents in combination with P. aeruginosa-specific phages from the families Siphoviridae and Podoviridae, and to elucidate the main reasons for the onset of synergism between them. 2. Material and methods 2.1. Bacterial strains Three P. aeruginosa strains were used in the study: a reference strain ATCC 9027 (external ear infection), PA-4U isolated from environmental biofilm and PA-M2 from river water. Bacteria were stored in LuriaeBertani broth (LB) containing glycerol (v⁄v 10%) at 70 C. For the experiments, they were inoculated in LB and incubated overnight at 37 C.
formazane, was considered as the MIC. The MIC determination was carried out in three replicates and three independent experiments. 2.3. Bacteriophages In order to determine antagonistic, additive and synergistic effects of subinhibitory concentrations of the antibiotics and phages, three previously isolated and characterized bacteriophages were used: d (family Podoviridae), s-1 and 001A (family Siphoviridae). Their hosts were PA-4U, ATCC 9027 and PA-M2, respectively. Pphages d and 001A possessed a broad activity range when tested against 33 P. aeruginosa strains, and were able to infect up to 72.7 and 75.8% of the examined strains, respectively, while phage s-1 exhibited a moderate lytic spectrum (plaque on 45.5% of strains). The first two phages possessed considerable lytic efficacy against host strains, while phage s-1 was not very effective. Their latent periods were approximately 60 min for phage d, 75 min for s-1 and 90 min for phage 001A. All three phages were clonally different, as confirmed previously by the RFLP method (Knezevic et al., 2009, 2011). The phages were multiplied, precipitated with NaCl and PEG6000, purified by equilibrium ultracentrifugation in CsCl and finally dialyzed (Sambrook and Russell, 2001). Plaqueforming units in the purified stocks were first determined by the spot method and then the corresponding dilutions were plated by an overlay agar method in triplicate for a more precise phage count estimation. The stocks were stored at 4 1 C. 2.4. Time kill curves
2.2. MIC determination The antimicrobial agents evaluated in the study included ceftriaxone, gentamicin (Galenika a.d., Serbia), ciprofloxacin (Zdravlje a.d., Serbia) and polymyxin B (HiMedia, India). Stock solutions were prepared at a concentration of 256 mg/l and used for preparation of twofold dilutions. MICs were determined using a slightly modified microtiter plate method developed by Rahman et al. (2004). Briefly, a bacterial suspension previously adjusted to match turbidity of a 0.5 McFarland Nefelometer standard (measured by absorbance at 625 nm 0.08e0.1; approx. 2 108 CFU/ml) was diluted in double-strength LB (1:100 v/v). Into each well, 100 ml of the inoculated double-strength medium and the same volume of twofold antibiotic dilutions were added to obtain final concentrations in the range 0.03e64 mg/ml. The microtiter plates were incubated for 18 h at 37 C without shaking, amended with a 0.22 mm filter-sterilized solution of 2,3,5triphenyltetrazolium chloride (final concentration 200 mg/ml) and incubated additionally for 3 h. Controls for plate sterility and bacterial growth without antibiotic were also included. Bacterial growth was estimated using a microtiter plate reader at 540 nm (Multiskan EX, Thermo-Labsystem, Vantaa, Finland). The lowest concentration of antibiotic that inhibited bacterial growth, which was visible as the absence of red
The effectiveness of combining phages and antibiotics was determined by the time-kill curve method (Verma, 2007). Changes in bacterial count were monitored in parallel in test tubes containing the following: only bacteria (approx. 1 106 CFU/ml); bacteria and a subinhibitory concentration of antibiotics (1/4 of MIC); bacteria and corresponding bacteriophages at a multiplicity of infection (MOI ¼ 0.01 for phage d; MOI ¼ 0.1 for 001A, and MOI ¼ 5e10 for s-1, determined according to their previously established bacteriolytic efficiency (Knezevic et al., 2011)); or bacteria, a subinhibitory concentration of antibiotics and bacteriophages (final volume 10 ml). The test tubes were incubated in a water bath at 37 C for 5 h and bacterial counts were determined after 0, 60, 90, 120, 240 and 300 min of incubation by spreading appropriate dilutions on MullereHinton agar (detection limit 102 CFU/ml). The plates were incubated at 37 C overnight and bacterial colonies were counted. The results from the experiments, carried out at least twice, were averaged and expressed as logarithms with corresponding standard errors (mean SE). If bacterial CFU/ml decreased by 2 log for the phage-antibiotic combination compared to the more active single agent (phage or antibiotic), as well as to the initial inoculum titer, the interaction was considered to be synergistic.
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2.5. Cell morphology In order to examine the importance of cell filamentation in case of synergism between antibiotics and P. aeruginosaspecific phages, we examined the morphology of bacterial cells of strains ATCC 9027 and PA-M2 with subinhibitory antibiotic concentrations (1/4 MIC) in parallel by bright field microscopy after staining with methylene blue and by epifluorescence microscopy (BX51, Olympus) after fixation, filtration onto 0.2-mm-pore-size polycarbonate filters and staining with 0.1% (w/v) DAPI (40 ,60 -diamidino-2-phenylindole). The length of randomly observed cells (at least 20 per field) was measured using software (Olympus cell B) and means were calculated. The statistical difference in cell length among treatments was evaluated using the ANOVA test and the Holm post-hoc test. The null hypothesis was that there was not a significant difference between cell length in the medium with and without subinhibitory concentrations of antibiotics, and it was rejected if P 0.05. The phenomenon in which cell dimensions were 5 mm was considered to be filamentation, while morphological changes wherein the cell length was <5 mm but significantly longer than regular (i.e. untreated) cells were denoted as elongation. 3. Results 3.1. MIC The determined MICs varied from 0.06 mg/ml of ciprofloxacin for strain PA-4U to 64 mg/ml of ceftriaxone for strain PA-M2 (Table 1). 3.2. Phage-antibiotic synergism According to the time-kill curves, when subinhibitory concentrations of gentamicin were tested with phages against corresponding P. aeruginosa strains, bacterial count reduction was not observed (Fig. 1A e aec). In contrast, the bacterial count was even greater, by 0.48e1.97 log compared to the initial count, indicating the absence of antimicrobial activity. The reduction in the initial bacterial count was also absent when subinhibitory concentrations of ciprofloxacin and bacteriophage were simultaneously used, though the bacterial count was lower by 0.13 and 0.15 log (001A vs. PA-M2 and d vs. PA-4U, respectively) in comparison to a more active single agent (Fig. 1B e b and c). Although a combination of ciprofloxacin and phage s-1 reduced the initial count of strain ATCC 9027 for 3.30 log, the bacterial count reduction was Table 1 MICs of antibiotics for P. aeruginosa strains. P. aeruginosa strains
ATCC 9027 PA-M2 PA-4U
MIC (mg/ml) GEN
CIP
CRO
PMB
2 2 1
1 1 0.06
8 64 16
4 4 2
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lower by 1.30 log in relation to that obtained with the applied concentration of antibiotic (Fig. 1B e a). Similarly, the absence of synergism was also observed in the phage and polymyxin B combination, since the bacterial count was greater than the inoculum count for 1.28e2.13 log, depending on the phage-host system (Fig. 1D e aec). However, after combining phages and subinhibitory concentrations of ceftriaxone, the bacterial count was reduced by 2.56 log (s-1 vs. ATCC 9027), 1.26 log (d vs. PA-4U) and 1.45 log (001A vs. PA-M2) in comparison to the more active single agent, while the initial count decreased by 2.12, 2.22, and 1.67 log, respectively (Fig. 1C e aec). 3.3. Cell length The average length of P. aeruginosa cells grown in LB was not statistically different from the average cell dimensions in the presence of 1/4 MIC of gentamicin and polymyxin B (P > 0.05) (Table 2). Cells of both strains were significantly shorter for bacteria grown in LB medium without antibiotic compared to those grown in medium with subinhibitory concentration of ciprofloxacin and ceftriaxone (P < 0.0001). In addition, the difference in cell length between bacteria grown in presence of subinhibitory concentrations of ciprofloxacin and ceftriaxone was not statistically significant, and neither phage presence nor absence in the medium had an effect on filamentation (P > 0.05). 4. Discussion Synergism between phages and antibiotics, noted earlier by other authors, has not been widely discussed. Although Himmelweit (1945) successfully combined phages and penicillin against Staphylococcus in 1945, his work had remained unnoted until recently. More than a half a century later, Huff et al. (2004) observed a similar phenomenon in E. colispecific phages and ciprofloxacin, but failed to provide an explanation. On the other hand, Hagens et al. (2006) speculated that, during filamentous phage progeny extrusion, the outer membrane was a less effective barrier for antibiotic penetration into a bacterial cell. This explanation for phageantibiotic synergy, however, would be acceptable for phages Pf1 and Pf3 belonging to the family Inoviridae and released from bacterial cells by extrusion, but not for phages belonging to the families Podoviridae and Siphoviridae released by cell lysis. Results clearly indicate that the combination of ceftriaxone and phages tended to reduce the bacterial host count for 5 h. A similar phenomenon was not observed when other antimicrobials were tested, and only a combination of phage s-1 and ceftriaxone against P. aeruginosa ATCC 9027 had a synergistic effect sensu stricto. In contrast to s-1, phages d and 001A failed to reduce the bacterial count for 2 log in the presence of subinhibitory concentrations of ceftriaxone, and the reasons for the absence of synergism for corresponding phage-host systems are unclear.
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Fig. 1. Time-kill curves of phage s-1 against ATCC 9027 (a), phage d against PA-4U (b) and phage 001A against strain PA-M2 (c) in the presence of subinhibitory concentrations of gentamicin (A), ciprofloxacin (B), ceftriaxone (C) and polymyxin B (D); (C) bacteria without treatment; (D) antibiotic; (>) phage and (-) phage and antibiotic.
Earlier systematic experiments on combining various antibiotics and phages against E. coli showed that cell filamentation, as a result of antibiotic subinhibitory concentrations presence in a nutritive medium, had a strong impact on synergy and phage plaque enlargement (Comeau
Table 2 Cell length in the presence of subinhibitory concentrations of antibiotics and/ or bacteriophages. Straina
Phage Cell length (mm) in the presence of antibiotics GEN
ATCC 9027 none s-1 PA-M2 none 001A
1.30 1.19 1.50 1.68
CIP
0.13 2.75 0.09 2.60 0.15 17.85 0.16 16.87
CRO 0.23 3.44 0.23 3.06 0.48 18.68 0.36 17.30
PMB 0.34 0.21 0.39 0.64
1.35 1.78 1.49 1.69
0.11 0.17 0.15 0.18
a Cell dimensions in LB without antibiotics: 1.14 0.08 mm for ATCC 9027 and 1.22 0.06 mm for PA-M2.
et al., 2007). Cell filamentation in the presence of subinhibitory concentrations of b-lactams and fluoroquinolones had been previously demonstrated in several studies and was explained by the fact that these antibiotics, although exhibiting different mechanisms of action, finally block bacterial cell division (Bergogne-Berezin, 1985; Drago et al., 2004; Rella and Haas, 1982). According to Comeau et al. (2007), phageantibiotic synergism is a result of a change in morphology that permits faster assembly of phages through altered or larger pools of precursors important to phage maturation, and accelerates the timing of cell lysis. Present results confirm that subinhibitory concentrations of ceftriaxone and ciprofloxacin cause significant elongation and filamentation of P. aeruginosa cells, while this was not observed with gentamicin and polymyxin B. It is also interesting to note that cells of strain ATCC 9027 in the presence of subinhibitory concentrations of ciprofloxacin and ceftriaxone were significantly shorter than those of PA-M2 (P < 0.0001),
P. Knezevic et al. / Research in Microbiology 164 (2013) 55e60
i.e. in the case of ATCC 9027, cells exposed to antibiotics were about 2e3 times longer, while in the case of PA-M2 they were 10e14 times longer. Since only cells longer than 5 mm can be considered filaments (Hahn et al., 1999), it is clear that the concentrations of ceftriaxone and ciprofloxacin used caused significant elongation of ATCC 9027, but not filamentation. In contrast, ceftriaxone and ciprofloxacin caused morphological changes corresponding to filamentation in PAM2, but no significant cell count reduction was observed after combining it with P. aeruginosa-specific bacteriophages. Taking into account results obtained and the fact that synergism was not proven in the absence of altered morphology either in the present study or in that of Comeau et al. (2007), cell elongation/filamentation appears to be a necessary but insufficient reason for phage-antibiotic synergy. It is interesting to note, regarding PA-M2 and ATCC 9027, that we did not establish such a link between changed cell length and reduction in bacterial number that would support the idea that the longer the cell, the more bacteria are efficiently destroyed. This suggests a more complex phenomenon, with other factors and mechanisms involved that should be further examined. For instance, phage s-1 belongs to the family Siphoviridae and is temperate, like all P. aeruginosa-specific phages from this family isolated to date. This implies possible involvement of phage induction by an antibiotic, following cell infection, in the onset of synergism. The mode of an effect of antibiotics on bacterial cells should also be taken into consideration to explain the obtained results. Namely, bacterial count reduction in combination with phages undoubtedly was influenced by the antibiotics used. It is widely known that ceftriaxone inhibits cell wall synthesis binding to PBP (Bergogne-Berezin, 1985), that ciprofloxacin inhibits DNA replication targeting DNA gyrase (Zhao et al., 1997) and that gentamicin inhibits protein synthesis binding to the 30S ribosomal subunit and disrupts cell envelopes (Kadurugamuwa et al., 1993), while polymyxin B disturbs membrane structures (Tam et al., 2005). Phage multiplication is not dependent on peptidoglycan synthesis; however, functional bacterial ribosomes, cell membrane integrity and functional DNA gyrase are necessary for Podoviridae and Siphoviridae successful multiplication (Constantinou et al., 1986; De Wyngaert and Hinkle, 1979; Hamatake et al., 1981). In addition, ceftriaxone has several other differences in comparison to ciprofloxacin, gentamicin and polymyxin B that might contribute to the phenomenon described. Ceftriaxone exhibits time-dependent killing, like all other cephalosporins (Perry and Schentag, 2001) and these pharmacological characteristics might also have an impact on phage-antibiotic synergy. Interestingly, ceftriaxone is the only agent used in the study against which P. aeruginosa strains frequently possess or acquire resistance, i.e. most strains of P. aeruginosa are intermediate or resistant to this cephalosporin (Wroblewska, 2006). This also emphasizes the importance of synergy, as it is possible to increase the sensitivity of P. aeruginosa to this agent using bacteriophages. In summary, among the tested antimicrobial agents, only ceftriaxone causes cell morphological changes and shows
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a mode of action excluding interference with phage multiplication. Since only the use of subinhibitory concentrations of ceftriaxone provided a synergistic effect with P. aeruginosaspecific bacteriophages, it can be assumed that both cell elongation and a specific mode of action are necessary prerequisites for synergy. Phage-antibiotic synergism offers a possible opportunity for developing treatment strategies for infections caused by P. aeruginosa in the future using corresponding antibiotics and phages which meet the prerequisites for therapeutic application. This approach offers additional advantages, including the rapid onset of synergism possibly contributing to a reduction in bacterial number in vivo to levels that the immune system can successfully cope with; moreover, use of subinhibitory concentrations of antibiotics results in avoidance of antibiotic side effects occurring after administration of high doses. Finally, in addition to further experiments designed to better understand the mechanisms of rapid synergism, the observed phenomenon should be assessed for possible applications in control of P. aeruginosa. Acknowledgments This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant OI 172058. The authors acknowledge Emilija Nikolic-Djoric, MSc (Faculty of Agriculture, University of Novi Sad) for help in statistical analysis and Ljiljana Knezevic, MA (Faculty of Sciences, University of Novi Sad) for language revision. References Adam, D., 1989. Use of quinolones in pediatric patients. Rev. Infect. Dis. 11, 1113e1116. Bergogne-Berezin, E., 1985. Antibacterial activity of ceftriaxone. Rev. Med. Interne. 6, 178e186. Comeau, A.M., Tetart, F., Trojet, S.N., Pre`re, M.F., Krisch, H.M., 2007. Phageantibiotic synergy (PAS): b-lactam and quinolone antibiotics stimulate virulent phage growth. PLoS One 2, e799. Constantinou, A., Voelker-Meiman, K., Sternglanz, R., McCorquodale, M.M., McCorquodale, J., 1986. Involvement of host DNA gyrase in growth of bacteriophage T5. J. Virol. 75, 875e882. De Wyngaert, M.A., Hinkle, D.C., 1979. Involvement of DNA gyrase in replication and transcrption of bacteriophage T7. J. Virol. 29, 529e535. Drago, L., De Vechii, E.D.E., Nicola, L., Tocalli, L., Gismondo, M.R., 2004. Effect of moxifloxacin on bacterial pathogenicity factors in comparison with amoxicillin, clarithromycin and ceftriaxone. J. Chemother. 16, 30e37. Giamarellou, H., Antoniadou, A., 2001. Antipseudomonal antibiotics. Med. Clin. North. Am. 85, 19e41. Hagens, S., Habel, A., Blasi, U., 2006. Augmentation of the antimicrobial efficacy of antibiotics by filamentous phage. Microb. Drug Resist. 12, 164e168. Hahn, M.W., Moore, E.R.B., Hofle, M.B., 1999. Bacterial filament formation, a defense mechanism against flagellate grazing, is growth rate controlled in bacteria of different phyla. Appl. Environ. Microbiol. 65, 25e35. Hamatake, R.K., Mukai, R., Hayashi, M., 1981. Role of DNA gyrase subunits in synthesis of bacteriophage VX174 viral DNA. Proc. Natl. Acad. Sci. U S A 78, 1532e1536.
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Heo, Y.-J., Lee, Y.-R., Jung, H.-H., Lee, J., Ko, G.G., Cho, Y.-H., 2009. Antibacterial efficacy of phages towards Pseudomonas aeruginosa infections in mice and Drosophila melanogaster. Antimicrob. Agents Chemother. 53, 2469e2474. Himmelweit, F., 1945. Combined action of penicillin and bacteriophage on Staphylococci. Lancet 246, 104e105. Hoiby, N., Krogh, J.H., Moser, C., Song, Z., Ciofu, O., Kharazmi, A., 2001. Pseudomonas aeruginosa and the in vitro and in vivo biofilm mode of growth. Microbes. Infect. 3, 23e35. Huff, W.E., Huff, G.R., Rath, N.C., Balog, J.M., Donoghue, A.M., 2004. Therapeutic efficacy of bacteriophage and baytril (enrofloxacin) individually and in combination to treat colibacillosis in broilers. Poult. Sci. 83, 1944e1947. Kadurugamuwa, J.L., Clarke, A.J., Beveridge, T.J., 1993. Surface action of gentamicin on Pseudomonas aeruginosa. J. Bacteriol. 175, 5798e5805. Knezevic, P., Kostanjsek, R., Obreht, D., Petrovic, O., 2009. Isolation of Pseudomonas aeruginosa specific bacteriophages with broad activity spectra. Curr. Microbiol. 59, 173e180. Knezevic, P., Obreht, D., Curcin, S., Petrusic, M., Aleksic, V., Kostanjsek, R., Petrovic, O., 2011. Phages of Pseudomonas aeruginosa: response to environmental factors and in vitro ability to inhibit bacterial growth and biofilm formation. J. Appl. Microbiol. 111, 245e254. Livermore, D.M., 2004. The need for new antibiotics. Clin. Microbiol. Infect. 10, 1e9. McKinnon, P.S., Davis, S.L., 2004. Pharmacokinetic and pharmacodynamic issues in the treatment of bacterial infectious diseases. Eur. J. Clin. Microbiol. Infect. Dis. 23, 271e288. McVay, C.S., Velasquez, M., Fralick, J.A., 2007. Phage therapy of Pseudomonas aeruginosa infection in a mouse burn wound model. Antimicrob. Agents Chemother. 51, 1934e1938. Nordmann, P., Naas, T., Fortineau, N., Poirel, L., 2007. Superbugs in the coming new decade; multidrug resistance and prospects for treatment of Staphylococcus aureus, Enterococcus spp. and Pseudomonas aeruginosa in 2010. Curr. Opin. Microbiol. 10, 436e440. Perry, T.R., Schentag, J.J., 2001. Clinical use of ceftriaxone: a pharmacokinetic-pharmacodynamic perspective on the impact of minimum
inhibitory concentration and serum protein binding. Clin. Pharm. 40, 685e694. Rahman, M., Kuhn, I., Rahman, M., Olsson-Liljequist, B., Mo¨llby, R., 2004. Evaluation of a scanner-assisted colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. Appl. Environ. Microbiol. 70, 2398e2403. Rella, M., Haas, D., 1982. Resistance of Pseudomonas aeruginosa PAO to nalidixic acid and low levels of b lactam antibiotics: mapping of chromosomal genes. Antimicrob. Agents Chemother. 22, 242e249. Russell, A.D., 1998. Types of antibiotics and synthetic antimicrobial agents. In: Hugo, W.B., Russell, A.D. (Eds.), Pharmacelutical Microbiology. Blackwell Science Ltd., pp. 91e130. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Tam, V.H., Schilling, A.N., Vo, G., Kabbara, S., Kwa, A.L., Wiederhold, N.P., Lewis, R.E., 2005. Pharmacodynamics of polymyxin B against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 49, 3624e3630. Verma, P., 2007. Methods for determining bactericidal activity and antimicrobial interactions: synergy testing, time-kill curves, and population analysis. In: Schwalbe, R., Steele-Moore, L., Goodwin, A.C. (Eds.), Antimicrobial Susceptibility Testing Protocols. CRC Press, pp. 275e299. Wang, J., Hu, B., Xu, M., Yan, Q., Liu, S., Zhu, X., Sun, Z., Reed, E., Ding, L., Gong, J., Li, Q.Q., Hu, J., 2006. Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosa. Int. J. Mol. Med. 17, 309e317. Watanabe, R., Matsumoto, T., Sano, G., Ishii, Y., Tateda, K., Sumiyama, Y., Uchiyama, J., Sakurai, S., Matsuzaki, S., Imai, S., Yamaguchi, K., 2007. Efficacy of bacteriophages therapy against gut-derived sepsis caused by Pseudomonas aeruginosa in mice. Antimicrob. Agents Chemother. 51, 446e452. Wroblewska, M., 2006. Novel therapies of multidrug-resistant Pseudomonas aeruginosa and Acinetobacter spp. Infections: State Art. Arch. Immunol. Ther. Ex. 54, 113e120. Zhao, X., Xu, C., Domagala, J., Drlica, K., 1997. DNA topoisomerse targets of the fluoroquinolones: a strategy for avoiding bacterial resistance. Proc. Natl. Acad. Sci. U S A 94, 13991e13996.
Phage-antibiotic synergism: a possible approach to combatting Pseudomonas aeruginosa Petar Knezevic a,*, Sanja Curcin b, Verica Aleksic a, Milivoje Petrusic a, Ljiljana Vlaski c a
Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 2, Novi Sad, Vojvodina, Serbia b Molecular Plant Physiology, Centre for Organismal Studies (COS) Heidelberg, INF 360 D-69120 Heidelberg, Germany c University ENT Clinic, Clinical Center of Vojvodina, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3, Novi Sad, Vojvodina, Serbia Received 19 March 2012; accepted 24 August 2012 Available online 7 September 2012
Abstract Pseudomonas aeruginosa is a highly resistant opportunistic pathogen and an important etiological agent of various types of infections. During the last decade, P. aeruginosa phages have been extensively examined as alternative antimicrobial agents. The aim of the study was to determine antimicrobial effectiveness of combining subinhibitory concentrations of gentamicin, ceftriaxone, ciprofloxacin or polymyxin B with P. aeruginosa-specific bacteriophages belonging to families Podoviridae and Siphoviridae. The time-kill curve method showed that a combination of bacteriophages and subinhibitory concentrations of ceftriaxone generally reduced bacterial growth, and synergism was proven for a Siphoviridae phage s-1 after 300 min of incubation. The detected alteration in morphology after ceftriaxone application, resulting in cell elongation, along with its specific mode of action, seemed to be a necessary but was not a sufficient reason for phage-antibiotic synergism. The phenomenon offers an opportunity for future development of treatment strategies for potentially lethal infections caused by P. aeruginosa. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Bacteriophages; Antibiotics; Synergism; Pseudomonas aeruginosa
1. Introduction Pseudomonas aeruginosa, one of the most common opportunistic pathogens in humans, is an important etiological agent of various types of infections and one of the main nosocomial pathogens. It stands out among Gram-negative rods as extremely resistant to a number of antimicrobials due to its intrinsic and acquired mechanisms of resistance, including an attached mode of growth in the form of a biofilm (Hoiby et al., 2001; Livermore, 2004). The resistance phenomenon in P. aeruginosa is a global health concern that is rapidly increasing in magnitude. Some groups of antibiotics can be successfully applied to infections caused by
* Corresponding author. E-mail addresses: [email protected] (P. Knezevic), sanja. [email protected] (S. Curcin), [email protected] (V. Aleksic), [email protected] (M. Petrusic), [email protected] (Lj. Vlaski).
this bacterium: some b lactams, aminoglycosides, fluoroquinolones and polymyxins (Giamarellou and Antoniadou, 2001). The prospects of novel anti-Pseudomonas agents are poor, since most do not represent new classes of antimicrobials, but rather, are derivatives of existing antibiotic molecules (Nordmann et al., 2007). In addition, almost all currently used anti-Pseudomonas agents are toxic, teratogenic or cause reactions of hypersensitivity (Adam, 1989; McKinnon and Davis, 2004; Russell, 1998; Tam et al., 2005). Thus, alternative treatments of potentially lethal P. aeruginosa infections must be sought. One solution involves a combination of two or more conventional antimicrobial agents (reviewed in Wroblewska, 2006). These treatments prolong the emergence of antibiotic-resistant strains and effectively kill bacteria, which is not the case when antimicrobials are used separately. During the last decade, bacteriophages have also been extensively examined as potential antimicrobial agents for therapy, including various P. aeruginosa-specific phages (Heo et al.,
0923-2508/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.resmic.2012.08.008
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2009; McVay et al., 2007: 20, Wang et al., 2006; Watanabe et al., 2007). However, few studies have dealt with combining phages and conventional antibiotics. Hagens et al. (2006) noted that filamentous phages could increase P. aeruginosa sensitivity to several antibiotics (gentamicin, tetracycline, chloramphenicol and carbenicillin), while Comeau et al. (2007) reported that among tetracyclines, gentamicin, quinolones and b-lactams (aztreonam and several cephalosporins), only the latter two groups gave phage-antibiotic synergy against Escherichia coli. Existing data are scarce and inconsistent, and the activity of some important groups of antibiotics such as polymyxins has not yet been examined in combination with phages. Furthermore, the phenomenon has not been confirmed for P. aeruginosa-specific phages of the order Caudovirales. The aim of the present study was to examine the efficacy of subinhibitory concentrations of conventional anti-Pseudomonas agents in combination with P. aeruginosa-specific phages from the families Siphoviridae and Podoviridae, and to elucidate the main reasons for the onset of synergism between them. 2. Material and methods 2.1. Bacterial strains Three P. aeruginosa strains were used in the study: a reference strain ATCC 9027 (external ear infection), PA-4U isolated from environmental biofilm and PA-M2 from river water. Bacteria were stored in LuriaeBertani broth (LB) containing glycerol (v⁄v 10%) at 70 C. For the experiments, they were inoculated in LB and incubated overnight at 37 C.
formazane, was considered as the MIC. The MIC determination was carried out in three replicates and three independent experiments. 2.3. Bacteriophages In order to determine antagonistic, additive and synergistic effects of subinhibitory concentrations of the antibiotics and phages, three previously isolated and characterized bacteriophages were used: d (family Podoviridae), s-1 and 001A (family Siphoviridae). Their hosts were PA-4U, ATCC 9027 and PA-M2, respectively. Pphages d and 001A possessed a broad activity range when tested against 33 P. aeruginosa strains, and were able to infect up to 72.7 and 75.8% of the examined strains, respectively, while phage s-1 exhibited a moderate lytic spectrum (plaque on 45.5% of strains). The first two phages possessed considerable lytic efficacy against host strains, while phage s-1 was not very effective. Their latent periods were approximately 60 min for phage d, 75 min for s-1 and 90 min for phage 001A. All three phages were clonally different, as confirmed previously by the RFLP method (Knezevic et al., 2009, 2011). The phages were multiplied, precipitated with NaCl and PEG6000, purified by equilibrium ultracentrifugation in CsCl and finally dialyzed (Sambrook and Russell, 2001). Plaqueforming units in the purified stocks were first determined by the spot method and then the corresponding dilutions were plated by an overlay agar method in triplicate for a more precise phage count estimation. The stocks were stored at 4 1 C. 2.4. Time kill curves
2.2. MIC determination The antimicrobial agents evaluated in the study included ceftriaxone, gentamicin (Galenika a.d., Serbia), ciprofloxacin (Zdravlje a.d., Serbia) and polymyxin B (HiMedia, India). Stock solutions were prepared at a concentration of 256 mg/l and used for preparation of twofold dilutions. MICs were determined using a slightly modified microtiter plate method developed by Rahman et al. (2004). Briefly, a bacterial suspension previously adjusted to match turbidity of a 0.5 McFarland Nefelometer standard (measured by absorbance at 625 nm 0.08e0.1; approx. 2 108 CFU/ml) was diluted in double-strength LB (1:100 v/v). Into each well, 100 ml of the inoculated double-strength medium and the same volume of twofold antibiotic dilutions were added to obtain final concentrations in the range 0.03e64 mg/ml. The microtiter plates were incubated for 18 h at 37 C without shaking, amended with a 0.22 mm filter-sterilized solution of 2,3,5triphenyltetrazolium chloride (final concentration 200 mg/ml) and incubated additionally for 3 h. Controls for plate sterility and bacterial growth without antibiotic were also included. Bacterial growth was estimated using a microtiter plate reader at 540 nm (Multiskan EX, Thermo-Labsystem, Vantaa, Finland). The lowest concentration of antibiotic that inhibited bacterial growth, which was visible as the absence of red
The effectiveness of combining phages and antibiotics was determined by the time-kill curve method (Verma, 2007). Changes in bacterial count were monitored in parallel in test tubes containing the following: only bacteria (approx. 1 106 CFU/ml); bacteria and a subinhibitory concentration of antibiotics (1/4 of MIC); bacteria and corresponding bacteriophages at a multiplicity of infection (MOI ¼ 0.01 for phage d; MOI ¼ 0.1 for 001A, and MOI ¼ 5e10 for s-1, determined according to their previously established bacteriolytic efficiency (Knezevic et al., 2011)); or bacteria, a subinhibitory concentration of antibiotics and bacteriophages (final volume 10 ml). The test tubes were incubated in a water bath at 37 C for 5 h and bacterial counts were determined after 0, 60, 90, 120, 240 and 300 min of incubation by spreading appropriate dilutions on MullereHinton agar (detection limit 102 CFU/ml). The plates were incubated at 37 C overnight and bacterial colonies were counted. The results from the experiments, carried out at least twice, were averaged and expressed as logarithms with corresponding standard errors (mean SE). If bacterial CFU/ml decreased by 2 log for the phage-antibiotic combination compared to the more active single agent (phage or antibiotic), as well as to the initial inoculum titer, the interaction was considered to be synergistic.
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2.5. Cell morphology In order to examine the importance of cell filamentation in case of synergism between antibiotics and P. aeruginosaspecific phages, we examined the morphology of bacterial cells of strains ATCC 9027 and PA-M2 with subinhibitory antibiotic concentrations (1/4 MIC) in parallel by bright field microscopy after staining with methylene blue and by epifluorescence microscopy (BX51, Olympus) after fixation, filtration onto 0.2-mm-pore-size polycarbonate filters and staining with 0.1% (w/v) DAPI (40 ,60 -diamidino-2-phenylindole). The length of randomly observed cells (at least 20 per field) was measured using software (Olympus cell B) and means were calculated. The statistical difference in cell length among treatments was evaluated using the ANOVA test and the Holm post-hoc test. The null hypothesis was that there was not a significant difference between cell length in the medium with and without subinhibitory concentrations of antibiotics, and it was rejected if P 0.05. The phenomenon in which cell dimensions were 5 mm was considered to be filamentation, while morphological changes wherein the cell length was <5 mm but significantly longer than regular (i.e. untreated) cells were denoted as elongation. 3. Results 3.1. MIC The determined MICs varied from 0.06 mg/ml of ciprofloxacin for strain PA-4U to 64 mg/ml of ceftriaxone for strain PA-M2 (Table 1). 3.2. Phage-antibiotic synergism According to the time-kill curves, when subinhibitory concentrations of gentamicin were tested with phages against corresponding P. aeruginosa strains, bacterial count reduction was not observed (Fig. 1A e aec). In contrast, the bacterial count was even greater, by 0.48e1.97 log compared to the initial count, indicating the absence of antimicrobial activity. The reduction in the initial bacterial count was also absent when subinhibitory concentrations of ciprofloxacin and bacteriophage were simultaneously used, though the bacterial count was lower by 0.13 and 0.15 log (001A vs. PA-M2 and d vs. PA-4U, respectively) in comparison to a more active single agent (Fig. 1B e b and c). Although a combination of ciprofloxacin and phage s-1 reduced the initial count of strain ATCC 9027 for 3.30 log, the bacterial count reduction was Table 1 MICs of antibiotics for P. aeruginosa strains. P. aeruginosa strains
ATCC 9027 PA-M2 PA-4U
MIC (mg/ml) GEN
CIP
CRO
PMB
2 2 1
1 1 0.06
8 64 16
4 4 2
57
lower by 1.30 log in relation to that obtained with the applied concentration of antibiotic (Fig. 1B e a). Similarly, the absence of synergism was also observed in the phage and polymyxin B combination, since the bacterial count was greater than the inoculum count for 1.28e2.13 log, depending on the phage-host system (Fig. 1D e aec). However, after combining phages and subinhibitory concentrations of ceftriaxone, the bacterial count was reduced by 2.56 log (s-1 vs. ATCC 9027), 1.26 log (d vs. PA-4U) and 1.45 log (001A vs. PA-M2) in comparison to the more active single agent, while the initial count decreased by 2.12, 2.22, and 1.67 log, respectively (Fig. 1C e aec). 3.3. Cell length The average length of P. aeruginosa cells grown in LB was not statistically different from the average cell dimensions in the presence of 1/4 MIC of gentamicin and polymyxin B (P > 0.05) (Table 2). Cells of both strains were significantly shorter for bacteria grown in LB medium without antibiotic compared to those grown in medium with subinhibitory concentration of ciprofloxacin and ceftriaxone (P < 0.0001). In addition, the difference in cell length between bacteria grown in presence of subinhibitory concentrations of ciprofloxacin and ceftriaxone was not statistically significant, and neither phage presence nor absence in the medium had an effect on filamentation (P > 0.05). 4. Discussion Synergism between phages and antibiotics, noted earlier by other authors, has not been widely discussed. Although Himmelweit (1945) successfully combined phages and penicillin against Staphylococcus in 1945, his work had remained unnoted until recently. More than a half a century later, Huff et al. (2004) observed a similar phenomenon in E. colispecific phages and ciprofloxacin, but failed to provide an explanation. On the other hand, Hagens et al. (2006) speculated that, during filamentous phage progeny extrusion, the outer membrane was a less effective barrier for antibiotic penetration into a bacterial cell. This explanation for phageantibiotic synergy, however, would be acceptable for phages Pf1 and Pf3 belonging to the family Inoviridae and released from bacterial cells by extrusion, but not for phages belonging to the families Podoviridae and Siphoviridae released by cell lysis. Results clearly indicate that the combination of ceftriaxone and phages tended to reduce the bacterial host count for 5 h. A similar phenomenon was not observed when other antimicrobials were tested, and only a combination of phage s-1 and ceftriaxone against P. aeruginosa ATCC 9027 had a synergistic effect sensu stricto. In contrast to s-1, phages d and 001A failed to reduce the bacterial count for 2 log in the presence of subinhibitory concentrations of ceftriaxone, and the reasons for the absence of synergism for corresponding phage-host systems are unclear.
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Fig. 1. Time-kill curves of phage s-1 against ATCC 9027 (a), phage d against PA-4U (b) and phage 001A against strain PA-M2 (c) in the presence of subinhibitory concentrations of gentamicin (A), ciprofloxacin (B), ceftriaxone (C) and polymyxin B (D); (C) bacteria without treatment; (D) antibiotic; (>) phage and (-) phage and antibiotic.
Earlier systematic experiments on combining various antibiotics and phages against E. coli showed that cell filamentation, as a result of antibiotic subinhibitory concentrations presence in a nutritive medium, had a strong impact on synergy and phage plaque enlargement (Comeau
Table 2 Cell length in the presence of subinhibitory concentrations of antibiotics and/ or bacteriophages. Straina
Phage Cell length (mm) in the presence of antibiotics GEN
ATCC 9027 none s-1 PA-M2 none 001A
1.30 1.19 1.50 1.68
CIP
0.13 2.75 0.09 2.60 0.15 17.85 0.16 16.87
CRO 0.23 3.44 0.23 3.06 0.48 18.68 0.36 17.30
PMB 0.34 0.21 0.39 0.64
1.35 1.78 1.49 1.69
0.11 0.17 0.15 0.18
a Cell dimensions in LB without antibiotics: 1.14 0.08 mm for ATCC 9027 and 1.22 0.06 mm for PA-M2.
et al., 2007). Cell filamentation in the presence of subinhibitory concentrations of b-lactams and fluoroquinolones had been previously demonstrated in several studies and was explained by the fact that these antibiotics, although exhibiting different mechanisms of action, finally block bacterial cell division (Bergogne-Berezin, 1985; Drago et al., 2004; Rella and Haas, 1982). According to Comeau et al. (2007), phageantibiotic synergism is a result of a change in morphology that permits faster assembly of phages through altered or larger pools of precursors important to phage maturation, and accelerates the timing of cell lysis. Present results confirm that subinhibitory concentrations of ceftriaxone and ciprofloxacin cause significant elongation and filamentation of P. aeruginosa cells, while this was not observed with gentamicin and polymyxin B. It is also interesting to note that cells of strain ATCC 9027 in the presence of subinhibitory concentrations of ciprofloxacin and ceftriaxone were significantly shorter than those of PA-M2 (P < 0.0001),
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i.e. in the case of ATCC 9027, cells exposed to antibiotics were about 2e3 times longer, while in the case of PA-M2 they were 10e14 times longer. Since only cells longer than 5 mm can be considered filaments (Hahn et al., 1999), it is clear that the concentrations of ceftriaxone and ciprofloxacin used caused significant elongation of ATCC 9027, but not filamentation. In contrast, ceftriaxone and ciprofloxacin caused morphological changes corresponding to filamentation in PAM2, but no significant cell count reduction was observed after combining it with P. aeruginosa-specific bacteriophages. Taking into account results obtained and the fact that synergism was not proven in the absence of altered morphology either in the present study or in that of Comeau et al. (2007), cell elongation/filamentation appears to be a necessary but insufficient reason for phage-antibiotic synergy. It is interesting to note, regarding PA-M2 and ATCC 9027, that we did not establish such a link between changed cell length and reduction in bacterial number that would support the idea that the longer the cell, the more bacteria are efficiently destroyed. This suggests a more complex phenomenon, with other factors and mechanisms involved that should be further examined. For instance, phage s-1 belongs to the family Siphoviridae and is temperate, like all P. aeruginosa-specific phages from this family isolated to date. This implies possible involvement of phage induction by an antibiotic, following cell infection, in the onset of synergism. The mode of an effect of antibiotics on bacterial cells should also be taken into consideration to explain the obtained results. Namely, bacterial count reduction in combination with phages undoubtedly was influenced by the antibiotics used. It is widely known that ceftriaxone inhibits cell wall synthesis binding to PBP (Bergogne-Berezin, 1985), that ciprofloxacin inhibits DNA replication targeting DNA gyrase (Zhao et al., 1997) and that gentamicin inhibits protein synthesis binding to the 30S ribosomal subunit and disrupts cell envelopes (Kadurugamuwa et al., 1993), while polymyxin B disturbs membrane structures (Tam et al., 2005). Phage multiplication is not dependent on peptidoglycan synthesis; however, functional bacterial ribosomes, cell membrane integrity and functional DNA gyrase are necessary for Podoviridae and Siphoviridae successful multiplication (Constantinou et al., 1986; De Wyngaert and Hinkle, 1979; Hamatake et al., 1981). In addition, ceftriaxone has several other differences in comparison to ciprofloxacin, gentamicin and polymyxin B that might contribute to the phenomenon described. Ceftriaxone exhibits time-dependent killing, like all other cephalosporins (Perry and Schentag, 2001) and these pharmacological characteristics might also have an impact on phage-antibiotic synergy. Interestingly, ceftriaxone is the only agent used in the study against which P. aeruginosa strains frequently possess or acquire resistance, i.e. most strains of P. aeruginosa are intermediate or resistant to this cephalosporin (Wroblewska, 2006). This also emphasizes the importance of synergy, as it is possible to increase the sensitivity of P. aeruginosa to this agent using bacteriophages. In summary, among the tested antimicrobial agents, only ceftriaxone causes cell morphological changes and shows
59
a mode of action excluding interference with phage multiplication. Since only the use of subinhibitory concentrations of ceftriaxone provided a synergistic effect with P. aeruginosaspecific bacteriophages, it can be assumed that both cell elongation and a specific mode of action are necessary prerequisites for synergy. Phage-antibiotic synergism offers a possible opportunity for developing treatment strategies for infections caused by P. aeruginosa in the future using corresponding antibiotics and phages which meet the prerequisites for therapeutic application. This approach offers additional advantages, including the rapid onset of synergism possibly contributing to a reduction in bacterial number in vivo to levels that the immune system can successfully cope with; moreover, use of subinhibitory concentrations of antibiotics results in avoidance of antibiotic side effects occurring after administration of high doses. Finally, in addition to further experiments designed to better understand the mechanisms of rapid synergism, the observed phenomenon should be assessed for possible applications in control of P. aeruginosa. Acknowledgments This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant OI 172058. The authors acknowledge Emilija Nikolic-Djoric, MSc (Faculty of Agriculture, University of Novi Sad) for help in statistical analysis and Ljiljana Knezevic, MA (Faculty of Sciences, University of Novi Sad) for language revision. References Adam, D., 1989. Use of quinolones in pediatric patients. Rev. Infect. Dis. 11, 1113e1116. Bergogne-Berezin, E., 1985. Antibacterial activity of ceftriaxone. Rev. Med. Interne. 6, 178e186. Comeau, A.M., Tetart, F., Trojet, S.N., Pre`re, M.F., Krisch, H.M., 2007. Phageantibiotic synergy (PAS): b-lactam and quinolone antibiotics stimulate virulent phage growth. PLoS One 2, e799. Constantinou, A., Voelker-Meiman, K., Sternglanz, R., McCorquodale, M.M., McCorquodale, J., 1986. Involvement of host DNA gyrase in growth of bacteriophage T5. J. Virol. 75, 875e882. De Wyngaert, M.A., Hinkle, D.C., 1979. Involvement of DNA gyrase in replication and transcrption of bacteriophage T7. J. Virol. 29, 529e535. Drago, L., De Vechii, E.D.E., Nicola, L., Tocalli, L., Gismondo, M.R., 2004. Effect of moxifloxacin on bacterial pathogenicity factors in comparison with amoxicillin, clarithromycin and ceftriaxone. J. Chemother. 16, 30e37. Giamarellou, H., Antoniadou, A., 2001. Antipseudomonal antibiotics. Med. Clin. North. Am. 85, 19e41. Hagens, S., Habel, A., Blasi, U., 2006. Augmentation of the antimicrobial efficacy of antibiotics by filamentous phage. Microb. Drug Resist. 12, 164e168. Hahn, M.W., Moore, E.R.B., Hofle, M.B., 1999. Bacterial filament formation, a defense mechanism against flagellate grazing, is growth rate controlled in bacteria of different phyla. Appl. Environ. Microbiol. 65, 25e35. Hamatake, R.K., Mukai, R., Hayashi, M., 1981. Role of DNA gyrase subunits in synthesis of bacteriophage VX174 viral DNA. Proc. Natl. Acad. Sci. U S A 78, 1532e1536.
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