EFFECTS OF BACTERIOPHAGES ON CLEARANCE OF PSEUDOMONAS AERUGINOSA AND STAPHYLOCOCCUS AUREUS AND SERUM CYTOKINE LEVELS IN INFECTED MICE

Michał Zimecki, Maja Kocięba, Beata Weber-Dąbrowska, Marzanna Łusiak-Szelachowska, Danuta Syper, Andrzej Górski
Institute of Immunology and Experimental Therapy of Polish Academy of Sciences, Wrocław, Poland

ABSTRACT

In this study we demonstrated that concomitant, intraperitoneal administration of T8 and A5 bacteriophages with lethal doses of Pseudomonas aeruginosa or Staphylococcus aureus, respectively, protected CBA mice against lethality and significantly reduced the number of CFUs in the organs 24h after infection. In the case of P. aeruginosa infection, the serum levels of tumor necrosis factor alpha and interleukin 6 were not significantly altered by bacteriophages. On the other hand, the levels of these cytokines were significantly elevated upon administration of bacteriophages during S. aureus infection. The relevance of the phage-induced changes in the cytokine levels to the protection of mice against Gram-negative and Gram-positive sepsis is discussed.

Key words: bacteriophages, Pseudomonas aeruginosa, Staphylococcus aureus, mice, TNF-alpha, interleukin-6.

INTRODUCTION

The increasing resistance of pathogenic bacteria to antibiotics has led to the reintroduction of phage therapy and intensification of research in the field of phage biology [2]. Phage therapy appears to be effective in combating suppurative infections and providing long-term resistance [15] to new infection. Our recent studies also demonstrated that successful phage therapy was associated with the normalization of cytokine production in cultures of peripheral blood of patients [16], increased output of neutrophils into the circulation [17], and regulation of mitogen-induced mouse splenocyte proliferation [18]. Cytokines, such as TNF-α and IL-6, play a crucial role in combating infection. TNF-α exhibits potent antibacterial properties [6], although excessive production of that cytokine may be detrimental [8]. IL-6, in turn, elicits the production of protective, acute-phase proteins [9], controls TNF-α production [13] and, most importantly, is responsible for mobilization of neutrophils [3], the major type of phagocyte [5] in the circulation. The aim of this investigation was to correlate the effectiveness of phage action on the clearance of Pseudomonas aeruginosa and Staphylococcus aureus in the organs of infected mice with changes in serum cytokine levels.

MATERIALS AND METHODS

Animals: CBA mice, 10-12 weeks old, were provided by the Institute of Immunology and Experimental Therapy (IIET), Wrocław, Poland. The mice had free access to water and standard rodent laboratory chow. All protocols were approved by the IIET Animal Care and Use Committee.

Determination of P. aeruginosa and S. aureus in the organs: Bacterial strains were obtained from the collection of microorganisms of IIET, Wrocław, Poland. Mice were injected i.p. with the bacteria in 0.2 ml of 0.9% saline solution. Bacterial cell numbers were determined colorimetrically at a wavelength of 600 nm, according to the previously prepared standards. The bacteriophages were administered i.p. in the amount of 10⁸ pfu/mouse immediately after bacteria injection. Twenty-four hours post-injection, the spleens, livers, and lungs were isolated and the organs were weighed. The organs were homogenized using a syringe piston and plastic screen in sterile PBS (1 g of wet tissue per 25 ml of PBS). Five- and fifty-fold dilutions of cell suspension were applied to McConkey and Chapman agar plates and incubated overnight, and colony-forming units (CFUs) were enumerated. The number of colonies was expressed as the number of CFUs per milligram of organ. The bacteriophages used in the study (A5 specific to S. aureus and T8 specific to P. aeruginosa) came from the Institute’s collection.

Determination of TNF-α and IL-6: Cytokine activity in the sera of mice bled at 2, 4, and 24 h after infection was determined by bioassays using specific indicator cell lines [4, 14].

Preparation and purification of phages: Preparation and purification of specific bacteriophages was described by us before [18].

Statistics: Groups of mice consisted of 6-8 mice. All data are expressed as mean values ± standard error (SE). Differences between groups were analyzed by the Student’s unpaired t-test. A P value of 0.05 or less was regarded as significant.

RESULTS

The effects of bacteriophages on the clearing rate of bacteria in the organs
Mice were treated with P. aeruginosa and phage T-8 as described above. After 24 h the CFUs were counted in the spleens, livers, and lungs of the mice infected with increasing numbers of bacteria (Tables 1-3). Treatment of mice with the T-8 phage resulted in full protection of the mice against death 24 h post-infection. In the control group infected with 2.5 x 10⁷ bacteria, one out of 5 mice survived, and in that with a 10⁸ bacterial dose, all the mice died. In addition, the bacterial clearance process in the studied organs was very effective (more than 99% in the case of the 2.5 x 10⁷ bacterial dose). The clearing rate of S. aureus (10⁸ dose) in the presence of A5 phage was, however, somewhat lower (82, 69, and 68.4% in spleen, liver, and lung, respectively).

Table 1. Effect of bacteriophages on the number of CFUs in the organs of mice infected with 107 Pseudomonas aeruginosa

Groups	Organ	CFU/organ	±SE	t-Student‘s test
P. aeruginosa	Spleen liver lungs	41800 4440 55840	2362.2 1023 2636.4
P. aeruginosa + bacteriophages	Spleen liver lungs	500 990 3080	151.6 145.7 972.3	<0.001 <0.01 <0.01

Table 2. Effect of bacteriophages on the number of CFUs in the organs of mice infected with 2.5 x 107 Pseudomonas aeruginosa

Groups	Organ	CFU/organ	±SE	t-Student’s test
P. aeruginosa	Spleen liver lungs	1698000 1092000 968000
P. aeruginosa + bacteriophages	spleen liver lungs	13520 5100 1500	8762 266 456	<0.001 <0.001 <0.001

Table 3. Effect of bacteriophages on the number of CFUs in the organs of mice infected with 108 Pseudomonas aeruginosa

Groups	Organ	CFU/organ	±SE	t-Student‘s test
P. aeruginosa	spleen liver lungs	Mice died after 24 h
P. aeruginosa + bacteriophages	spleen liver lungs	40880 3720 5720	5244 1095 3751

Table 4. Effect of bacteriophages on the number of CFUs in the organs of mice infected with 108 Staphylococcus aureus

Groups	Organ	CFU/organ	±SE	t-Student’s test
S. aureus	spleen liver lungs	38580 26844 4744	4696 4307 2341
S. aureus + bacteriophages	spleen liver lungs	6960 8280 1500	1431 2222 408	<0.001 <0.01 NS

The effects of infection and bacteriophage treatment on cytokine serum levels
The levels of TNF-α and IL-6, determined by bioassays, were highest at 2 h post-infection and correlated with the dose of bacteria (Tables 5, 6, and 7). In the Pseudomonas model, TNF-α serum levels were lowered in the presence of the phages, but IL-6 levels were sometimes increased (Table 5, 4-h determination). On the other hand, infection with S. aureus led to more marked changes in the cytokine levels (Table 8). TNF-α level was enhanced 2-fold at 2 h post-infection and IL-6 serum concentration was elevated particularly strongly (18-fold and 3.4-fold at 2 and 4 h, respectively).

Table 5. Serum levels of TNF-α and IL-6 in mice infected with 107 Pseudomonas aeruginosa and treated with 108 bacteriophages

Determined	Groups	TNF-α			IL-6
after		(pg ml-1)	±SE	P	(pg ml-1)	+SE	P
	P. aeruginosa	446	115		1985	672
2 h	P. aeruginosa + bacteriophages	493	124	NS	1151	200	NS
	P. aeruginosa	206	4.5		239	99
4 h	P. aeruginosa + bacteriophages	242	22	NS	1044	253	<0.02
	P. aeruginosa	205	8		9	2
24 h	P. aeruginosa + bacteriophages	196	12	NS	10.3	3	NS

Table 6. Serum levels of TNF-α and IL-6 in mice infected with 2.5 x 107 Pseudomonas aeruginosa and treated with 108 bacteriophages

Determined	Groups	TNF-α			IL-6
after		(pg ml-1)	±SE	P	(pg ml-1)	+SE	P
	P. aeruginosa	557	123		5159	1217
2 h	P. aeruginosa + bacteriophages	311	60	NS	3533	651	NS
	P. aeruginosa	224	17		2288	500
4 h	P. aeruginosa + bacteriophages	218	14	NS	3163	428	NS
	P. aeruginosa	168*			154
24 h	P. aeruginosa + bacteriophages	214	10		274	81

*one mouse (remaining mice died)

Table 7. Serum levels of TNF-α and IL-6 in mice infected with 108 Pseudomonas aeruginosa and treated with 108 bacteriophages

Determined	Groups	TNF-α			IL-6
after		(pg ml-1)	±SE	P	(pg ml-1)	+SE	P
	P. aeruginosa	1147	326		6535	1503
2 h	P. aeruginosa + bacteriophages	732	248	NS	8023	751	NS
	P. aeruginosa	591	320		4564	942
4 h	P. aeruginosa + bacteriophages	200	22	NS	4368	1129	NS
	P. aeruginosa	mice died
24 h	P. aeruginosa + bacteriophages	222	23		2443	1030

Table 8. Serum levels of TNF-α and IL-6 in mice infected with 108 Staphylococcus aureus and treated with 108 bacteriophages

Determined	Groups	TNF-α			IL-6
after		(pg ml-1)	±SE	P	(pg ml-1)	+SE	P
	S. aureus	129.2	30.6		100	17.54
2 h	S. aureus + bacteriophages	264	65.4	<0.05	1864	697.9	<0.01
	S. aureus	65.2	15.1		85.4	30.25
4 h	S. aureus + bacteriophages	88.4	11.2	NS	294.2	52.9	<0.01
	S. aureus	72.2	2.0		4.8	1.19
24 h	S. aureus + bacteriophages	43.4	8.8	<0.01	12.0	2.80	<0.05

DISCUSSION

The results of our study clearly demonstrated the effectiveness of specific bacteriophages in protecting mice against Gram-negative and Gram-positive sepsis. That was evidenced by the survival of all the mice treated with even lethal doses of bacteria and by the almost complete clearance of bacteria from the studied organs. Our results contradict other findings showing no protection of mice against S. aureus by a staphylococcal phage [11], which could be due to a lack of specificity of the phage towards that bacterial strain. Nevertheless, our study showed that the staphylococcal phage (A5) was less efficient than the P. aeruginosa-specific phage. Furthermore, our results showed that the serum levels of TNF-α and IL-6 directly correlated with the increasing numbers of P. aeruginosa administered. At a high number of bacteria (10⁸), bacteriophages regulated the cytokine levels, i.e. TNF-α levels decreased (2- and 4-h measurement) and IL-6 concentration increased (2-h measurement). This finding could reflect the decreased bacteria burden, but also an elevated concentration of lipopolysaccharide released by phage-mediated lysis. IL-6 is a good marker of inflammation [10]; it elicits neutrophil recruitment [3] and induces the production of protective acute-phase proteins [9], so that its role in the resolution of sepsis and tempering the consequences of endotoxemia cannot be underestimated. More evident changes in the cytokine levels were observed in the case of S. aureus infection. The two-fold elevation of TNF-α may be of importance in maintaining an appropriate level of this bactericidal cytokine, and the strong increase in IL-6 may be a result of the initially very low levels of these cytokines in the bacteria-treated control group. The low levels of these cytokines released upon S. aureus infection could result from a lower ability of cell wall constituents, e.g. lipoteicholic acid and proteoglycan, to induce TNF-α as compared with LPS [12]. Nevertheless, the serum IL-6 concentration increased very strongly (18-fold) upon bacteriophage treatment. This phenomenon could be likely due to the destruction of bacterial walls and the release of compounds constituting the cell walls of Gram-positive bacteria, such as lipoteichoic acid and peptidoglycan, which are potent IL-6 inducers [7]. Significant increase in IL-6 level may, in turn, be essential for the rapid mobilization of neutrophils [3], which are major phagocytic cells. The murine bone marrow contains, in addition, a reservoir of mature neutrophils which may be released into the circulation upon infection [1]. Therefore, the staphylococcal phages may also increase antibacterial resistance indirectly by eliciting neutrophil output from this reservoir. It should also be mentioned (data not shown) that administration of both A5 and T8 phages alone (10⁸/mouse, i.p.) did not lead to increased serum levels of TNF alpha or IL-6 at 2 h post-injection, indicating that these phage preparations were not pyrogenic and could not, therefore, affect the immune system by means of desensitization to bacterial products.

In conclusion, the investigation showed the efficacy of applying specific phages in clearing Gram-negative and Gram-positive bacteria in the organs of infected mice and indicated that during the initial phase of bacterial destruction by phages, characteristic changes in the serum levels of TNF-α and IL-6 occur which may be significant in the development of defense against infection.

ACKNOWLEDGMENTS

This study was supported in part by the grant from the Polish Ministry of Education No. PBZ-MIN-007/PO4/2003.

REFERENCES

1. Boxio R., Bossenmeyer-Pourie C., Steinckwich N., Dournon C., Nusse O., 2004. Mouse bone marrow contains large numbers of functionally competent neutrophils. J. Leukoc. Biol. 75, 604-611.

2. Carlton R.M., 1999. Phage therapy: past history and future prospects. Arch. Immunol. Ther. Exp. 47, 267-274.

3. Dalrymple S.A., Slattery R., Aud D.M., Krishna M., Lucian L.A., Murray R., 1996. Interleukin-6 is required for a protective immune response to systemic Escherichia coli infection. Infect. Immun. 64, 3231-3235.

4. Espevik T., Nissen-Meyer J., 1986. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immunol. Methods 95, 99-105.

5. Gregory S.H., Sagnimeni A.J., Wing E.J., 1996. Bacteria in the bloodstream are trapped in the liver and killed by immigrating neutrophils. J. Immunol. 157, 2514-2520.

6. Havell E.A., 1989. Evidence that tumor necrosis factor has an important role in antibacterial resistance. J. Immunol. 143, 2894-2899.

7. Leemans J.C., Vervoordeldonk M.J., Florquin S., Kessel K.P., van der Poll T., 2002. Differential role of interleukin 6 in lung inflammation induced by lipoteichoic acid and peptidoglycan from Staphylococcus aureus. Am. J. Respir. Crit. Care Med. 165, 1445-1450.

8. Marks J.D., Marks C.B., Luce J.M., Montgomery A.B., Turner J., Metz C.A., Murray J.F., 1990. Plasma tumor necrosis factor in patients with septic shock. Mortality rate, incidence of adult respiratory distress syndrome, and effects of methylprednisolone administration. Am. Rev. Respir. Dis. 141, 94-97.

9. Nishimoto N., Yoshizaki K., Tagoh H., Monden M., Kishimoto S., Hirano T., Kishimoto T., 1989. Elevation of serum interleukin 6 prior to acute phase proteins on the inflammation by surgical operation. Clin. Immunol. Immunopathol 50, 399-401.

10. Revel M., 1989. Host defense against infections and inflammations: role of the multifunctional IL-6/IFN-beta 2 cytokine. Experientia 45, 549-557.

11. Soothill J.S., 1992. Treatment of experimental infections of mice with bacteriophages. J. Med. Microbiol. 37, 258-261.

12. Sriskandan S., Cohen J., 1999. Gram-positive sepsis. Mechanisms and differences from gram-negative sepsis. Infect. Dis. Clin. North Am. 13, 397-412.

13. Tilg H., Trehu E., Atkins M.B., Dinarello C.A., Mier J.W., 1994. Interleukin-6 (IL-6) as an anti-inflammatory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood 83, 113-118.

14. Van Snick J., Cayphas S., Vink A., Uyttenhove C., Coulie P.G., Rubira M.R., Simpson R.J., 1986. Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc. Natl. Acad. Sci. USA 83, 9679-9683.

15. Weber-Dšbrowska B., Mulczyk M., Górski A., 2000. Bacteriophage therapy of bacterial infections: an update of our Institute’s experience. Arch. Immunol. Ther. Exp. 48, 547-551.

16. Weber-Dabrowska B., Zimecki M., Mulczyk M., 2000. Effective phage therapy is associated with normalization of cytokine production by blood cell cultures. Arch. Immunol. Ther. Exp. 48, 31-37.

17. Weber-Dšbrowska B., Zimecki M., Mulczyk M., Górski A., 2002. Effect of phage therapy on the turnover and function of peripheral neutrophils. FEMS Immunol. Med. Microbiol. 34, 135-138.

18. Zimecki M., Weber-Dšbrowska B., Łusiak-Szelachowska M., Mulczyk M., Boratyński J., Pozniak G., Syper D., Górski A., 2003. Bacteriophages provide regulatory signals in mitogen-induced murine splenocyte proliferation. Cell. Mol. Biol. Lett. 8, 699-711.

 

Accepted for print: 29.06.2007

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Michał Zimecki
Institute of Immunology and Experimental Therapy
of Polish Academy of Sciences, Wrocław, Poland
R. Weigla 12, 53-114 Wrocław, Poland
email: zimecki@immuno.iitd.pan.wroc.pl

Maja Kocięba
Institute of Immunology and Experimental Therapy
of Polish Academy of Sciences, Wrocław, Poland
R. Weigla 12, 53-114 Wrocław, Poland

Beata Weber-Dąbrowska
Institute of Immunology and Experimental Therapy
of Polish Academy of Sciences, Wrocław, Poland
R. Weigla 12, 53-114 Wrocław, Poland
Phone: (+4871) 3371172
Fax: (+4871) 3371382
email: weber@iitd.pan.wroc.pl

Marzanna Łusiak-Szelachowska
Institute of Immunology and Experimental Therapy
of Polish Academy of Sciences, Wrocław, Poland
R. Weigla 12, 53-114 Wrocław, Poland


Danuta Syper
Institute of Immunology and Experimental Therapy
of Polish Academy of Sciences, Wrocław, Poland
R. Weigla 12, 53-114 Wrocław, Poland


Andrzej Górski
Institute of Immunology and Experimental Therapy
of Polish Academy of Sciences, Wrocław, Poland
R. Weigla 12, 53-114 Wrocław, Poland

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