Inhibitory Effect of Breast Milk Against Pediatric Bacterial Infection

Darstan J. Garib Akhter A. Ahmed and Abdulilah S. Ismaeil Department of Biology,College of Science, Salahaddin University- Hawler

:Received :Accepted

Abstract This study presents the inhibitory effect of breast milk and formula milk against some pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus spp and pseudomonas aeruginosa) isolated from infants suffering from different infections. The results showed that the breast milk had antibacterial activity against tested bacteria in different ranges, and pseudomonas aeruginosa was mostly affected followed by Staphylococcus aureus and Escherichia coli(stool), whereas formula milk had no inhibitory effect but enhances the growth of the tested bacteria.

Keywords: Breast milk, Formula milk, Pediatric, Antibacterial effect, Bacterial infection.

Introduction uman milk is the optimum food for the human infant. The intrinsic components of milk or partially digested products of human milk, which have local antipathogenic Heffects that supplement the infant's innate immunity.This includes substances that function as prebiotics such as free fatty acids (FFA), monoglycerides, antimicrobial peptides, and glycans, which bind diarrheal' pathogens [1,2,3]. In addition to these, there are other factors within breast milk that support or act in concert with the infant's innate immune system including bifidus factor, lysozyme, Lactoperoxidase, lactoferrin, lipoprotein lipase and even epidermal growth factor, which may stimulate the maturation of the gastrointestinal epithelium as a barrier[4]. Breast feeding can reduce the incidence and the severity of gastrointestinal and respiratory infections in the suckling neonate by providing additional protective factors to the infant's mucosal surfaces. Human milk provides protection against a abroad array of infectious agents through redundancy. Protective factors in milk can target multiple early steps in pathogen replication and target each step with more than one antimicrobial compound [5,6]. The antimicrobial activity in human milk results from protective factors working not only individually but also additively and synergistically. Pathogens can be inactivated directly by antimicrobial lipids, antimicrobial peptides, antibodies, and lysozymes and can be prevented from binding to cellular receptors and coreceptors by fucosylated oligosaccharides and milk glycoproteins such as lactoferrin and lactoherin [7,8,9]. The protective effect of breast feeding takes a greater magnitude when its implication in reducing morbidity and mortality of infectious diseases, particularly in the third world countries is taken into consideration, diarrhea is one of the most infectious disease causing an estimated five million deaths per year in children under five years of age, 80% of which occur in the first two years of life [10]. Enteropathogenic bacteria have been found to be inhibited by breast milk when tested on in vitro assay systems [11,12,13]. The objective of this study is to demonstrate whether the human breast milk exerts any inhibitory effect on pediatric bacterial pathogens using an in vitro assay system and to compare the antibacterial effect of breast milk with formula milk (bottle milk).

Materials and methods Collection of samples Samples of milk were collected from healthy nursing mothers in Erbil city. All donors were in good health and had no clinical evidence of mastitis nor tuberculosis and with no in take of medications. After hand washing and cleansing of the breast with soap and tap water, milk samples were collected manually into sterile caped tubes and kept in cool condition. The samples were tested for their antimicrobial activity during 24 hours. A commercially powdered infant formula milk (Dielac, Vitnam Dairy Products Joint Stock Company) was used to compare it's antimicrobial effect with human breast milk.

Bacterial isolates The clinical bacterial isolates were collected from infants associated with different bacterial infections at Raparen Pediatric Hospital in Erbil city. The bacterial isolates were: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus spp and pseudomonas aeruginosa . All isolates were identified using the following testes: coagulase test, mannitol fermentation, hemolysis on blood agar, Indol production, colony characteristic on Eosine methylene blue, citrate utilization, urease production, oxidase test and triple sugar agar test [14]. . Antibacterial activity test For the in vitro assay, identified baeterial isolates were activated in 5 ml of nutrient broth (oxoid) and incubated for 18 hours at 37oC. using a calibrated wire loop (0.01 ml) two loopfuls from each bacterial culture was added to (1ml) of human milk or infant formula. Tenfold dilutions from both the milk and formula were made and ( 0.01 ml )of each dilution was plated in triplicate onto appropriate plates for growth of the test organisms,( Mannitol salt agar for Staph aureus and MacConky agar for gram negative bacteria) at 0 and after 3 hours of incubation at 37oC. All plates were incubated at 37oC for 24 hours. The incubation period of 3 hours was chosen to approximate physiologic conditions in the feeding infant. After incubation period, the colony count of each pathogen at the 0 time and 3 hours was determined by colony counting method [15].

Result and Discussion Table(1) shows the mean colony count at 0 and 3 hours of the different clinical bacterial isolates in breast milk and formula milk. It is obvious that there are significant increases in 3 hours colony count of the formula milk in all tested bacteria, which are also illustrated in figures(1-6), from the same table it can be observed that the number of colonies decreased in most of tested bacteria when incubated with breast milk, both gram-positive and gram- negative bacteria were inhibited in various degrees. Statistical analysis (Duncan test) shows significant decrease in the growth of Stahylococcus aureus figure(1). It has been demonstrated by that there is a thermostable anti-staphylococcal factor in human milk, this factor appears to be pre-antibiotic which has a therapeutic effect upon staphylococcal infection [16]. Isolates of Escherieshia coli figure(2) also exhibit a significant decrease in growth with breast milk. The breast milk inhibited the growth of enteric pathogens proving the protective role of human breast milk against gastrointestinal infections which is confirmed by WHO reports of reduced incidence and severity of diarrheal diseases among breast feeding infants in the first six months of life unlike artificially feeding infants [17]. Oligosaccharides either free or conjugated with proteins or lipids, may directly inhibit the adhesion of diarrheal pathogens and protect the infant against infectious diarrhea, or indirectly produce a protective and immunomodulatory result through a prebiotic effect on the infant intestinal microflora [18, 10, 20]. Breast milk was also effective against Pseudomonas aeroginosa isolated from eye swab showing a significant decrease in the number of colonies as shown in figure(3), there is evidence that breast milk could help to care conjunctivitis or can potentially combat some of the bacteria known to cause neonatal eye infection. Many cultures have considered that human milk has special medicinal and nutritional properties as for treating the pink eye or conjunctivitis [21,22]. Slight decrease was observed in the growth of urinary isolates of Escherieshia coli in the breast milk, reports have been referred to the protective role of the breast milk against urinary tract infections (UTI) [23,24]. There was a slight increase in the number of Klebsiella pneumoniae and Proteus spp. colonies, this may be attributed to the presence of polysaccharide capsule which acts as a protective barrier in Klebsiella pneumoniae. Recently it has been suggested that the immunological factors of breast milk may contribute to one of the most important and peculiar characteristics of human milk, that is the dynamic variability of its nutritive, bioactive and functional components[23]. Colostrums of human milk contain an abundant amount of IgA [25]. Which are produced by mother when microbes, food and other antigenic mater passing through the gut, IgA prevents the attachment of bacteria and viruses to the mucosal epithelial cells that would potentially cause infection[26]. In addition lactoferrin accounts as an important compound of breast milk being the main protein in human milk, it acts as a microbicidal agent killing bacteria and viruses therefore add to the protective factor of breast milk[17]. In general there are two mechanisms proposed to explain the inhibitory effect of human breast milk in which specific components may protect the infant from infection. One is the interaction between specific constitute in milk with epithelial surfaces or with specific substances in the gastrointestinal lumen during digestion of milk while the second proposed mechanism is the possible modulation of infants immune system by protective factors in the milk which results in selective production of immune factors in the infant [16,27].

. References 1. Salminen SJ, Gueimonde M, Isolauri E.(2005) Probiotics that modify disease risk. J Nutr ; 135(5):1294-8. 2. Isaacs CE.(2005) Human milk inactivates pathogens individually, additively, and synergistically. J Nutr ;135(5):1286-8. 3. Phadke SM, Deslouches TA. Antimicrobial peptides in mitigating infection.(2005)J Nutr;135(5):1289-93. 4. Morrow AL, Ruiz-Palacios GM, Jiang X, Newburg DS.(2005) Human-milk glycans that inhibit pathogen binding protect breast-feeding infants against infectious diarrhea. J Nutr ; 135(5):1304-7. 5. Howie, P. W., Forsyth, J. S., Ogston, S. A., Clark, A. & Florey, C du V. (1990) Protective effect of breast feeding against infection. Br. Med. J. 300:11-16. 6. Cunningham, A. S., Jelliffe, D. B. & Jelliffe, E.F.P. (1991) Breast-feeding and health in the 1980s: Aglobal epidemiologic review. J. Pediatr. 118:659-666. 7. Crane, J. K., Azar, S. S., Stam, A. & Newburg, D. S. (1994) Oligosaccharides from human milk block binding and activity of the Escherichia coli heat-stable enterotoxin (sta) in T84 intestinal cells. J. Nutr. 124:2358-2364 . 8. Nozaki, A., Ikeda, M., Naganuma, A., Nakamura, T., Inudoh, M., Tanaka, K. & Kato, N. (2003) Identification of a lactoferrin-derived peptide possessing binding activity to hepatitis C virus E2 envelope protein. J. Biol. Chem. 278:10162-10173 . 9. Newburg, D. S., Peterson, J. A., Ruiz-Palacios, G. M., Matson, D. O., Morrow, A. L., Shults, J., Guerrero, M. L., Chaturvedi, P. & Newburg, S. O. (1998) Role of human-milk lactadherin in protection against symptomatic rotavirus infection. Lancet 351:1160-1164 10. Synder JD, Metson MH. (1982)The magnitude of the global problem of acute diarrheal disease: a review of active surveillance data. Bull WHO ; 60:605-613. 11. Dolan S, Finkelstein M, Finkelstein R.(1986) Antimicrobial activity of human milk against pediatric pathogens. J Infect Dis ; 154(4):722-725. 12. Bjorck L, et al.(1975) Antibacterial activity of the lactoperoxidase system in milk against pseudomonads and other gram negative bacteria. Applied Microbiol ; 30(2):199-204. 13. Dolby J, et al.(1977) Bacteriostasis of E. coli by milk: Effect of bicarbonate and transferrin on the activity of infant feeds. J Hygiene ; 78:235-242. 14. Forbes, Betty A., Danniel F. Sahm and Alice S. weissfeld (2007) Bailey and Scott's Diagnostic Microbiology 12th ed. Mosby Elsevier, ch16,22,24,pp.254,323,340. 15. Esperanza F. Rivera, M.D. and Ricarchito B. Manera, M.D.(1989) Antimicrobial activity of breastmilk against common pediatric pathogens. Phil J Microbiol Infect Dis ; 18(2):67-74. 16. Rivera, E. F. and Manera, R. B. (1989) Antimicrobial activity of breastmilk against common pediatric pathogens. Phil J Microbiol Infect Dis ; 18(2):67-74. 17. Chantry, C. J. , Howard, C. R. and Auinger, P. Full breastfeeding duration and associated decrease in respiratory tract infection in US children. (2006)Pediatrics. 117:425-32. 18. Coppa, G. V. , Zampini, L. , Galeazzi, T. , Facinelli, B. , Ferrante, L. , Capretti, R. and Orazio, G. (2006) Human milk oligosaccharides inhibit the adhesion to Caco-2 cells of diarrheal pathogens: Escherichia coli, Vibrio cholerae and Salmonella fyris. Pediatr Res.; 59:377-82. 19. Arifeen, S. , Black, R. E. , Antelman, G. , Baqui, A. , Caulfield, L. and Becker, S. (2001) Exclusive breastfeeding reduces acute respiratory infections and diarrhea deaths among infants in Dhaka slums. Pediatrics ; 108: 67. 20. Boehm, G. , Jelinek, J. , Knol, J. , M,Rabet, L. Stahl, B. , Vos, P. and Garseen, J. (2004) Prebiotic and immune responses. J. Pediatr Gastroenterol Nutr. ; 39:S772-3. 21. Singn, M. , Sugathan, P. S. and Buhujwala, R. A. (1982) Human colostrums for prophylaxis against sticky eyes and conjunctivitis in the newborn. J. tropical pediatrics;28: 22.J Trop Pediatr. 2007 Feb;53(1):68-9. 23. Lawrence, R. M. and Pane, C. A. (2007) Human breast milk: current concepts of immunology and infectious disease. Curr Probl Pediatr Adolesc Health Care ; 37:7-37. 24. Hanson, L. A. (2004) Protective effects of breastfeeding against urinary tract infection. Acta paediatr ; 93:154-6. 25. Islam, S. K. , Ahmed, L. , Khan, N. I. , Huque, S. , Begum, A. and Yunus, A. B. (2006) Immune components (IgA, IgM, IgG, immune cells) of colostrums of Bangladeshi mothers. Pediatr int. ; 48:543-8. 26. Hanson, L. A. and Korotkova, M. (2002) The role of breastfeeding in prevention of neonatal infection. Sem neonatal ; 7:275-81. 27. Hanson, L. A. , Korotkova, M. , Haversen, L. Mattsby-Baltzer, I. , Hahn-Zoric, M. and Silfverdal, S. (2002) Breastfeeding, a complex support system for offspring. Pediatr Int. ; 44:347-52.

كاريطةرى دذةبةكتيرى شيرى دايك لة سةر بةكتيرياي هةوكردن لة َمندال ثوختة ىىى ىىىىىىىىىىىىى ىىىىىىىى ىىى ىىىىىىىى ىىىى ىىىى ى ىىىى ىىىىىىى (ىىىى) ىىى ىىى ىىىىىىىىىىىى ىى ىىىىى ىىى ىىىىىىى ىى ىىىىىى ىىىىىىى ىىىىىىى ىىىىى ىىىى: (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus spp and pseudomonas aeruginosa) ىى ىىىىىىىى ىىىىىىى ىى ىىىى ىىىى ىىىىىىىىىىى ىىى ى ىىىى ىىىىىىىى ىىىى ىىىىى ىىىىىىىىىى ىى ىىىىىىى ىىىىىىى ىى ىىىىىىى ىىىىىىىى ىىىىىىى ىىىىى pseudomonas aeruginosa ىىىىى Staphylococcus aureus ى ىىىىى Escherichia coli(stool) ىى ىىىىىىى ىىىىىىى ىىىى ىىىى ىىىى ىىىىىىىى ىىى ىىىىىىىى ىى ىى ىىىىى ىىىىىىى ىى ىىىىى ىىىىىىىىىى.

التاثير المثبط لحليب الم على البكتريا المسببة لللتهابات عند الطفال الخلصة تت مقارنة الفعالية الضادة لكل من حليب الم و الليب الصناعى ضد مموعة من العزلت البكتيية السببة لللتهابات الختلفة ( Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus spp and pseudomonas aeruginosa) ف الطفال الرضع . بينت النتائج ان لليب الم تأثي مضاد على البكتييا الستخدمة ف الدراسة بنسب متفاوتة وكانت لة فعالية تثبيط كبية ضد كل من العزلت البكتيية (Staphylococcus aureus, Escherichia coli (stool) and pseudomonas aeruginosa ) بينما ل يظهر الليب الصناعى اى تأثيتثبيطي على العزلت الختلفة بل على العكس ساعدت على زيادة نوها جيعا.

Table(1):Mean colony count at 0 & 3 hrs. incubation of different bacterial isolates in formula & breast milk Mean±Std. Error of Mean±Std Error Statistical Bacteria Type of milk bacteria before of bacteria after 3 hours decision incubation * 105 incubation * 105 Formula milk 9.333±2.289 52.45±7.06 P< 0.001 Staphylococcus aureus Breast milk 4.633±0.687 2.783±0.516 P< 0.034 Formula milk 10.35±1.713 54.083±5.995 P< 0.0001 E. coli (stool) Breast milk 7.366±1.803 5.366±1.362 P< 0.05 Formula milk 7.066±1.565 53.533±8.840 P< 0.01 E. coli (urine) Breast milk 5.833±0.574 5.5±0.435 N.S.* Formula milk 15.3±1.876 80.95±3.841 P< 0.0001 Proteus sp. Breast milk 9.316±0.611 15.916±4.789 P< 0.253 Formula milk 15.183±2.733 96.533±6.1 P< 0.0001 Pseudomonas aeruginosa Breast milk 24.5±6.43 3.1±0.994 P< 0.018 Formula milk 7.35±0.822 68.766±12.348 P< 0.05 Klebsiella pneumoniae Breast milk 7.933±0.78 9.85±2.687 N.S.*

* N.S. = Non Significant 430.0P > 100.0P > 6 07 5 06 1 1

* 05 * 0 0

4 0 0 0 0 0 0 04 0 3 0 03 2 02 1 01

0 N u noitabucnIN .rh 0 noitabucnI .srh 3

noitabucnI .rh 0 noitabucnI .srh 3 u m b m e b r

Formula milk o Breast milk b f

a c b t a e c r i t a e

Figure(1): Effect of formula & breast milk on ther growth of Staphylococcus aureus i a

P 50.0> 1000.0P > 01 07 9 06 8 1 1

* 05 0 * 0 7

0 0 0 0

0 04 0 0 5 03 4 3 02 2 01 1 0 0

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o Formula milk o Breast milk f f

b b a a c c t t e (Figure(2): Effect of formula & breast milk on the growthe of E. coli (stool r r i i a a

810.0P > 1000.0P > 53 021 03 001 1 1 * 0

* 52 0

0 08 0 0 0 0 0 02 0 0 06 51 04 01 5 02 0 0

N noitabucnIN .rh 0 noitabucnI .srh 3 noitabucnI .rh 0 noitabucnI .srh 3 u u m m b b e e r r

o Formula milk o Breast milk f f

b b a a c c t t e e r r i i a a

Figure(3): Effect of formula & breast milk on the growth of Pseudomonas aeruginos

60 P< 0.01 0 0 0 0

0 50 1

i 40 r e t c a 30 b

e 20 b m u

0 Before incubation After incubation

Figure(4): Effect of formula milk on the growth of E. coli (urine)

90 80 0 0 0

0 P< 0.0001 1

50 a i r e t

c 40 a b

f 30 o

b 20 m u

N 10 0 Before incubation After incubation

Figure(5): Effect of formula milk on the growth of Proteus sp.

90 P< 0.05

0 80 0 0 0

0 70 1

50 a i r e

t 40 c a b

f 30 o

e 20 b m

u 10 N

0 Before incubation After incubation Figure(6): Effect of formula milk on the growth of Klebsiella sp.