Combined effects of antibiotics and acidified nitrite on biofilm formation by beta-lactamase-producing uropathogenic bacteria


  • Hozan Yousif Hassan Department of Pharmacognosy, College of Pharmacy, Hawler Medical University, Erbil, Iraq.
  • Safaa Toma Hanna Aka Department of Pharmacognosy, College of Pharmacy, Hawler Medical University, Erbil, Iraq.
  • Aras Najmaddin Hamad Department of Pharmaceutical Chemistry, College of Pharmacy, Hawler Medical University, Erbil, Iraq.
  • Salah Tofik Jalal Balaky Department of Medical Microbiology, College of Health Sciences, Hawler Medical University, Erbil, Iraq.



Beta - lactumase, Acidified nitrite, Biofilm, UTI


Background and objective: The increased prevalence of extended- spectrum beta-lactamase producing Enterobacteriaceae has increased the use of last-resort antimicrobial drugs like Carbapenems. An alternative idea is to use new combinations of common antibiotics. The aim of the present study is to examine whether acidified nitrite has the ability to enhance the activity of beta-lactam antibiotics against the biofilm formation and bacterial growth of beta-lactamase- producing uropathogenic isolates.

Methods: In this cross-sectional study, a total of 37 beta-lactamase- producing uropathogens were collected from patients at Urology Department at Rizgary Teaching Hospital in Erbil/Iraq. Biofilm formation was determined using a 96-well tissue culture plate assay. The ability to produce beta-lactamase production was detected by a phenotypic confirmatory combination disk diffusion test. The sub-minimal inhibitory concentration of antibiotics alone and in combination with each of acidified nitrite, ascorbic acid and sodium nitrite towards biofilm formation were observed.

Results: The combination of cefotaxime with each of acidified nitrite (P <0.001), ascorbic acid (P <0.001) and sodium nitrite (P = 0.003) significantly enhanced the effect of cefotaxime against the biofilm producing activity of beta-lactamase producing uropathogens. Furthermore, with ceftazidime identical synergistic results were obtained with ascorbic acid (P = 0.001) and acidified nitrite (P = 0.007).

Conclusion: Acidified nitrite significantly improved the activity of beta-lactam antibiotics against the biofilm mass of beta-lactamase producing uropathogens.


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Foxman B. The epidemiology of urinary tract infection. Nat Rev Urol. 2010; 7(12):653.

Shakya P, Shrestha D, Maharjan E, Sharma VK, Paudyal R. ESBL Production among E. coli and Klebsiella spp. causing urinary tract infection: A Hospital Based Study. Open Microbiol J. 2017; 11:23.

Svensson L, Poljakovic M, Demirel I, Sahlberg C, Persson K. Host-Derived Nitric Oxide and Its Antibacterial Effects in the Urinary Tract. Adv Microb Physiol. 2018; 73:1–62.

Di Domenico EG, Toma L, Provot C, Ascenzioni F, Sperduti I, Prignano G, et al. Development of an in vitro assay, based on the biofilm ring test, for rapid profiling of biofilm-growing bacteria. Front Microbiol. 2016; 7:1429.

Fux CA, Costerton JW, Stewart PS, Stoodley P. Survival strategies of infectious biofilms. Trends Microbiol. 2005; 13(1):34–40.

Poovendran P, Vidhya N, Murugan S. Antimicrobial activity of Coccinia grandis against biofilm and ESBL producing uropathogenic E. coli. Glob J Pharmacol. 2011; 5(1):23–6.

Tezel BU, Akçelik N, Yüksel FN, Karatuğ NT, Akçelik M. Effects of sub-MIC antibiotic concentrations on biofilm production of Salmonella Infantis. Biotechnol Biotechnol Equip. 2016; 30(6):1184–91. 13102818.2016.1224981

Allan RN, Morgan S, Brito-Mutunayagam S, Skipp P, Feelisch M, Hayes SM, et al. Low concentrations of nitric oxide modulate Streptococcus pneumoniae biofilm metabolism and antibiotic tolerance. Antimicrob Agents Chemother. 2016; 60(4):2456–66. 10.1128/AAC.02432-15

Lundberg JO, Weitzberg E, Cole JA, Benjamin N. Nitrate, bacteria and human health. Nat Rev Microbiol. 2004; 2(7):593.

Bang CS, Kinnunen A, Karlsson M, Önnberg A, Söderquist B, Persson K. The antibacterial effect of nitric oxide against ESBL-producing uropathogenic E. coli is improved by combination with miconazole and polymyxin B nonapeptide. BMC Microbiol. 2014; 14(1):65.

Zemke AC, Shiva S, Burns JL, Moskowitz SM, Pilewski JM, Gladwin MT, et al. Nitrite modulates bacterial antibiotic susceptibility and biofilm formation in association with airway epithelial cells. Free Radic Biol Med. 2014; 77:307–16. 011

Namivandi-Zangeneh R, Sadrearhami Z, Bagheri A, Sauvage-Nguyen M, Ho KKK, Kumar N, et al. Nitric Oxide-Loaded Antimicrobial Polymer for the Synergistic Eradication of Bacterial Biofilm. ACS Macro Lett. 2018; 7(5):592–7.

Waheda N, Al-Bazzaz PH, Mansoor EY, Toma S. The antibacterial effect of acidified nitrite on uropathogenic Escherichia coli: In vitro study. Zanco J Med Sci. 2010; 14(1).

Ormerod AD, Shah AA, Li H, Benjamin NB, Ferguson GP, Leifert C. An observational prospective study of topical acidified nitrite for killing methicillin-resistant Staphylococcus aureus (MRSA) in contaminated wounds. BMC Res Notes. 2011; 4(1):458.

Jardeleza C, Foreman A, Baker L, Paramasivan S, Field J, Tan LW, et al. The effects of nitric oxide on Staphylococcus aureus biofilm growth and its implications in chronic rhinosinusitis. In: International forum of allergy & rhinology. Wiley Online Library; 2011. P. 438–44.

Arora DP, Hossain S, Xu Y, Boon EM. Nitric oxide regulation of bacterial biofilms. Biochemistry. 2015; 54(24):3717–28. https://doi. org/10.1021/bi501476n

Kishikawa H, Ebberyd A, Römling U, Brauner A, Lüthje P, Lundberg JO, et al. Control of pathogen growth and biofilm formation using a urinary catheter that releases antimicrobial nitrogen oxides. Free Radic Biol Med. 2013; 65:1257–64.

Regev-Shoshani G, Ko M, Miller C, Av-Gay Y. Slow release of nitric oxide from charged catheters and its effect on biofilm formation by Escherichia coli. Antimicrob Agents Chemother. 2010; 54(1):273–9. 00511-09

Greenberg D, Mizrahi M, Av-Gay Y, Margel D. Nitric Oxide Charged Catheters as a Potential Strategy for Prevention of Hospital Acquired Infections. In: Open Forum Infectious Diseases. Oxford University Press. 2016. 10.1093/ofid/ofw172.256

Margel D, Mizrahi M, Regev-Shoshani G, Mary KO, Moshe M, Ozalvo R, et al. Nitric oxide charged catheters as a potential strategy for prevention of hospital acquired infections. PloS One. 2017; 12(4):e0174443. 10.1371/journal.pone.0174443

Barraud N, J Kelso M, A Rice S, Kjelleberg S. Nitric oxide: a key mediator of biofilm dispersal with applications in infectious diseases. Curr Pharm Des. 2015; 21(1):31–42.

Sadeghi M, Sedigh Ebrahim-Saraie H, Mojtahedi A. Prevalence of ESBL and AmpC genes in E. coli isolates from urinary tract infections in the north of Iran. New Microbes New Infect. 2021; 45:100947.

Haji SH, Jalal ST, Omer SA, Mawlood AH. Molecular detection of SHV-Type ESBL in E. coli and K.pneumoniae and their antimicrobial resistance profile. Zanco J Med Sci. 2018; 22(2):262–72.

Stepanović S, Vuković D, Dakić I, Savić B, Švabić-Vlahović M. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods. 2000; 40(2):175–9.

Cursino L, Chartone-Souza E, Nascimento AMA. Synergic interaction between ascorbic acid and antibiotics against Pseudomonas aeruginosa. Braz Arch Biol Technol. 2005; 48(3):379–84. 07

Dong L, Tong Z, Linghu D, Lin Y, Tao R, Liu J, et al. Effects of sub-minimum inhibitory concentrations of antimicrobial agents on Streptococcus mutans biofilm formation. Int J Antimicrob Agents. 2012; 39(5):390–5.

Yang B, Lei Z, Zhao Y, Ahmed S, Wang C, Zhang S, et al. Combination Susceptibility Testing of Common Antimicrobials in Vitro and the Effects of Sub-MIC of Antimicrobials on Staphylococcus aureus Biofilm Formation. Front Microbiol. 2017; 8:2125.

Wojnicz D, Tichaczek-Goska D. Effect of sub-minimum inhibitory concentrations of ciprofloxacin, amikacin and colistin on biofilm formation and virulence factors of Escherichia coli planktonic and biofilm forms isolated from human urine. Braz J Microbiol. 2013; 44(1):259–65. 37

Knight J, Madduma-Liyanage K, Mobley JA, Assimos DG, Holmes RP. Ascorbic acid intake and oxalate synthesis. Urolithiasis. 2016; 44(4):289–97.

Ren H, Wu J, Colletta A, Meyerhoff ME, Xi C. Efficient eradication of mature Pseudomonas aeruginosa biofilm via controlled delivery of nitric oxide combined with antimicrobial peptide and antibiotics. Front Microbiol. 2016; 7:1260.

Helgadóttir S, Pandit S, Mokkapati VR, Westerlund F, Apell P, Mijakovic I. Vitamin C pretreatment enhances the antibacterial effect of cold atmospheric plasma. Front Cell Infect Microbiol. 2017; 7:43.

Zemke AC, Gladwin MT, Bomberger JM. Sodium nitrite blocks the activity of aminoglycosides against Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2015; AAC–00546.

Vumma R, Bang CS, Kruse R, Johansson K, Persson K. Antibacterial effects of nitric oxide on uropathogenic Escherichia coli during bladder epithelial cell colonization—a comparison with nitrofurantoin. J Antibiot (Tokyo). 2016; 69(3):183.

Beloin C, Renard S, Ghigo J-M, Lebeaux D. Novel approaches to combat bacterial biofilms. Curr Opin Pharmacol. 2014; 18:61–8.




How to Cite

Hassan, H. Y., Hanna Aka, S. T., Najmaddin Hamad , A. ., & Jalal Balaky, S. T. (2023). Combined effects of antibiotics and acidified nitrite on biofilm formation by beta-lactamase-producing uropathogenic bacteria. Zanco Journal of Medical Sciences (Zanco J Med Sci), 27(2), 145–154.



Original Articles