The effect of Lactobacillus reuteri on the biofilm formation of some bacterial species isolated from children with diarrhea
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Abstract
Biofilm formation is a key factor in bacterial infections and persistence in various settings. This study aimed to assess the antibacterial and antibiofilm properties of both the supernatant and pellet of Lactobacillus reuteri against diarrheagenic bacteria. Seventy-six isolates were obtained from 50 children (aged 3 weeks to 9 years) with diarrhea from hospitals in Erbil/Iraq, including Escherichia coli 37 (48.68%), enterohemorrhagic E.coli 4 (5.26%), Klebsiella pneumonia 16 (21.05%), Enterobacter cloacae 7 (9.21%), Proteus mirabilis 7 (9.21%), Pseudomonas aeruginosa 1 (1.32%), Acinetobacter baumannii 1 (1.32%), Shigella sonnei 1 (1.32%), Klebsiella oxytoca 1 (1.32%), and Enterobacter aerogenes 1 (1.32%). This is the first report of A. baumannii detection in pediatric diarrhea in Iraq. L. reuteri was isolated from a single infant formula milk, which may not represent all formula types. Gram staining showed that all pathogens were Gram-negative, while L. reuteri was Gram-positive. We confirmed identification by culture on MacConkey, Eosin methylene blue, and MacConkey sorbitol agar, followed by the Vitek-2 system and Molecular diagnosis using 16S rRNA sequencing. Biofilm formation was not detected on Congo red agar, but the microtiter plate method confirmed biofilm production. Both supernatant and pellet of L. reuteri significantly inhibited biofilm formation in several diarrheagenic bacteria (p < 0.05), while no significant effect was observed against K. pneumoniae. These findings support the potential use of L. reuteri as an alternative therapy against diarrheagenic bacteria.
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References
1. Thaer Nejres M, Abdulkhaliq Awadh H. Prevalence and Gene Expression Variation of Pathogenic Hemolysin Producing E. coli Isolated From Patients with Urinary Tract Infections and Diarrhea. Tikrit Journal of Pure Science. 2025;30(2):22-33. https://doi.org/10.25130/tjps.v30i2.1739
2. Makki SZ, Fadhil JH, Qader AHA, Naji HR, Mohammed NT, Naji SE. Molecular detection of bacteria causing diarrhea in humans. Research Journal of Biotechnology Vol. 2024;19:11. https://doi.org/10.25303/1911rjbt1470151
3. Safa Ali N, Halah Abdulkhaliq A. A Study of Urinary Tract Infection Prevalence, Antibiotic Resistance, and Biofilm Formation Capability of the Bacterial Causal Agents. Tikrit Journal of Pure Science. 2022;27(6):11-7. https://doi.org/10.25130/tjps.v27i6.765
4. Noor Hussein A, Ghada AM. Evaluation of Some Materials to Inhibit Biofilm Formed by Acinetobacter baumannii Isolates. Tikrit Journal of Pure Science. 2019;24(4):19-24. https://tjpsj.org/index.php/tjps/article/view/391
5. Azagra-Boronat I, Massot-Cladera M, Knipping K, Garssen J, Ben Amor K, Knol J, et al. Strain-Specific Probiotic Properties of Bifidobacteria and Lactobacilli for the Prevention of Diarrhea Caused by Rotavirus in a Preclinical Model. Nutrients. 2020;12(2):498. https://doi.org/10.3390/nu12020498
6. Jasim HM, AL Zubaidy ZM. Antibacterial and Anti-Biofilm Formation Using Lactobacillus plantarum and L. retueri Against MRSA Isolates. Diyala Journal for Pure Science. 2022;18(4). https://doi.org/10.24237/djps.1804.598B
7. Dawoud AM, Salman SS, Sleem AS. Assessment of antibacterial and anti-biofilm activity of probiotic Lactobacillus against biofilm-forming Proteus mirabilis clinical isolates. Microbes and Infectious Diseases. 2023;4(4):1369-82. https://doi.org/10.21608/mid.2023.223993.1567
8. Mu Q, Tavella VJ, Luo XM. Role of Lactobacillus reuteri in human health and diseases. Frontiers in Microbiology. 2018;9:757. https://doi.org/10.3389/fmicb.2018.00757
9. Saviano A, Brigida M, Migneco A, Gunawardena G, Zanza C, Candelli M, et al. Lactobacillus Reuteri DSM 17938 (Limosilactobacillus reuteri) in Diarrhea and Constipation: Two Sides of the Same Coin? Medicina. 2021;57(7):643. https://doi.org/10.3390/medicina57070643
10. Peng Y, Ma Y, Luo Z, Jiang Y, Xu Z, Yu R. Lactobacillus reuteri in digestive system diseases: focus on clinical trials and mechanisms. Frontiers in cellular and infection microbiology. 2023;13:1254198. https://doi.org/10.3389/fcimb.2023.1254198
11. Khan R, Wali S, Khan S, Munir S, Pari B, Yousuf AM, et al. Isolation and characterization of pathogenic Klebsiella pneumoniae strains from lettuce: A potential source of antibiotic resistance and development of a mathematical model for ANOVA results. Frontiers in Microbiology. 2024;15:1473055. https://doi.org/10.3389/fmicb.2024.1473055
12. Garba DE, Dibua UM-E, Attah F, Oka CU, Ozoadibe YO. Isolation and characterization of multidrug-resistant bacterial pathogens from diarrhoeagenic stool in hospitals in Lokoja, Kogi State. 2024. https://doi.org/10.21203/rs.3.rs-4514529/v1
13. Jose NM, Bunt CR, Hussain MA. Comparison of microbiological and probiotic characteristics of lactobacilli isolates from dairy food products and animal rumen contents. Microorganisms. 2015;3(2):198-212. https://doi.org/10.3390/microorganisms3020198
14. AL-Hilali RM, Al-Mozan HDK. Isolation and identification of Gram-negative bacteria that cause diarrhea. University of Thi-Qar Journal of Science. 2023;10(1):175-80. https://doi.org/10.32792/utq/utjsci/v10i1.1059
15. Tola MA, Abera NA, Gebeyehu YM, Dinku SF, Tullu KD. High prevalence of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae fecal carriage among children under five years in Addis Ababa, Ethiopia. PLOS ONE. 2021;16(10):e0258117. https://doi.org/10.1371/journal.pone.0258117
16. Keywan Jebrail C, Sawsan Mohammed S. Effect of Enterococcus avium and Enterococcus casseliflavus supernatant and pellet on Klebsiella pneumoniae biofilm formation. Tikrit Journal of Pure Science. 2023;28(3):10-20. https://doi.org/10.25130/tjps.v28i3.1420
17. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 33rd Edition (M100-Ed33) ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2023.
18. Hassan PA, Khider AK. Correlation of biofilm formation and antibiotic resistance among clinical and soil isolates of Acinetobacter baumannii in Iraq. Acta Microbiologica et Immunologica Hungarica. 2019:1-10. https://doi.org/10.1556/030.66.2019.026
19. Er S. Comparison of the Microtiter Plate Method and the Congo Red Agar Technique in the determination of Staphylococcal Biofilm. Mugla Journal of Science and Technology. 2024;10(1):128-32. https://doi.org/10.22531/muglajsci.1493167
20. Kamali E, Jamali A, Ardebili A, Ezadi F, Mohebbi A. Evaluation of antimicrobial resistance, biofilm-forming potential, and the presence of biofilm-related genes among clinical isolates of Pseudomonas aeruginosa. BMC Research Notes. 2020;13(1):27. https://doi.org/10.1186/s13104-020-4890-z
21. Ali SH, Babakir-Mina M, Soor TAH, Ahmad HA. Molecular Characterization of Escherichia coli Isolated from Children Who Have Acute Diarrhea in Different Iraqi Kurdistan Hospitals. Egyptian Academic Journal of Biological Sciences, E Medical Entomology & Parasitology. 2022;14(2):75-85. https://doi.org/10.21608/eajbse.2022.264062
22. Alzubaidi AFA, Abbas MA. Microbial etiology of acute diarrhea in children under ten years of age in Diyala, Iraq/hospital-based study. Indian Journal of Forensic Medicine & Toxicology. 2020;14(4):1516-22. https://doi.org/10.37506/ijfmt.v14i4.11758
23. Altaie HAA, Gdoura Ben Amor M, Mohammed BA, Gdoura R. Detection and Characterization of Escherichia coli and Escherichia coli O157: H7 in Human, Animal, and Food Samples from Kirkuk Province, Iraq. Microbiology Research. 2025;16(1):20. https://doi.org/10.3390/microbiolres16010020
24. Salihi KA. Isolation and identification of bacterial causes of diarrhea in children less than 2 years old in Al-Muthanna Governorate. Muthanna Medical Journal. 2025;10(1):1-13. https://doi.org/10.52113/1/1/2023-1-13
25. Rashwan RS, Galal SM, Abd EL-kareem DM, Ghandour A, Abd El-Hamid RF. Extended-spectrum beta-lactamase genes among Gram-negative bacilli isolates from Egyptian children with diarrhea. Microbes and Infectious Diseases. 2023;4(4):1312-24. https://doi.org/10.21608/mid.2023.222631.1563
26. Okeke IN, Lamikanra A, Steinrück H, Kaper JB. Characterization of Escherichia coli strains from cases of childhood diarrhea in provincial southwestern Nigeria. Journal of Clinical Microbiology. 2000;38(1):7-12. https://doi.org/10.1128/jcm.38.1.7-12.2000
27. Balázs B, Tóth Z, Nagy JB, Majoros L, Tóth Á, Kardos G. Fecal carriage of carbapenem-resistant Acinetobacter baumannii: comparison to clinical isolates from the same period (2017–2019). Pathogens. 2022;11(9):1003. https://doi.org/10.3390/pathogens11091003
28. Niamah AK, Abdulmuttaleb AM, Hussain NA. Antibacterial spectrum of produced reuterin from new isolates of Lactobacillus reuteri. Journal of microbiology, biotechnology and food sciences. 2020;10(1):134-9. https://doi.org/10.15414/jmbfs.2020.10.1.134-139
29. Ali MS, Lee E-B, Lim S-K, Suk K, Park S-C. Isolation and identification of Limosilactobacillus reuteri PSC102 and evaluation of its potential probiotic, antioxidant, and antibacterial properties. Antioxidants. 2023;12(2):238. https://doi.org/10.3390/antiox12020238
30. Abdullah ZH, Merza NS. Phenotypic and Molecular detection of Acinetobacter baumannii isolated from patients in Duhok city, Iraq. Science Journal of the University of Zakho. 2019;7(4):132-7. https://doi.org/10.25271/sjuoz.2019.7.4.644
31. Cheng J, Cho J-H, Suh J-W. Characterization of Human Breast Milk-Derived Limosilactobacillus reuteri MBHC 10138 with Respect to Purine Degradation, Anti-Biofilm, and Anti-Lipid Accumulation Activities. Antibiotics. 2024;13(10):964. https://doi.org/10.3390/antibiotics13100964
32. Sinkiewicz G, Ljunggren L. Occurrence of Lactobacillus reuteri in human breast milk. Microbial ecology in health and disease. 2008;20(3):122-6. https://doi.org/10.1080/08910600802341007
33. Issa FA. ANTIBIOTIC RESISTANCE PATTERNS OF COMMON UROPATHOGENS ISOLATED FROM FEMALES AT ZAKHO CITY, KURDISTAN REGION, IRAQ. Science Journal of the University of Zakho. 2024;12(4):490-6. https://doi.org/10.25271/sjuoz.2024.12.4.1395
34. Källman O, Fendukly F, Karlsson I, Kronvall G. Contribution of efflux to cefuroxime resistance in clinical isolates of Escherichia coli. Scandinavian journal of infectious diseases. 2003;35(8):464-70. https://doi.org/10.1080/00365540310014639
35. Schumacher H, Skibsted U, Skov R, Scheibel J. Cefuroxime resistance in Escherichia coli: resistance mechanisms and prevalence. Apmis. 1996;104(7‐8):531-8. https://doi.org/10.1111/j.1699-0463.1996.tb04908.x.
36. Wu D, Ding Y, Yao K, Gao W, Wang Y. Antimicrobial resistance analysis of clinical Escherichia coli isolates in neonatal ward. Frontiers in Pediatrics. 2021;9:670470. https://doi.org/10.3389/fped.2021.670470
37. Al-Tuhmazi HA, Al-Hisnawi AA. Determination of the Activity of Antibiotic Resistance in Bacterial Causative Agents of Diarrhea among Children Under the Age of 5 Years. Medical Journal of Babylon. 2024;21(3):527-32. https://doi.org/10.4103/MJBL.MJBL_436_23
38. Hamza OA, Omran R. Multidrug resistance of Escherichia coli and Klebsiella isolated from Iraqi patients and microbiota. Journal of Biosciences and Medicines. 2022;10(11):240-52. https://doi.org/10.4236/jbm.2022.1011019
39. Kiiru S, Maina J, Mwaniki JN, Songoro E, Kariuki S. Enteric bacterial agents associated with diarrhea and their antimicrobial sensitivity profiles in children under 5 years from Mukuru informal settlement, Nairobi, Kenya. BMC Infectious Diseases. 2024;24(1):237. https://doi.org/10.1186/s12879-024-09114-5
40. Halim RMA, Kassem NN, Mahmoud BS. Detection of biofilm-producing staphylococci among different clinical isolates and its relation to methicillin susceptibility. Open access Macedonian journal of medical sciences. 2018;6(8):1335. https://doi.org/10.3889/oamjms.2018.246
41. Jabir DM. Evaluation of the ability of some bacterial species isolated from UTI to form biofilm. Al-Qadisiyah Journal of Pure Science. 2022;27(1):8-14. https://doi.org/10.29350/qjps.2022.27.1.1492
42. Alamri AM, Alsultan AA, Ansari MA, Alnimr AM. Biofilm formation in clonally unrelated multidrug-resistant Acinetobacter baumannii isolates. Pathogens. 2020;9(8):630. https://doi.org/10.3390/pathogens9080630
43. Arciola CR, Campoccia D, Gamberini S, Cervellati M, Donati E, Montanaro L. Detection of slime production by means of an optimized Congo red agar plate test based on a colorimetric scale in Staphylococcus epidermidis clinical isolates genotyped for ica locus. Biomaterials. 2002;23(21):4233-9. https://doi.org/10.1016/S0142-9612(02)00171-0
44. Al-Shamiri MM, Zhang S, Mi P, Liu Y, Xun M, Yang E, et al. Phenotypic and genotypic characteristics of Acinetobacter baumannii and the relationship among antibiotic resistance, biofilm formation and motility. Microbial Pathogenesis. 2021;155:104922. https://doi.org/10.1016/j.micpath.2021.104922
45. Ebrahem AM, Jameel SK, Abbas AH. Investigation of biofilm formation and antibiotic resistance of bacteria isolated from septic neonates. J Med Chem Sci. 2023;6:816-26. https://doi.org/10.26655/JMCHEMSCI.2023.4.13
46. Lysitsas M, Triantafillou E, Chatzipanagiotidou I, Antoniou K, Spyrou V, Billinis C, et al. Phenotypic investigation and detection of biofilm-associated genes in Acinetobacter baumannii isolates obtained from Companion animals. Tropical Medicine and Infectious Disease. 2024;9(5):109. https://doi.org/10.3390/tropicalmed9050109
47. Ballash GA, Mollenkopf DF, Diaz-Campos D, van Balen JC, Cianciolo RE, Wittum TE. Pathogenomics and clinical recurrence influence biofilm capacity of Escherichia coli isolated from canine urinary tract infections. PLOS ONE. 2022;17(8):e0270461. https://doi.org/10.1371/journal.pone.0270461
48. Capita R, Castaño-Arriba A, Rodríguez-Melcón C, Igrejas G, Poeta P, Alonso-Calleja C. Diversity, antibiotic resistance, and biofilm-forming ability of enterobacteria isolated from red meat and poultry preparations. Microorganisms. 2020;8(8):1226. https://doi.org/10.3390/microorganisms8081226
49. Flamerz RA, Obid SS, Jasim WM. Study the Effect of Biofilm Production on Antibiotic Resistance in Proteus mirabilis Isolated from Clinical Samples in Kirkuk City. NTU Journal of Pure Sciences. 2023;2(1). https://doi.org/10.56286/ntujps.v2i1.339
50. Piatek J, Krauss H, Ciechelska-Rybarczyk A, Bernatek M, Wojtyla-Buciora P, Sommermeyer H. In vitro growth inhibition of bacterial pathogens by probiotics and a synbiotic: product composition matters. International journal of environmental research and public health. 2020;17(9):3332. https://doi.org/10.3390/ijerph17093332
51. Mirzaei A, Habibi M, Bouzari S, Asadi Karam MR. Characterization of antibiotic-susceptibility patterns, virulence factor profiles and clonal relatedness in Proteus mirabilis isolates from patients with urinary tract infection in Iran. Infection and Drug Resistance. 2019:3967-79. https://doi.org/10.2147/IDR.S230303
52. Xu Y, Li D, Ding X, Wang Y, Liang S, Xie L, et al. Probiotic characterization and comparison of broiler-derived Lactobacillus strains based on a technique for order preference by similarity to ideal solution analysis. Poultry Science. 2023;102(5):102564. https://doi.org/10.1016/j.psj.2023.102564
53. Singh TP, Kaur G, Malik RK, Schillinger U, Guigas C, Kapila S. Characterization of intestinal Lactobacillus reuteri strains as potential probiotics. Probiotics and antimicrobial proteins. 2012;4(1):47-58. https://doi.org/10.1007/s12602-012-9090-2
54. Al-Shamiri MM, Wang J, Zhang S, Li P, Odhiambo WO, Chen Y, et al. Probiotic Lactobacillus species and their biosurfactants eliminate Acinetobacter baumannii biofilm in various ways. Microbiology Spectrum. 2023;11(2):e04614-22. https://doi.org/10.1128/spectrum.04614-22
55. Al-Dhabi N, Valan Arasu M, Vijayaraghavan P, Esmail G, Duraipandiyan V, Kim Y, et al. Probiotic and antioxidant potential of Lactobacillus reuteri LR12 and Lactobacillus lactis LL10 isolated from pineapple puree and quality analysis of pineapple-flavored goat milk yogurt during storage. Microorganisms 8 (10): 1461. BMC Compliment Altern Med. 2020;16:264. https://doi.org/10.3390/microorganisms8101461
56. Siddique A, Azim S, Ali A, Adnan F, Arif M, Imran M, et al. Lactobacillus reuteri and Enterococcus faecium from poultry gut reduce mucin adhesion and biofilm formation of cephalosporin- and fluoroquinolone-resistant Salmonella enterica. Animals. 2021;11(12):3435. https://doi.org/10.3390/ani11123435
57. Wasfi R, Abd El‐Rahman OA, Zafer MM, Ashour HM. Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries‐inducing Streptococcus mutans. Journal of cellular and molecular medicine. 2018;22(3):1972-83. https://doi.org/10.1111/jcmm.13496.
58. Shaaban M, Abd El-Rahman OA, Al-Qaidi B, Ashour HM. Antimicrobial and antibiofilm activities of probiotic Lactobacilli on antibiotic-resistant Proteus mirabilis. Microorganisms. 2020;8(6):960. https://doi.org/10.3390/microorganisms8060960
59. Sun M-C, Chen Y-X, Fan X-J, Wang Y-D, Zhang T, Zhao C. The anti-biofilm mechanism of reuterin against Staphylococcus aureus and its potential application on milk-contact material surfaces. Food Bioscience. 2025;63:105744. https://doi.org/10.1016/j.fbio.2024.105744