Research Article|Articles in Press, 101819

Ciprofloxacin resistance and tolerance of Pseudomonas aeruginosa ocular isolates

Published:February 01, 2023DOI:



      Tolerance to antibiotics may occur due to changes in bacterial growth patterns and can be a precursor to development of resistance. However, there is a lack of information on the ability of ocular bacteria isolates to develop tolerance. This paper explores the tolerance to 8 different antibiotics of 61 microbial keratitis isolates of Pseudomonas aeruginosa from Australia and India using the MBC/MIC ratio, with tolerance defined by a ratio 32, and tolerance to ciprofloxacin by an agar diffusion assay.


      Antibiotics used were ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. Isolates were sourced from microbial keratitis infections in Australia and India. Minimum bactericidal and minimum inhibitory concentration (MBC and MIC) were obtained using broth microdilution and compared to breakpoints from the Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) to determine bacterial susceptibility. Tolerance was assessed as MBC/MIC ≥ 32. An alternative method for tolerance detection (TD) was assessed with 13P. aeruginosa sensitive isolates by agar disk diffusion assay of ciprofloxacin followed by application of glucose to the agar and observation of re-growth of colonies.


      Thirty-three isolates were resistant to imipenem, 20 to ciprofloxacin, 14 to tobramycin and piperacillin, 12 to levofloxacin and ceftazidime, 8 to gentamicin, and 5 to polymyxin B. The percentage of strains resistant to levofloxacin (7 vs 30 %; p = 0.023), gentamicin (0 vs 24 %; p = 0.005) and tobramycin (4 vs 33 %; p = 0.004) was significantly greater in isolates from India. On average, strains from India exhibited notably greater MIC and MBC values compared to strains obtained from Australia. Out of 61 isolates, none displayed an MBC/MIC ratio 32. However, three sensitive isolates had low tolerance, nine had medium tolerance and one had high tolerance to ciprofloxacin with the TDtest.


      This study used two methods to determine whether P. aeruginosa strains could show tolerance to antibiotics. Using the MBC/MIC criteria no strain was considered tolerant to any of the eight antibiotics used. When 13 strains were tested for tolerance against ciprofloxacin, the most commonly used monotherapy for keratitis, one had high tolerance and nine had medium tolerance. This demonstrates the capacity of P. aeruginosa to develop tolerance which may result in therapeutic failures if inappropriate dosing regimens are used to treat keratitis.


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        • Meredith H.R.
        • Srimani J.K.
        • Lee A.J.
        • Lopatkin A.J.
        • You L.
        Collective antibiotic tolerance: mechanisms, dynamics and intervention.
        Nat Chem Biol. 2015; 11: 182-188
        • Ezisi C.
        • Ogbonnaya C.
        • Okoye O.
        • Ezeanosike E.
        • Ginger-Eke H.
        • Arine O.
        Microbial keratitis – A review of epidemiology, pathogenesis, ocular manifestations, and management.
        Niger J Ophthalmol. 2018; 26: 13-23
        • Upadhyay M.P.
        • Srinivasan M.
        • Whitcher J.P.
        Diagnosing and managing microbial keratitis.
        Community Eye Health. 2015; 28: 3-6
        • Stapleton F.
        Contact lens-related corneal infection in Australia.
        Clin Exp Optom. 2020; 103: 408-417
        • Willcox M.D.P.
        Contact lens –related keratitis and ocular microbiology: a review of the latest research related to the microbiota of the ocular surface.
        Contact Lens Spectrum. 2017; 32: 2
        • Keay L.
        • Edwards K.
        • Naduvilath T.
        • Taylor H.R.
        • Snibson G.R.
        • Forde K.
        • et al.
        Microbial keratitis predisposing factors and morbidity.
        Ophthalmology. 2006; 113: 109-116
        • Willcox M.D.
        Management and treatment of contact lens-related Pseudomonas keratitis.
        Clin Ophthalmol. 2012; 6: 919-924
        • Austin A.
        • Schallhorn J.
        • Geske M.
        • Mannis M.
        • Lietman T.
        • Rose-Nussbaumer J.
        Empirical treatment of bacterial keratitis: an international survey of corneal specialists.
        BMJ Open Ophthalmol. 2017; 2
        • Green M.
        • Apel A.
        • Stapleton F.
        Risk factors and causative organisms in microbial keratitis.
        Cornea. 2008; 27: 22-27
        • Gupta S.
        • Mittal S.
        • Nayak N.
        • Satpathy G.
        • Khokhar S.
        • Agarwal T.
        In vitro antibiotic susceptibility of Pseudomonas aeruginosa corneal ulcer Isolates.
        Ocul Immunol Inflamm. 2015; 23: 252-255
        • Hsu D.I.
        • Okamoto M.P.
        • Murthy R.
        • Wong-Beringer A.
        Fluoroquinolone-resistant Pseudomonas aeruginosa: risk factors for acquisition and impact on outcomes.
        J Antimicrob Chemother. 2005; 55: 535-541
        • Stower H.
        Antibiotic tolerance leads to antibiotic resistance.
        Nat Med. 2020; 26: 163
        • Brauner A.
        • Fridman O.
        • Gefen O.
        • Balaban N.Q.
        Distinguishing between resistance, tolerance and persistence to antibiotic treatment.
        Nat Rev Microbiol. 2016; 14: 320-330
        • Kester J.C.
        • Fortune S.M.
        Persisters and beyond: mechanisms of phenotypic drug resistance and drug tolerance in bacteria.
        Crit Rev Biochem Mol Biol. 2014; 49: 91-101
        • Anderl J.N.
        • Zahller J.
        • Roe F.
        • Stewart P.S.
        Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin.
        Antimicrob Agents Chemother. 2003; 47: 1251-1256
        • Finkel S.E.
        Long-term survival during stationary phase: evolution and the GASP phenotype.
        Nat Rev Microbiol. 2006; 4: 113-120
        • Fridman O.
        • Goldberg A.
        • Ronin I.
        • Shoresh N.
        • Balaban N.Q.
        Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations.
        Nature. 2014; 513: 418-421
        • Zhu K.
        • Chen S.
        • Sysoeva T.A.
        • You L.
        Universal antibiotic tolerance arising from antibiotic-triggered accumulation of pyocyanin in Pseudomonas aeruginosa.
        PLoS Biol. 2019; 17: e3000573
        • Hall C.W.
        • Mah T.F.
        Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria.
        FEMS Microbiol Rev. 2017; 41: 276-301
        • Spoering A.L.
        • Lewis K.
        Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials.
        J Bacteriol. 2001; 183: 6746-6751
        • Hu Y.
        • Coates A.
        Nonmultiplying bacteria are profoundly tolerant to antibiotics.
        Handb Exp Pharmacol. 2012; 211: 99-119
        • Walters 3rd, M.C.
        • Roe F.
        • Bugnicourt A.
        • Franklin M.J.
        • Stewart P.S.
        Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin.
        Antimicrob Agents Chemother. 2003; 47: 317-323
        • Gefen O.
        • Chekol B.
        • Strahilevitz J.
        • Balaban N.Q.
        TDtest: easy detection of bacterial tolerance and persistence in clinical isolates by a modified disk-diffusion assay.
        Sci Rep. 2017; 7: 41284
        • Khan M.
        • Stapleton F.
        • Willcox M.D.P.
        Susceptibility of Contact Lens-Related Pseudomonas aeruginosa Keratitis Isolates to Multipurpose Disinfecting Solutions, Disinfectants, and Antibiotics.
        Transl Vis Sci Technol. 2020; 9: 2
        • Weinstine M.P.
        • Lewis II, J.S.
        • Bobenchik A.M.
        • Campeau S.
        • Cullen S.K.
        • Galas M.F.
        • et al.
        CLSI M100-ED30: 2020 Performance Standards for Antimicrobial Susceptibility Testing.
        30th Ed. Clinical & Laboratory Standards Institute, 2020
      1. Breakpoint tables for interpretation of MICs and zone diameters. 10th ed.: European Committee on Antimicrobial Susceptibility Testing (EUCAST). 2020.

        • Pasticci M.B.
        • Moretti A.
        • Stagni G.
        • Ravasio V.
        • Soavi L.
        • Raglio A.
        • et al.
        Bactericidal activity of oxacillin and glycopeptides against Staphylococcus aureus in patients with endocarditis: looking for a relationship between tolerance and outcome.
        Ann Clin Microbiol Antimicrob. 2011; 10: 26
        • Kotkova H.
        • Cabrnochova M.
        • Licha I.
        • Tkadlec J.
        • Fila L.
        • Bartosova J.
        • et al.
        Evaluation of TD test for analysis of persistence or tolerance in clinical isolates of Staphylococcus aureus.
        J Microbiol Methods. 2019; 167105705
        • Cabrera-Aguas M.
        • Khoo P.
        • Watson S.L.
        Infectious keratitis: A review.
        Clin Exp Ophthalmol. 2022;
        • Tuomanen E.
        • Durack D.T.
        • Tomasz A.
        Antibiotic tolerance among clinical isolates of bacteria.
        Antimicrob Agents Chemother. 1986; 30: 521-527
        • May J.
        • Shannon K.
        • King A.
        • French G.
        Glycopeptide tolerance in Staphylococcus aureus.
        J Antimicrob Chemother. 1998; 42: 189-197
        • Azuma M.
        • Murakami K.
        • Murata R.
        • Kataoka K.
        • Fujii H.
        • Miyake Y.
        • et al.
        Clinical significance of carbapenem-tolerant Pseudomonas aeruginosa isolated in the respiratory tract.
        Antibiotics (Basel). 2020; 9
        • Sherris J.C.
        Problems in in vitro determination of antibiotic tolerance in clinical isolates.
        Antimicrob Agents Chemother. 1986; 30: 633-637
        • Pearce J.G.
        • Essex R.W.
        • Maddess T.
        The clinical treatment of bacterial keratitis: A review of drop instillation regimes.
        Cont Lens Anterior Eye. 2022; 101725
        • Su H.C.
        • Ramkissoon K.
        • Doolittle J.
        • Clark M.
        • Khatun J.
        • Secrest A.
        • et al.
        The development of ciprofloxacin resistance in Pseudomonas aeruginosa involves multiple response stages and multiple proteins.
        Antimicrob Agents Chemother. 2010; 54: 4626-4635
        • Levin-Reisman I.
        • Ronin I.
        • Gefen O.
        • Braniss I.
        • Shoresh N.
        • Balaban N.Q.
        Antibiotic tolerance facilitates the evolution of resistance.
        Science. 2017; 355: 826-830
        • Khan M.
        • Stapleton F.
        • Summers S.
        • Rice S.A.
        • Willcox M.D.P.
        Antibiotic resistance characteristics of Pseudomonas aeruginosa isolated from keratitis in Australia and India.
        Antibiotics (Basel). 2020; 9
        • Budhiraja G.
        • Acharya M.
        • Kumar M.
        Role of topical high concentration levofloxacin 1.5% in bacterial keratitis.
        Indian Journal of Ophthalmology Case Reports. 2021; 1: 643-647
        • Acharya M.
        • Farooqui J.H.
        • Gaba T.
        • Gandhi A.
        • Mathur U.
        Delhi Infectious Keratitis Study: Update on clinico-microbiological profile and outcomes of infectious keratitis.
        J Curr Ophthalmol. 2020; 32: 249-255