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. aمل خالیا حیة لبكتریا 0.25ام ، بعدها حقنت بخمسة أی eruginosa )10 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 P . aساعة من حقنها ببكتریا 12الفئران بعد تتم قتل eruginosa ،د حسبت أعدا .النامیة 5تم حقن اذ M( ألعداد بمعاملة السیطرة ، كذلك تم حساب نسبة الخالیا البلعمیة المستعمرات النامیة وقورنت ا acrop hage ( في . 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. S CI 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 Departme nt of Biology , College of Science, Unive rsity of Al- Mustansiriya Abstract The effect of local Lactobacillus gasseri filtrate against Pseudomonas aeruginosa infection in mice was st udied . 0.25 ml of concentrated filtrate Lactobacillus gasseri was injected in intrap eritoneally ( I.P.) 5 day s before challen ge with 0.2 ml viab le P. aeruginosa ( 10 8 cell/ ml). Animals were sacrificed after 12 h. from challen ge by cutt ing the femor al artery . To follow bacterial growt h in the p eritoneal cav ity , its contents were washed out with 5 ml of PBS .The fluid was diluted, 0.1 ml from each dilution and was sp read on culture media. The number of colonies in 5 ml of harvested fluid was exp ressed as Log 10 CFU ,and the p ercentage of M acrop hage in the peritoneal cavity was counted. Growth of P. aeruginosa in the p eritoneal cavity was markedly inhib ited in Lb. gasseri p retreated mice, wherease such inhibition of bacterial growt h was not observed in another group ( mice wer e not t reated with Lb. gasseri ) The p ercentage of macrophage detectable in the p eritoneal cavity was 18% in control and 27% in test . It was suggested that macrophages activated with Lb. gasseri were involved in the p rotective to P. aeruginosa. Introduction Lactic Acid Bacteria ( LAB) are gram-p ositive bacteria with cell wall components such as p ep tidoglycan, p olysaccharide and teichoic acid, all of whi ch have showed to own immunostimulatory p rop erties. In addition to cell wall comp onents, immunostimulatory effects were observed with antigens originated from the cy top lasms of some strains of LAB [ 1 ] . Certain sp ecific p robiotic strains (for examp le, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus johnsoni i h ave we ll d efined and proved the clinical effects for the treatment and/or p revention of diseases of intestinal and extraintestinal origin [ 1 ] . And have immunostimulatory p rop erties, including modulation of cytokine p roduction, increased p hago cytic activity of p oly morp hs, adjuvant effects on sp ecific humoral resp onses, T- ly mphocytic function, and NK activity [2][3]. Probiotic bacteria are showed that they would p romote the endogenous host defense mechan isms. In addition to the effects of p robiotics on nonimmunolo gic gut defense, which is characterized by st abilization of the gut microflora , p robiotic bacteria have showed their ability to enhance hu moral immune resp onses and thereby p romote the intest ine's immunolo gic barrier. M oreover, p robiotic bacteria have showed their ability to st imulate nonsp ecific host resistance to microbial p athogens .Thereby they would have the ability to aid in immune elimination, and to modulate the host 's immune resp onses to p otentially harmful antigens with a potential to down-regulate hypersensitivity reactions [4] [5]. Oral introduction of Lactobacillus casei and Lactobacillus bulgaricus activated the p roduction of macrophages and administration of L. casei and Lactobacillus acidophilus activated p hagocytosis in mice , enhanced p hagocytosis was also rep orted in humans by L. acidophilus[2]. De Simon e et a l [6 ] st udied the influence of a y ogurt-supp lemented diet on IBN AL- HAITHAM J. FOR PURE & APPL. S CI 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)-containin g y ogurt for 7 and 14 day s had a higher percenta ge of B lymphocytes than did mice fed a control diet supp lemented with a cow milk. In a similar exp eriment, Puri et al [7]showed that intestinal lymp hocy tes from mice fed live LAB- containin g y ogurt had a higher p roliferative r esp onse to LPS than did mice fed milk after a challen ge with S. typhimurium. Although the mechanism(s) of action involv ed in the inhib ition of P. aeruginosa was not invest igated in the p resent study , it is therefore believ ed that some factors may include bacteriocins,hy drogen p eroxide, diacety l and CO2 , or enzymes by the selected Lactic Acid Bacteria [ 8]. Finally , L. casei st rain Shirota administration before or after an initial challen ge dramatically inhibited E. co li growt h in a murin e model of urinary tract infection [9]. M ost st udies invest igated the effects of Lactobacillus on p athogenic bacteria in vitro, whereas very few studies have invest igated the effects of Lactobacillus in vivo. The aim of this work was to st udy the effect of Lac tobacillus gasseri f iltrate on p revention of P. aeruginosa infection in mice. Material and Methods Ani mals : Balblc- M ale mice were obtained from Dep artment of Biology – College of Science - AL- M ust ansiriy a University . M ice were used in exp erimentals at 7 to 9 weeks of age , 25-30 gram weight ( Five mice for each group ). Bacterial isolates : 1) Lb. gasseri ( maintained from Dep artment of Biology – College of Science - AL- M ust ansiriy a University ) was cultured on De M an Regosa Sh arpe medium ( M RS) at 37 C for 48 hrs ,T he Lb. gasseri filtrate was p repare accordin g to [ 9]. 2) A clinical isolate of P. aeruginosa was isolated from wound infection and identified accordin g to [10]. Bacterial infection: In the exp eriment t o test the p rotective effect of Lb. gasseri filtrate against P. aeruginosa in mice , 0.25 ml of Lb. gasseri f iltrate was injected in intrap eritoneally ( I.P.) 5 days before challen ged with 0.2 ml viable P. aeruginosa ( 10 8 cell/ ml). [11]. De termination of bacterial growth: The challen ge 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 challen ge , an imals were sacrificed by cutting the femoral artery . To follow bacterial growt h 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 sp read on nutrient agar p lates ( containin g 0.4% glucose ) . The number of colon ies in 5 ml of harvested fluid was exp ressed as Log 10 CFU [ 11] . Counting of WBCs in peritoneal cavity : Smear sp ecimens for differential counts were p rep ared for Giemsa st aining and examined [12]. Results and Disscussion In our st udy the protective effect of Lb. gasseri filtrate against P. aeruginosa infection in mice was st udied . PB S treated control mice and those p retreated with 0.25 ml of Lb. gasseri filtrate 5 days earlier wer e ino culated I.P. with 0.2 ml viab le P. aeruginosa and the growt h of the bacteria in the p eritoneal cav ity was followed . The number of the P. aeruginosa in the p eritoneal cavity decreased gr adually to about 10 6 CFU by 12 h. after challenge in control mice Fi g. (1). In mice p retreated with Lb. gasseri filtrate , however,the bacteria wer e reduced rapidly to 10 3 CFU in 12 h. . Although the mechanism(s) of action involved in the inhibition of P. aeruginosa was not invest igated in the p resent study , it is therefore believed IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 that some factors may include bacteriocins,hy drogen p eroxide, diacety l and CO2 , or enzy mes by the selected Lactic Ac id Bacteria [ 8]. Differential cell counts of p eritoneal leuko cytes were st udied consecutively after treatment with Lb. gasseri filtrate . The p ercentage of macrop hage detectable in the p eritoneal cavity was in control and 27% in test. M acrop hages were char acterist ically increased in Lb. gasseri f iltrate treated mice. These findings are in agreement with the p reviously rep orted result which showed that the administration of Lactobacillus or y ogurt to y oung mice enhanced lung clearan ce of P. a eruginosa and p hagocytic activity of alveolar macrophages [13]. Villena et al [14] found that p neumococcal colonization of lung and bacteremia wer e significantly gr eater in control group mice compared with the Lb. casei p retreated group . Although the number of bacteria in lungs and blood st ream tended to decrease (P < 0.05) during infection in Lb. casei p retreated group mice, they suggested that the addition of L. casei to the rep letion diet has a beneficial effect because it accelerates the recovery of the innate immun e resp onse and improves the sp ecific immun e mechanisms against Streptococcus pneumoniae resp iratory infection in malnourished mice. A limited number of animal st udies were conducted on the effect of LAB on macrophages. Goulet et al [ 15 ] found that p hagocytic activity of alveolar macrophages was significantly (P < 0.05) higher in mice f ed milk fermented with L. acidophilus and L. casei than in control mice fed ultrahigh-temp erature-treated milk. Perdigon et a l [2] showed that feeding milk (100 µg p rotein/d) fermented with L. casei and L. ac idophilus, or both for 8 d. increased the in vitro and in vivo p hagocytic activity of p eritoneal macrophages . Ot her st udies in which reconst itut ed lyop hilized LAB were administered or ally or intrap eritoneally showed enhancement of macrop hage activation by LAB [ 16 ]. These observations reviewed together suggest ed that sp ecific immunomodulatory p rop erties of p robiotic bacteria should be characterized during the development of clinical applications for extended target p op ulations. Further exp eriments ar e requ ired to establish the mechan ism by which Lb. gasseri filtrate affects P. aeruginosa p athogenicity . In the future , the immunological asp ects of the protective role of Lb. gasseri filtrate should be studied . Re ferences 1. M eydani, S.N. and Ha, W.K. (2000), Immunologic effects of y ogurt , Americ. J. of Clinic. Nut r.71 ( 4): 861-872, 2. Perdigon , G. ; de M acías, M E. ; Alvarez ,S.; Oliver, G . and Holgado ,AP. (1998 ), Sy st emic augmentation of the immune resp onse in mice by feeding f ermented milks with Lactobacillus case i and Lactobacillus acidophilus , Immunolo gy , 63: 17–23,. 3. Anderson,A.D. ; M cNaught,C.E. ; Jain, P.K. and M acFie,J.( 2004 ), Randomised clinical trial of sy nbiotic therapy in elective surgical patients, Gut , 53 : 241-245. 4. Kaila, M .; Isolauri, E.; Sop p i, E.; Virtanen, E.; Laine, S. and Arvi lommi, H. (1992).Enhancement of the cir culatin g antibody secreting cell resp onse in human d iarrhea by a human lactobacillus strain. Pediatr. Res.; 32:141–4 , . 5. Isolauri, E.; and Sütas, Y. ; Kankaanp aa,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 Ne gri , R. (1987). Enhan cement of immune r esp onse of murine Pey er's p atches by a diet supp lemented with y ogurt, Immunop harmacol Immunotoxicol , 9 : 87–100. 7. 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Yo gurt: nutritional and health p rop erties, M cLean, VA: Kirby Lithogr aphics, 187–200. 16. Kitazawa, H.; M ataumura ,K. ;Itoh, T. and Yamauchi ,T. (1992).Interferon induction in murine p eritoneal macrophage by stimulation with Lactobacillus acidophilus , M icrobiol.Immunol.36,311–5. 0 1 2 3 4 5 6 non tre ated 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 bacte ria Afte r 12 h. (log 10)