2010) 1( 23مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد ضد األصابة ببكتریا gasseri Lactobacillus تأثیر بكتریا Pseudomonas aeruginosa في الفئران محمد فرج المرجاني ، رواء عبد األمیر عبد الجبار و علي مرتضى حسن لیة العلوم ، الجامعة المستنصریة قسم علوم الحیاة ، ك الخالصة في Pseudomonas aeruginosaضد األصابة ببكتریا Lactobacillus gasseriدرس تأثیر راشح بكتریا مدة Lb. gasseriمل من راشح بكتریا 0.25 الفئران ، اذ حقنت مجموعة من الفئران داخل الغشاء البریتوني ب P. aeruginosa )10مل خالیا حیة لبكتریا 0.25ام ، بعدها حقنت بخمسة أی 8 ء ) مل / خلیة داخل الغشا .مل من دارئ الفوسفات 0.2البریتوني ، بینما حقنت مجموعة السیطرة ب 0.1 رع تم عمل تخافیف من محتویات البریتون ، وز . مل دارئ الفوسفات داخل الغشاء البریتوني وأخذت محتویاته مل على األوساط الزرعیة لحساب عدد المستعمرات البكتیریة د حسبت أعدا .النامیة 5تم حقن اذ، P . aeruginosaساعة من حقنها ببكتریا 12الفئران بعد تتم قتل في ) Macrophage( ألعداد بمعاملة السیطرة ، كذلك تم حساب نسبة الخالیا البلعمیة المستعمرات النامیة وقورنت ا . محتویات البریتون ا P. aeruginosaعند تعریضها لبكتریا Lb. gasseri أظهرت النتائج عدم تأثر الفئران المعاملة براشح بكتریا خالف 18دون حقنها بالبكتریا الواقیة وكانت نسبة الخالیا البلعمیة هي من فقط P. aeruginosللمجموعة التي عرضت لبكتریا .Lbفي مجموعة األختبار ، مما یدل على التأثیر الوقائي لبكتریا % 27بینما كانت نسبتها ،في مجموعة السیطرة% gasseri في الحمایة من األصابة ببكتریاP. aeruginosa . IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.23 (1) 2010 The Effect of Lactobacillus gasseri Against Pseudomonas aeruginosa Infection in Mice. M. F. AL- Marjani , R. A.A. Abdul-Jabbar and A.M. Hassan Department of Biology , College of Science, University of Al- Mustansiriya Abstract The effect of local Lactobacillus gasseri filtrate against Pseudomonas aeruginosa infection in mice was studied . 0.25 ml of concentrated filtrate Lactobacillus gasseri was injected in intraperitoneally ( I.P.) 5 days before challenge with 0.2 ml viable P. aeruginosa ( 10 8 cell/ ml). Animals were sacrificed after 12 h. from challenge by cutting the femoral artery . To follow bacterial growth in the peritoneal cavity , its contents were washed out with 5 ml of PBS .The fluid was diluted, 0.1 ml from each dilution and was spread on culture media. The number of colonies in 5 ml of harvested fluid was expressed as Log 10 CFU ,and the percentage of Macrophage in the peritoneal cavity was counted. Growth of P. aeruginosa in the peritoneal cavity was markedly inhibited in Lb. gasseri pretreated mice, wherease such inhibition of bacterial growth was not observed in another group ( mice were not treated with Lb. gasseri ) The percentage of macrophage detectable in the peritoneal cavity was 18% in control and 27% in test . It was suggested that macrophages activated with Lb. gasseri were involved in the protective to P. aeruginosa. Introduction Lactic Acid Bacteria ( LAB) are gram-positive bacteria with cell wall components such as peptidoglycan, polysaccharide and teichoic acid, all of which have showed to own immunostimulatory properties. In addition to cell wall components, immunostimulatory effects were observed with antigens originated from the cytoplasms of some strains of LAB [ 1 ] . Certain specific probiotic strains (for example, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus johnsonii have well defined and proved the clinical effects for the treatment and/or prevention of diseases of intestinal and extraintestinal origin [ 1 ] . And have immunostimulatory properties, including modulation of cytokine production, increased phagocytic activity of polymorphs, adjuvant effects on specific humoral responses, T- lymphocytic function, and NK activity [2][3]. Probiotic bacteria are showed that they would promote the endogenous host defense mechanisms. In addition to the effects of p robiotics on nonimmunologic gut defense, which is characterized by stabilization of the gut microflora , probiotic bacteria have showed their ability to enhance humoral immune responses and thereby promote the intestine's immunologic barrier. Moreover, probiotic bacteria have showed their ability to stimulate nonspecific host resistance to microbial pathogens .Thereby they would have the ability to aid in immune elimination, and to modulate the host 's immune responses to potentially harmful antigens with a potential to down-regulate hypersensitivity reactions [4] [5]. Oral introduction of Lactobacillus casei and Lactobacillus bulgaricus activated the production of macrophages and administration of L. casei and Lactobacillus acidophilus activated phagocytosis in mice , enhanced phagocytosis was also reported in humans by L. acidophilus[2]. De Simone et al [6 ] studied the influence of a yogurt-supplemented diet on IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.23 (1) 2010 the immunocompetence and survival of animals subsequently infected with Salmonella typhimurium. De Simone et al. reported that mice fed live LAB (L. bulgaricus and Streptococcus thermophilus)-containing yogurt for 7 and 14 days had a higher percentage of B lymphocytes than did mice fed a control diet supplemented with a cow milk. In a similar experiment, Puri et al [7]showed that intestinal lymphocytes from mice fed live LAB- containing yogurt had a higher proliferative response to LPS than did mice fed milk after a challenge with S. typhimurium. Although the mechanism(s) of action involved in the inhibition of P. aeruginosa was not investigated in the present study , it is therefore believed that some factors may include bacteriocins,hydrogen peroxide, diacety l and CO2 , or enzymes by the selected Lactic Acid Bacteria [ 8]. Finally , L. casei strain Shirota administration before or after an initial challenge dramatically inhibited E. coli growth in a murine model of urinary tract infection [9]. Most studies investigated the effects of Lactobacillus on pathogenic bacteria in vitro, whereas very few studies have investigated the effects of Lactobacillus in vivo. The aim of this work was to study the effect of Lactobacillus gasseri filtrate on prevention of P. aeruginosa infection in mice. Material and Methods Animals : Balblc- Male mice were obtained from Department of Biology – College of Science - AL- Mustansiriya University . Mice were used in experimentals at 7 to 9 weeks of age , 25-30 gram weight ( Five mice for each group). Bacterial isolates : 1) Lb. gasseri ( maintained from Department of Biology – College of Science - AL- Mustansiriya University ) was cultured on De Man Regosa Sharpe medium ( MRS) at 37 C for 48 hrs ,The Lb. gasseri filtrate was prepare according to [ 9]. 2) A clinical isolate of P. aeruginosa was isolated from wound infection and identified according to [10]. Bacterial infection: In the experiment to test the protective effect of Lb. gasseri filtrate against P. aeruginosa in mice , 0.25 ml of Lb. gasseri filtrate was injected in intraperitoneally ( I.P.) 5 days before challenged with 0.2 ml viable P. aeruginosa ( 10 8 cell/ ml). [11]. Determination of bacterial growth: The challenge dose of bacteria was injected I.P. to control mice and mice that had been treated with Lb. gasseri filtrate 5 days earlier. Twelve hour. after the challenge , animals were sacrificed by cutting the femoral artery . To follow bacterial growth in the peritoneal cavity , its contents were washed out with 5 ml of PBS . The fluid was diluted 10- fold with PBS , 0.1 ml from each dilution was spread on nutrient agar plates ( containing 0.4% glucose ) . The number of colonies in 5 ml of harvested fluid was expressed as Log 10 CFU [ 11] . Counting of WBCs in peritoneal cavity : Smear specimens for differential counts were prepared for Giemsa staining and examined [12]. Results and Disscussion In our study the protective effect of Lb. gasseri filtrate against P. aeruginosa infection in mice was studied . PBS treated control mice and those pretreated with 0.25 ml of Lb. gasseri filtrate 5 days earlier were inoculated I.P. with 0.2 ml viable P. aeruginosa and the growth of the bacteria in the peritoneal cavity was followed . The number of the P. aeruginosa in the peritoneal cavity decreased gradually to about 10 6 CFU by 12 h. after challenge in control mice Fig. (1). In mice pretreated with Lb. gasseri filtrate , however,the bacteria were reduced rapidly to 10 3 CFU in 12 h. . Although the mechanism(s) of action involved in the inhibition of P. aeruginosa was not investigated in the present study , it is therefore believed IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.23 (1) 2010 that some factors may include bacteriocins,hydrogen peroxide, diacety l and CO2 , or enzymes by the selected Lactic Acid Bacteria [ 8]. Differential cell counts of peritoneal leukocytes were studied consecutively after treatment with Lb. gasseri filtrate . The percentage of macrophage detectable in the peritoneal cavity was in control and 27% in test. Macrophages were characteristically increased in Lb. gasseri filtrate treated mice. These findings are in agreement with the previously reported result which showed that the administration of Lactobacillus or yogurt to young mice enhanced lung clearance of P. aeruginosa and phagocytic activity of alveolar macrophages [13]. Villena et al [14] found that pneumococcal colonization of lung and bacteremia were significantly greater in control group mice compared with the Lb. casei pretreated group. Although the number of bacteria in lungs and blood stream tended to decrease (P < 0.05) during infection in Lb. casei pretreated group mice, they suggested that the addition of L. casei to the repletion diet has a beneficial effect because it accelerates the recovery of the innate immune response and improves the specific immune mechanisms against Streptococcus pneumoniae respiratory infection in malnourished mice. A limited number of animal studies were conducted on the effect of LAB on macrophages. Goulet et al [ 15 ] found that phagocytic activity of alveolar macrophages was significantly (P < 0.05) higher in mice fed milk fermented with L. acidophilus and L. casei than in control mice fed ultrahigh-temperature-treated milk. Perdigon et al [2] showed that feeding milk (100 µg protein/d) fermented with L. casei and L. acidophilus, or both for 8 d. increased the in vitro and in vivo phagocytic activity of peritoneal macrophages . Other studies in which reconstituted lyophilized LAB were administered orally or intraperitoneally showed enhancement of macrophage activation by LAB [ 16 ]. These observations reviewed together suggested that specific immunomodulatory properties of probiotic bacteria should be characterized during the development of clinical applications for extended target populations. Further experiments are required to establish the mechanism by which Lb. gasseri filtrate affects P. aeruginosa pathogenicity . In the future , the immunological aspects of the protective role of Lb. gasseri filtrate should be studied . References 1. Meydani, S.N. and Ha, W.K. (2000), Immunologic effects of yogurt , Americ. J. of Clinic. Nutr.71 ( 4): 861-872, 2. Perdigon , G. ; de Macías, ME. ; Alvarez ,S.; Oliver, G . and Holgado ,AP. (1998 ), Systemic augmentation of the immune response in mice by feeding fermented milks with Lactobacillus casei and Lactobacillus acidophilus , Immunology, 63: 17–23,. 3. Anderson,A.D. ; McNaught,C.E. ; Jain, P.K. and MacFie,J.( 2004 ), Randomised clinical trial of synbiotic therapy in elective surgical patients, Gut , 53 : 241-245. 4. Kaila, M.; Isolauri, E.; Soppi, E.; Virtanen, E.; Laine, S. and Arvilommi, H. (1992).Enhancement of the circulating antibody secreting cell response in human diarrhea by a human lactobacillus strain. Pediatr. Res.; 32:141–4 , . 5. Isolauri, E.; and Sütas, Y. ; Kankaanpaa,P. ; Arvilommi,H. and Salminen,S. “ Probiotics: effects on immunity " American J. of Clinical Nutr., 73( 2) : 444S-450s, 2001. 6. De Simone, C. ; Vesely, R . and Negri , R. (1987). Enhancement of immune response of murine Peyer's patches by a diet supplemented with yogurt, Immunopharmacol Immunotoxicol , 9 : 87–100. 7. Puri ,P. ; Mahapatra, SC. ; Bijlani ,RL. ;Prasad ,HK. and Nath, I. (1994).Feed efficiency and splenic lymphocyte proliferation response in yogurt- and milk-fed mice , Int J Food Sci Nutr. , 45 (23) : 1–5. 8. Brashears , M.M. ; Jaroni , D. and Trimble , J. (2003) . Isolation, Selection , and characteriztion of lactic acid bacteria for a competitive exclusion product to reduce shedding of E.coli O157:H7 in cattle .J. Food . Protection ., 66(3) : 355 – 363 . IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.23 (1) 2010 9. Martinez – Gonzalez , B. ; Eriotou , E.; Michopoules , S.; Kolantzopoulos , G. ; Tsakalidou , E. and Mentis , A. In Vitro and In Vivo inhibition of Helicobacter pylori by Lactobacillus casei strain shirota . Appl . Environ . Microbiol ., 70(1) :518 – 526 , 2004. 10. Greenwood, D.; Slack, R.C. and Peutherer, J.F. (2002), M edical Microbiology. (Sixteenth ed.). Churchill Livingston. 11. Miake,S.; Nomoto,K. ;Yokokura,T.; Yoshikai,Y.; Mutai,M. and Nomoto,K. (1985) ,Protective effect of Lactobacillus casei on Pseudomonas aeruginosa infection in mice , Infect Immun. , 48(2): 480–485. 12. John,I;Gallin,M. and Anthony,S. (1982).Advances in host Defence Mechanisms . 1. 13. Alvarez, S.; Herrero,C.; Bru,E. and Perdigon,G. (2001). Effect of Lactobacillus casei and yogurt administration on prevention of Pseudomonas aeruginosa infection in young mice , J.Food.Prot. 64(11) : 1768-74. 14. Villena, J. ; Racedo, S.;Aguero , G. ;Bru , E.; Medina , M. and Alvarez , S. (2005). Lactobacillus casei Improves Resistance to Pneumococcal Respiratory Infection in Malnourished Mice" , J. Nutr. 135 : 1462-1469, 15. Goulet, J. ; Saucier, L. and Moineau, S. (1989).Stimulation of the non-specific immune response of mice by fermented milks. In: National Yogurt Association, ed. Yogurt: nutritional and health properties, McLean, VA: Kirby Lithographics, 187–200. 16. Kitazawa, H.; Mataumura ,K. ;Itoh, T. and Yamauchi ,T. (1992).Interferon induction in murine peritoneal macrophage by stimulation with Lactobacillus acidophilus , Microbiol.Immunol.36,311–5. 0 1 2 3 4 5 6 non treated Lb.pretret عمودي ثالث�ي األبع��اد ١ Fig.(1): The growth of the bacteria in the peritoneal cavity after 12 h. - No. of viable bacteria After 12 h. (log 10)