Assessing the Influence of Urine pH on the Efficacy of Ciprofloxacin and Fosfomycin in Immunocompetent and Immunocompromised Murine Models of ['Escherichia coli', 'Klebsiella pneumoniae'] and

Citation: Herrera-Espejo, S.; Carretero-Ledesma, M.; Bahamonde-García, M.A.; Cordero, E.; Pachón, J.; Pachón-Ibáñez, M.E. Assessing the Influence of Urine pH on the Efficacy of Ciprofloxacin and Fosfomycin in Immunocompetent and Immunocompromised Murine Models of Escherichia coli and Klebsiella pneumoniae Infection in the Lower Urinary Tract. Antibiotics 2024,13, 827. https://doi.org/10.3390/ antibiotics13090827 Academic Editor: Marc Maresca Received: 2 August 2024 Revised: 26 August 2024 Accepted: 30 August 2024 Published: 1 September 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antibiotics Article Assessing the Influence of Urine pH on the Efficacy of Ciprofloxacin and Fosfomycin in Immunocompetent and Immunocompromised Murine Models of Escherichia coli and Klebsiella pneumoniae Infection in the Lower Urinary Tract Soraya Herrera-Espejo 1 , Marta Carretero-Ledesma 1 , Manuel Anselmo Bahamonde-García 1 , Elisa Cordero 1,2,3, * , Jerónimo Pachón2,4,* and María Eugenia Pachón-Ibáñez 1,3 1 Clinical Unit of Infectious Diseases, Microbiology and Parasitology, Institute of Biomedicine of Seville (IBiS), Virgen del Rocio University Hospital/CSIC/University of Seville, 41013 Seville, Spain; [email protected] (S.H.-E.); mcarr[email protected] (M.C.-L.); [email protected] (M.A.B.-G.); [email protected] (M.E.P.-I.) 2Department of Medicine, School of Medicine, University of Seville, 41004 Seville, Spain 3CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, 28029 Madrid, Spain 4Institute of Biomedicine of Seville (IBiS), Virgen del Rocio University Hospital/CSIC/University of Seville, 41013 Seville, Spain *Correspondence: [email protected] (E.C.); [email protected] (J.P.) Abstract: In vitro studies have suggested that acidic pH may reduce and increase the efficacy of ciprofloxacin and fosfomycin, respectively, when used to treat Escherichia coli and Klebsiella pneumoniae infections. We assessed the effects of acidic, neutral, and alkaline urine pH on the efficacy of optimized ciprofloxacin and fosfomycin dosages in UTI murine model of E. coli and K. pneumoniae. Immunocompetent and immunocompromised mice with adjusted urine pH were inoculated with E. coli and K. pneumoniae strains, and the efficacy was assessed based on the bacterial concentrations in tissues and fluids at 72 h, with respect to untreated controls. At acidic urine pH, both antimicrobials were effective, achieving similar reductions in E. coli concentrations in the kidneys in immunocompetent and immunocompromised mice and in K. pneumoniae in immunocompetent mice. At a neutral urine pH, both therapies reduced the presence of E. coli in the kidneys of immunocompetent mice. However, in immunocompromised mice, antimicrobials were ineffective at treating E. coli infection in the kidneys at a neutral urine pH and showed reduced efficacy against K. pneumoniae at both acidic and neutral urine pH. The results showed no correlation between urine pH and antimicrobial efficacy, suggesting that the reduced effectiveness is associated with the animals’ immunocompetence status. Keywords: Escherichia coli;Klebsiella pneumoniae; urinary tract infection; urine pH; ciprofloxacin; fosfomycin; experimental murine model 1. Introduction Urinary tract infections (UTIs) are highly prevalent among kidney transplant recipients (KTRs), representing nearly half of all infectious complications in this population [ 1 ]. As is the case in the general population, Escherichia coli is the most frequent cause of UTI in KTRs [ 1 , 2 ], followed by Klebsiella pneumoniae [ 3 ]. Antimicrobial resistance among Gramnegative bacilli (GNB) has increased during the last decade, and this may be associated with risk factors commonly observed in the transplant setting [ 1 , 4 , 5 ]. Effective selection of antimicrobial agents is crucial for resolving these infections and mitigating the emergence of bacterial resistance [ 6 ]. The antibiotics most frequently used to treat Enterobacterales infections include β -lactams, β -lactams/ β -lactamase inhibitors, fluoroquinolones, fosfomycin, and aminoglycosides, among others [ 7 ]. A recent study carried out in six European countries highlighted the highest resistance rates for antibiotics recommended for E. coli UTI Antibiotics 2024,13, 827. https://doi.org/10.3390/antibiotics13090827 https://www.mdpi.com/journal/antibiotics Antibiotics 2024,13, 827 2 of 18 treatment: 22.4% for trimethoprim/sulfamethoxazole, 16.7% for amoxicillin/clavulanic acid, and 15.1% for ciprofloxacin [8]. Ciprofloxacin is a second-generation fluoroquinolone antibiotic that interacts with DNA gyrase to influence transcription and DNA replication [ 9 , 10 ]. Its excellent bioavailability and high concentration in urine make it a good option for ambulatory management of UTIs [ 11 , 12 ]. However, in November 2018, the European Medicines Agency published a review detailing various serious, disabling, and potentially permanent side effects of fluoroquinolone antibiotics and recommended that they should not be used to treat bacterial infections in patients with kidney disease or in organ transplant recipients [13]. Fosfomycin, an antibiotic that is often prescribed to treat uncomplicated lower UTIs, has garnered attention due to its favorable pharmacokinetic and pharmacodynamics properties, including its high concentrations in urine [ 14 , 15 ]. Although E. coli’s resistance to fosfomycin remains relatively low (typically less than 3% [ 16 ]), recent reports indicate a concerning trend of rising resistance rates [ 17 ]. UTIs caused by extended-spectrum β - lactamase-producing Enterobacterales, such as E. coli, have similarly poor resistance to fosfomycin. Fosfomycin also exhibits activity against K. pneumoniae, with a susceptibility rate of 62% [18]. These findings underscore its potential as a valuable treatment for UTIs. Information regarding the use of fosfomycin in immunocompromised patients, such as KTRs, is scarce. To our knowledge, thus far, only Rivera-Sánchez et al. have described fosfomycin as one of the main therapeutic options for treating UTIs in KTRs caused by ciprofloxacin-resistant Gram-negative isolates [ 19 ]. As such, there is a pressing need to assess alternative treatment options and understand the factors that affect antibiotic efficacy in the urinary tract microenvironment. The microenvironment of the urinary tract may significantly influence the efficacy of antimicrobial agents [ 20 – 23 ]. While in vitro investigations have suggested that acidic pH or anaerobic conditions may adversely affect the bactericidal activity of ciprofloxacin [ 20 , 24 ], fosfomycin has demonstrated increased activity under such conditions [ 25 ]. However, there remains a notable gap in our understanding, as few in vivo studies have explored the impact of urine pH on the effectiveness of antibiotics in treating GNB UTIs [ 26 , 27 ]. In a recent observational study carried out in KTRs prescribed fosfomycin or ciprofloxacin therapy to treat UTI episodes caused by E. coli and K. pneumoniae, acidic urine pH was associated with symptomatic UTI episodes at one-month follow-up; this was particularly noticeable in patients who received fosfomycin therapy for E. coli episodes. Furthermore, at acidic pH, the minimum inhibitory concentration of ciprofloxacin against 90% of strains (MIC 90 ) increased from 8 to >8 mg/L in E. coli and from 4 to >8 mg/L in K. pneumoniae, whereas the fosfomycin MIC 90 decreased from 8 to 4 mg/L in E. coli and from 512 to 128 mg/L in K. pneumoniae [ 26 ]. In addition, acidic pH conditions increased the cells invasion by E. coli in human renal cell cultures, and such conditions were associated with enhanced bacterial concentrations and pyelonephritis-like symptoms in the kidneys in a lower UTI murine model of E. coli and K. pneumoniae infection [28]. To better characterize the association between different urine pHs, especially the acidic condition, and the antimicrobial efficacy, in the present study, we evaluated treatment with ciprofloxacin and fosfomycin in both immunocompetent and immunocompromised mice with E. coli or K. pneumoniae infections in their lower urinary tracts, considering the influence of different urinary pH conditions. By elucidating the impact of urinary pH on antibiotic efficacy, we aimed to provide valuable insights that will help to optimize strategies for treating UTIs. 2. Results 2.1. In Vitro Assays 2.1.1. Time–Kill Curve Assays At maximum serum concentration (mice C max , Supplementary Table S1), both antibiotics showed bactericidal activity against all E. coli strains and K. pneumoniae strains from 2 to 24 h and from 6 to 24 h, respectively; these effects were independent of the pH and Antibiotics 2024,13, 827 3 of 18 growth media (Müller–Hinton Broth (MHB) or urine) (Figures 1and 2and Supplementary Figures S1 and S2). No differences were observed in strain control growths between MHB or urine across the different pH conditions, although at 24 h, they were approximately 1 log 10 CFU/mL lower in the urine experiments than in MHB. Among the K. pneumoniae strains, HUVR5 (which is resistant to both antimicrobials) showed regrowth in the fosfomycin assay at alkaline broth pH at 24 h, while HUVR91 (which is resistant to ciprofloxacin) exhibited regrowth in the ciprofloxacin assay from 6 to 24 h at acidic broth pH. 2.1.2. Pharmacodynamics of Ciprofloxacin and Fosfomycin The pharmacokinetics data, namely C max , t 1/2 , and AUC 0–24 , of ciprofloxacin and fosfomycin and their pharmacodynamics variables, namely AUC 0–24 /MIC, C max /MIC, and ∆ t/MIC, for the E. coli and K. pneumoniae strains are listed in Supplementary Table S1. 2.2. In Vivo Assays 2.2.1. Impact of Acidic Urine pH and Immunocompromise on the Bacterial Concentrations in the Kidneys in a Lower UTI Model of E. coli In immunocompetent and immunocompromised, untreated control mice infected with the four E. coli strains, the bacterial concentrations in the kidneys after 72 h were greater in the groups with acidic urine pH than in those with a neutral urine pH in a range from +1.75 to +2.19 and from +3.41 to +5.51 log 10 CFU/g, respectively, and in the Nu14 strain with alkaline urine pH in immunocompromised mice (+3.11 log 10 CFU/g; Supplementary Figures S3 and S4). The immunocompromised, untreated control mice had higher bacterial concentrations in the kidneys at acidic urine pH than the immunocompetent mice infected with the E. coli Nu 14 and HUVR 94 strains (+2.2 and +2.25 log 10 CFU/g, respectively). Conversely, at a neutral urine pH, the bacterial concentrations in the kidneys were lower in immunocompromised than in immunocompetent mice infected with the E. coli Nu14 gyrA D87G and Nu14 glpT missense mutation strains ( − 2.99 and − 2.59 log 10 CFU/g, respectively). There were no differences in the bacterial concentrations in the kidneys with regard to the immunocompetence at alkaline urine pH or in the blood stream infection (BSI) and mortality rates at any pH condition (Supplementary Tables S2–S4). 2.2.2. Efficacy of Ciprofloxacin and Fosfomycin in an Immunocompetent Lower UTI Model of E. coli At acidic urine pH, ciprofloxacin reduced the bacterial concentrations of E. coli Nu14, HUVR94, and Nu14 glpT strains in the kidneys by − 1.08, − 4.08, and − 2.44 log 10 CFU/g, respectively, whereas fosfomycin reduced the bacterial concentrations of E. coli Nu14, HUVR94, and Nu14 glpT strains in the kidneys by − 0.97, − 1.54, and − 1.88 log 10 CFU/g, respectively. Ciprofloxacin reduced the bacterial concentrations of E. coli Nu14, HUVR94, Nu14 gyrA, and Nu14 glpT strains in the urine by − 2.70, − 3.63, − 5.05, and − 3.62 log 10 CFU/mL, respectively, compared with untreated controls, while fosfomycin also reduced the concentrations of E. coli HUVR94 and Nu14 gyrA in the urine by − 4.15 and − 4.28 log 10 CFU/mL, respectively (Figure 3and Supplementary Figure S3). Ultimately, ciprofloxacin was more effective than fosfomycin when it came to reducing the bacterial concentration of E. coli Nu14 ( − 1.64 log 10 CFU/mL) in urine. No differences were observed in the BSI and mortality rates in the control groups after both treatments (Supplementary Figure S5). Antibiotics 2024,13, 827 4 of 18 Antibiotics 2024, 13, x FOR PEER REVIEW 4 of 19 Figure 1. Bactericidal activity of ciprofloxacin and fosfomycin at Cmax concentrations in MHB and urine, determined at pH 5 (pink bars), pH 7 (white bars), and pH 8 (purple bars), against Escherichia coli Nu14 (A), HUVR94 (B), Nu14 gyrA (C), and Nu14 glpT (D) strains. Results are represented as differences (log10 CFU/mL) relative to the initial timepoint (0 h). Striped bar: growth control; empty bar: antimicrobial condition at the Cmax levels reached in C57BL/6J mice plasma (13.22 mg/L for ciprofloxacin and 1354.09 mg/L for fosfomycin). Differences in bacterial concentrations across pH levels at the same time point were compared using analysis of variance (ANOVA) followed by Dunnett’s and Tukey’s post hoc tests. Significant differences were observed between the antimicrobial condition and its corresponding growth control at all time points; however, the figure highlights only the statistically significant differences in pH within each time point. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Growth control curves for each strain and pH condition are represented in Supplementary Figure S1. Figure 1. Bactericidal activity of ciprofloxacin and fosfomycin at C max concentrations in MHB and urine, determined at pH 5 (pink bars), pH 7 (white bars), and pH 8 (purple bars), against Escherichia coli Nu14 (A), HUVR94 (B), Nu14 gyrA (C), and Nu14 glpT (D) strains. Results are represented as differences (log 10 CFU/mL) relative to the initial timepoint (0 h). Striped bar: growth control; empty bar: antimicrobial condition at the C max levels reached in C57BL/6J mice plasma (13.22 mg/L for ciprofloxacin and 1354.09 mg/L for fosfomycin). Differences in bacterial concentrations across pH levels at the same time point were compared using analysis of variance (ANOVA) followed by Dunnett’s and Tukey’s post hoc tests. Significant differences were observed between the antimicrobial condition and its corresponding growth control at all time points; however, the figure highlights only the statistically significant differences in pH within each time point. *: p< 0.05; **: p< 0.01; ***: p< 0.001; ****: p< 0.0001. Growth control curves for each strain and pH condition are represented in Supplementary Figure S1. Antibiotics 2024,13, 827 5 of 18 Antibiotics 2024, 13, x FOR PEER REVIEW 5 of 19 Figure 2. Bactericidal activity of ciprofloxacin and fosfomycin at Cmax concentrations in MHB and urine, determined at pH 5 (pink bars), pH 7 (white bars), and pH 8 (purple bars), against Klebsiella pneumoniae HUVR42 (A), HUVR5 (B), HUVR110 (C), and HUVR91 (D) strains. Results are represented as differences (log10 CFU/mL) relative to the initial timepoint (0 h). Striped bar: growth control; empty bar: antimicrobial condition at the Cmax levels reached in C57BL/6J mice plasma (13.22 mg/L for ciprofloxacin and 1354.09 mg/L for fosfomycin). Differences in bacterial concentrations across pH levels at the same time point were compared using analysis of variance (ANOVA) followed by Dunnett’s and Tukey’s post hoc tests. Significant differences were observed between the antimicrobial condition and its corresponding growth control at all time points; however, the figure highlights only the statistically significant differences in pH within each time point. *: p < 0.05; **: p < 0.01; ***: p Figure 2. Bactericidal activity of ciprofloxacin and fosfomycin at C max concentrations in MHB and urine, determined at pH 5 (pink bars), pH 7 (white bars), and pH 8 (purple bars), against Klebsiella pneumoniae HUVR42 (A), HUVR5 (B), HUVR110 (C), and HUVR91 (D) strains. Results are represented as differences (log 10 CFU/mL) relative to the initial timepoint (0 h). Striped bar: growth control; empty bar: antimicrobial condition at the C max levels reached in C57BL/6J mice plasma (13.22 mg/L for ciprofloxacin and 1354.09 mg/L for fosfomycin). Differences in bacterial concentrations across pH levels at the same time point were compared using analysis of variance (ANOVA) followed by Dunnett’s and Tukey’s post hoc tests. Significant differences were observed between the antimicrobial condition and its corresponding growth control at all time points; however, the figure highlights only the statistically significant differences in pH within each time point. *: p< 0.05; **: p< 0.01; ***: p< 0.001; ****: p< 0.0001. Growth control curves for each strain and pH condition are represented in Supplementary Figure S2. Antibiotics 2024,13, 827 6 of 18 At neutral urine pH, ciprofloxacin decreased the bacterial concentrations of E. coli Nu14, HUVR94, and Nu14 gyrA strains in the kidneys by − 1.94, − 2.25, and − 3.39 log 10 CFU/g, respectively, and fosfomycin reduced the concentrations of E. coli HUVR94, Nu14 gyrA, and Nu14 glpT strains by − 2.72, − 3.39, and − 1.50 log 10 CFU/g, respectively. Moreover, ciprofloxacin outperformed fosfomycin in reducing the bacterial concentration in the kidneys of mice infected with E. coli Nu14 ( − 1.60 log 10 CFU/g). Ciprofloxacin also reduced the bacterial concentrations of E. coli Nu14, HUVR94, Nu14 gyrA, and Nu14 glpT strains in the bladder by − 4.57, − 2.65, − 1.21, and − 2.67 log 10 CFU/g, respectively, and it reduced the concentrations of E. coli strains in the urine by − 3.35, − 3.46, − 4.69, and − 4.21 log 10 CFU/mL, respectively, compared with untreated controls. Fosfomycin reduced the bacterial concentrations of E. coli Nu14, Nu14 gyrA, and Nu14 glpT strains in the bladder by − 4.39, − 1.69, and − 2.34 log 10 CFU/g, respectively, compared to untreated controls, and it reduced E. coli Nu14 and Nu14 gyrA strains in the bladder by − 4.66 and − 3.55 log 10 CFU/mL (Figure 3and Supplementary Figure S3). Antibiotics 2024, 13, x FOR PEER REVIEW 6 of 19 < 0.001; ****: p < 0.0001. Growth control curves for each strain and pH condition are represented in Supplementary Figure S2. CFU/mL, respectively (Figure 3 and Supplementary Figure S3). Ultimately, ciprofloxacin was more effective than fosfomycin when it came to reducing the bacterial concentration of E. coli Nu14 (−1.64 log10 CFU/mL) in urine. No differences were observed in the BSI and mortality rates in the control groups after both treatments (Supplementary Figure S5). At neutral urine pH, ciprofloxacin decreased the bacterial concentrations of E. coli Nu14, HUVR94, and Nu14 gyrA strains in the kidneys by −1.94, −2.25, and −3.39 log10 CFU/g, respectively, and fosfomycin reduced the concentrations of E. coli HUVR94, Nu14 gyrA, and Nu14 glpT strains by −2.72, −3.39, and −1.50 log10 CFU/g, respectively. Moreover, ciprofloxacin outperformed fosfomycin in reducing the bacterial concentration in the kidneys of mice infected with E. coli Nu14 (−1.60 log10 CFU/g). Ciprofloxacin also reduced the bacterial concentrations of E. coli Nu14, HUVR94, Nu14 gyrA, and Nu14 glpT strains in the bladder by −4.57, −2.65, −1.21, and −2.67 log10 CFU/g, respectively, and it reduced the concentrations of E. coli strains in the urine by −3.35, −3.46, −4.69, and −4.21 log10 CFU/mL, respectively, compared with untreated controls. Fosfomycin reduced the bacterial concentrations of E. coli Nu14, Nu14 gyrA, and Nu14 glpT strains in the bladder by −4.39, −1.69, and −2.34 log10 CFU/g, respectively, compared to untreated controls, and it reduced E. coli Nu14 and Nu14 gyrA strains in the bladder by −4.66 and −3.55 log10 CFU/mL (Figure 3 and Supplementary Figure S3). Figure 3. In vivo efficacy of ciprofloxacin and fosfomycin for the experimental urinary tract infection in immunocompetent mice model by E. coli Nu14 (A), HUVR94 (B), Nu14 gyrA (C), and Nu14 glpT (D) strains at acidic (1), neutral (2), and alkaline (3) urine pH. Ciprofloxacin− and fosfomycin−treated groups are represented as difference with respect to mean of bacterial concentration in control mice groups (log10 CFU/mL; Supplementary Figure S3). Empty bar: ciprofloxacin−treated mice; striped bar: fosfomycin−treated mice. The Mann–Whitney U test was used to compare quantitative Figure 3. In vivo efficacy of ciprofloxacin and fosfomycin for the experimental urinary tract infection in immunocompetent mice model by E. coli Nu14 (A), HUVR94 (B), Nu14 gyrA (C), and Nu14 glpT (D) strains at acidic (1), neutral (2), and alkaline (3) urine pH. Ciprofloxacin − and fosfomycin − treated groups are represented as difference with respect to mean of bacterial concentration in control mice groups (log 10 CFU/mL; Supplementary Figure S3). Empty bar: ciprofloxacin − treated mice; striped bar: fosfomycin − treated mice. The Mann–Whitney U test was used to compare quantitative variables. *: p< 0.05 compared to their control mice group; **: p< 0.01 compared to their control mice group; ***: p< 0.001 compared to their control mice group; # :p< 0.05 compared to fosfomycin − treated mice group; ##:p< 0.01 compared to fosfomycin−treated mice group. At alkaline pH, in mice infected with E. coli Nu14, both antibiotics effectively decreased the bacterial concentrations in the bladder and urine compared with those of the untreated control groups, from − 3.37 to − 3.42 log 10 CFU/g and from − 3.74 to − 5.23 log 10 CFU/mL, Antibiotics 2024,13, 827 7 of 18 respectively (Figure 3and Supplementary Figure S3). The BSI and mortality rates were 0% in both the untreated control and the ciprofloxacin − or fosfomycin − treated groups infected with E. coli Nu14 at alkaline pH. 2.2.3. Efficacy of Ciprofloxacin and Fosfomycin in an Immunocompromised Lower UTI Model of E. coli At acidic urine pH, ciprofloxacin significantly reduced the bacterial concentrations of E. coli Nu14, HUVR94, Nu14 gyrA, and Nu14 glpT strains in the kidneys by − 4.37, − 3.15, − 3.54, and − 2.46 log 10 CFU/g, respectively, while fosfomycin reduced the presence of E. coli Nu14, HUVR94, Nu14 gyrA, and Nu14 glpT strains in the kidneys by − 5.58, − 4.17, − 5.49, and − 3.13 log 10 CFU/g, respectively. In immunocompromised mice, ciprofloxacin was less effective than fosfomycin at reducing the bacterial concentrations of E. coli HUVR94 in the kidneys, achieving a reduction of − 1.02 log 10 CFU/g. Ciprofloxacin also reduced the bacterial concentration of E. coli HUVR94 and Nu14 glpT in the bladder by − 3.41 and − 0.93 log 10 CFU/g, respectively, and reduced the concentrations of E. coli Nu14 and HUVR94 strains in the urine by − 3.47 and − 4.51 log 10 CFU/mL, respectively, compared with untreated controls. Fosfomycin reduced the concentrations of HUVR94, Nu14 gyrA, and Nu14 glpT in the bladder by − 3.43, − 0.72, and − 1.08 log 10 CFU/g, respectively, and reduced the bacterial concentration of Nu14 in the urine by − 4.07 log 10 CFU/mL (Figure 4 and Supplementary Figure S4). In accordance with the results of the immunocompetent mice, ciprofloxacin demonstrated better efficacy than fosfomycin in reducing the bacterial concentration of E. coli HUVR94 in the urine ( − 3.61 log 10 CFU/mL). No differences were observed in the BSI and mortality rates after both treatments with respect to the control groups (Supplementary Figure S5). Antibiotics 2024, 13, x FOR PEER REVIEW 8 of 19 Figure 4. In vivo efficacy of ciprofloxacin and fosfomycin for the experimental urinary tract infection in immunocompromised mice model by E. coli Nu14 (A), HUVR94 (B), Nu14 gyrA (C), and Nu14 glpT (D) strains at acidic (1), neutral (2), and alkaline (3) urine pH. Ciprofloxacin− and fosfomycin−treated groups are represented as difference with respect to mean bacterial concentration of control mice group (log10 CFU/mL; Supplementary Figure S4). Empty bar: ciprofloxacin−treated mice; striped bar: fosfomycin−treated mice. The Mann–Whitney U test was used to compare quantitative variables. *: p < 0.05 compared to their control mice group; **: p < 0.01 compared to their control mice group; #: p < 0.05 compared to fosfomycin−treated mice group; ##: p < 0.01 compared to fosfomycin−treated mice group. 2.2.4. Impact of Acidic Urine pH and Immunocompromise on the Bacterial Concentrations in the Kidneys in a Lower UTI Model of K. pneumoniae In immunocompetent and immunocompromised, untreated control mice infected with K. pneumoniae, the bacterial concentrations in the kidneys after 72 h were greater in the groups with acidic pH urine than in those with neutral urine pH, in a range from +1.90 to +4.43 and from +3.95 to +4.28 log10 CFU/g, and in the HUVR42 strain group with alkaline urine pH from +3.48 to +4.84 log10 CFU/g (Supplementary Figures S7 and S8). There were no differences in the bacterial concentrations in the kidneys at acidic and neutral urine pH when we compared the immunocompromised and immunocompetent, untreated control mice infected with K. pneumoniae strains. Only the HUVR42 strain differed, showing a higher bacterial concentration in the kidneys of immunocompromised mice (+1.64 log10 CFU/g) at neutral urine pH condition. In addition, at acidic urine pH, HUVR91 produced 100% BSI in immunocompromised mice vs. 0% in the immunocompetent mice. Immunocompetence did not affect the mortality rates of the mice at any pH condition (Supplementary Tables S2–S4). Figure 4. In vivo efficacy of ciprofloxacin and fosfomycin for the experimental urinary tract infection in immunocompromised mice model by E. coli Nu14 (A), HUVR94 (B), Nu14 gyrA (C), and Nu14 glpT (D) strains at acidic (1), neutral (2), and alkaline (3) urine pH. Ciprofloxacin − and fosfomycin − treated groups are represented as difference with respect to mean bacterial concentration of control mice group (log 10 CFU/mL; Supplementary Figure S4). Empty bar: ciprofloxacin − treated mice; striped bar: fosfomycin − treated mice. The Mann–Whitney U test was used to compare quantitative variables. *: p< 0.05 compared to their control mice group; **: p< 0.01 compared to their control mice group; #:p< 0.05 compared to fosfomycin − treated mice group; ## :p< 0.01 compared to fosfomycin − treated mice group. Antibiotics 2024,13, 827 8 of 18 At neutral urine pH, both antimicrobials were ineffective at reducing bacterial concentrations in the kidneys compared with untreated controls, which exhibited low bacterial concentrations in the kidneys, with means from 0.66 to 3.75 log 10 CFU/g. However, both ciprofloxacin and fosfomycin effectively reduced the bacterial concentrations of the four E. coli strains in the bladder and the urine. Thus, ciprofloxacin reduced the bacterial concentrations of Nu14, HUVR94, Nu14 gyrA, and Nu14 glpT strains in the bladder by − 3.03, − 4.65, − 5.26, and − 6.75 log 10 CFU/g, respectively, while fosfomycin reduced the concentrations by − 4.54, − 1.90, − 6.60, and − 6.46 log 10 CFU/g, respectively. Additionally, ciprofloxacin decreased the bacterial concentrations of Nu14, HUVR94, Nu14 gyrA, and Nu14 glpT in the urine by − 6.37, − 8.74, − 7.71, and − 8.37 log 10 CFU/mL, respectively, while fosfomycin reduced their concentrations by − 5.52, − 3.83, − 6.14, and − 6.19 log 10 CFU/mL. Ciprofloxacin also outperformed fosfomycin in terms of bladder and urine clearance in mice infected with E. coli HUVR94 (−2.75 log10 CFU/g and −3.21 log10 CFU/mL, respectively). At alkaline pH, in mice infected with E. coli Nu14, similar to the findings in the immunocompetent mice, both antimicrobials reduced the bacterial concentrations in the bladder ( − 5.41 and − 5.31 log 10 CFU/g) and urine ( − 5.37 and − 5.28 log 10 CFU/mL) with respect to those of the untreated mice (Figure 4and Supplementary Figure S4). Additionally, the BSI and mortality rates were 0% for both the untreated control and the ciprofloxacin − or fosfomycin − treated groups, although BSI was observed in one out of the five mice in the ciprofloxacin−treated group. 2.2.4. Impact of Acidic Urine pH and Immunocompromise on the Bacterial Concentrations in the Kidneys in a Lower UTI Model of K. pneumoniae In immunocompetent and immunocompromised, untreated control mice infected with K. pneumoniae, the bacterial concentrations in the kidneys after 72 h were greater in the groups with acidic pH urine than in those with neutral urine pH, in a range from +1.90 to +4.43 and from +3.95 to +4.28 log 10 CFU/g, and in the HUVR42 strain group with alkaline urine pH from +3.48 to +4.84 log10 CFU/g (Supplementary Figures S7 and S8). There were no differences in the bacterial concentrations in the kidneys at acidic and neutral urine pH when we compared the immunocompromised and immunocompetent, untreated control mice infected with K. pneumoniae strains. Only the HUVR42 strain differed, showing a higher bacterial concentration in the kidneys of immunocompromised mice (+1.64 log 10 CFU/g) at neutral urine pH condition. In addition, at acidic urine pH, HUVR91 produced 100% BSI in immunocompromised mice vs. 0% in the immunocompetent mice. Immunocompetence did not affect the mortality rates of the mice at any pH condition (Supplementary Tables S2–S4). 2.2.5. Efficacy of Ciprofloxacin and Fosfomycin in an Immunocompetent Lower UTI Model of K. pneumoniae At acidic urine pH, compared with untreated controls, ciprofloxacin reduced the bacterial concentrations of K. pneumoniae HUVR42, HUVR5, HUVR110, and HUVR91 strains in the kidneys by − 2.94, − 5.86, − 4.54, and − 4.69 log 10 CFU/g, respectively, while fosfomycin reduced the concentrations of K. pneumoniae HUVR42, HUVR5, and HUVR91 by − 3.57, − 4.93, and − 4.65 log 10 CFU/g, respectively. Ciprofloxacin reduced the bacterial concentrations of HUVR42, HUVR5, and HUVR91 in the bladder by − 1.57, − 4.07, and − 3.79 log 10 CFU/g, respectively, and reduced the concentrations of HUVR42 and HUVR5 strains in the urine by − 1.93 and − 3.03 log 10 CFU/mL, respectively. Fosfomycin reduced the bacterial concentrations of HUVR42, HUVR5, and HUVR91 in the urine by − 2.20, − 3.23, and − 2.93 log 10 CFU/g, respectively, and reduced the concentrations of K. pneumoniae HUVR42 in the urine by − 1.58 log 10 CFU/g (Figure 5and Supplementary Figure S7). Additionally, ciprofloxacin showed superior efficacy to fosfomycin when it came to reducing the bacterial concentration of K. pneumoniae HUVR5 in the urine ( − 2.77 log 10 CFU/mL). No differences were observed in the BSI and mortality rates after both treatments with respect to the control groups (Supplementary Figure S5). Antibiotics 2024,13, 827 9 of 18 Antibiotics 2024, 13, x FOR PEER REVIEW 9 of 19 2.2.5. Efficacy of Ciprofloxacin and Fosfomycin in an Immunocompetent Lower UTI Model of K. pneumoniae At acidic urine pH, compared with untreated controls, ciprofloxacin reduced the bacterial concentrations of K. pneumoniae HUVR42, HUVR5, HUVR110, and HUVR91 strains in the kidneys by −2.94, −5.86, −4.54, and −4.69 log10 CFU/g, respectively, while fosfomycin reduced the concentrations of K. pneumoniae HUVR42, HUVR5, and HUVR91 by −3.57, −4.93, and −4.65 log10 CFU/g, respectively. Ciprofloxacin reduced the bacterial concentrations of HUVR42, HUVR5, and HUVR91 in the bladder by −1.57, −4.07, and −3.79 log10 CFU/g, respectively, and reduced the concentrations of HUVR42 and HUVR5 strains in the urine by −1.93 and −3.03 log10 CFU/mL, respectively. Fosfomycin reduced the bacterial concentrations of HUVR42, HUVR5, and HUVR91 in the urine by −2.20, −3.23, and −2.93 log10 CFU/g, respectively, and reduced the concentrations of K. pneumoniae HUVR42 in the urine by −1.58 log10 CFU/g (Figure 5 and Supplementary Figure S7). Additionally, ciprofloxacin showed superior efficacy to fosfomycin when it came to reducing the bacterial concentration of K. pneumoniae HUVR5 in the urine (−2.77 log10 CFU/mL). No differences were observed in the BSI and mortality rates after both treatments with respect to the control groups (Supplementary Figure S5). Figure 5. In vivo efficacy of ciprofloxacin and fosfomycin for the experimental urinary tract infection in immunocompetent mice model by K. pneumoniae HUVR42 (A), HUVR5 (B), HUVR110 (C), and HUVR91 (D) strains at acidic (1), neutral (2), and alkaline (3) urine pH. Ciprofloxacin− and fosfomycin−treated groups are represented as difference with respect to mean bacterial concentration of control mice group (log10 CFU/mL; Supplementary Figure S7). Empty bar: ciprofloxacin−treated mice; striped bar: fosfomycin−treated mice. The Mann–Whitney U test was used to compare quantitative variables. *: p < 0.05 compared to their control mice group; **: p < 0.01 compared to their control mice group; ***: p < 0.001 compared to their control mice group; #: p < 0.05 compared to fosfomycin−treated mice group; ###: p < 0.001 compared to fosfomycin−treated mice group. Figure 5. In vivo efficacy of ciprofloxacin and fosfomycin for the experimental urinary tract infection in immunocompetent mice model by K. pneumoniae HUVR42 (A), HUVR5 (B), HUVR110 (C), and HUVR91 (D) strains at acidic (1), neutral (2), and alkaline (3) urine pH. Ciprofloxacin − and fosfomycin − treated groups are represented as difference with respect to mean bacterial concentration of control mice group (log 10 CFU/mL; Supplementary Figure S7). Empty bar: ciprofloxacin − treated mice; striped bar: fosfomycin − treated mice. The Mann–Whitney U test was used to compare quantitative variables. *: p< 0.05 compared to their control mice group; **: p< 0.01 compared to their control mice group; ***: p< 0.001 compared to their control mice group; # :p< 0.05 compared to fosfomycin−treated mice group; ###:p< 0.001 compared to fosfomycin−treated mice group. At neutral urine pH, ciprofloxacin reduced the bacterial concentrations of K. pneumoniae HUVR42 and HUVR91 in the kidneys by − 0.84 and − 3.12 log 10 CFU/g, while fosfomycin achieved a similar reduction for K. pneumoniae HUVR42 alone ( − 0.55 log 10 CFU/g). Both antimicrobials significantly decreased the bacterial concentrations of K. pneumoniae HUVR42, HUVR5, HUVR110, and HUVR91 in the bladder and in the urine. Ciprofloxacin reduced the bacterial concentrations in the bladder by − 3.18, − 5.01, − 3.98, and − 5.57 log 10 CFU/g, respectively, and reduced the urine bacterial concentrations of the same four K. pneumoniae strains by − 4.56, − 4.29, − 5.90, and − 8.04 log 10 CFU/mL, respectively, compared with untreated controls. Fosfomycin reduced the bacterial concentrations of HUVR42, HUVR5, HUVR110, and HUVR91 in the bladder by − 4.59, − 2.02, − 5.66, and − 3.69 log 10 CFU/g, respectively, and in the bladder, it reduced the concentrations of the same four K. pneumoniae strains by − 4.48, − 4.89, − 6.83, and − 8.44 log 10 CFU/mL, respectively, compared with untreated controls. Moreover, ciprofloxacin outperformed fosfomycin in reducing the bacterial concentration of K. pneumoniae HUVR5 in the bladders of infected mice (−2.99 log10 CFU/g). At alkaline pH, ciprofloxacin reduced the bacterial concentration of HUVR42 in the kidneys with respect to the concentrations observed in untreated controls ( − 1.62 log 10 CFU/g). Both antibiotics decreased the bacterial concentrations in the bladder ( − 2.85 Antibiotics 2024,13, 827 16 of 18 supervision, J.P., E.C. and M.E.P.-I.; project administration, M.E.P.-I.; funding acquisition, E.C. and M.E.P.-I. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (PI17/01405 and PI20/01255), Subdirección General de Redes y Centros de Investigación Cooperativa, Consejería de Economía, Industria y Competitividad US-1381501 US/JUNTA/FEDER, UE and co-financed by European Development Regional Fund: “A way to achieve Europe”, Operative program Intelligent Growth 2014–2020. Institutional Review Board Statement: The animal study protocol was approved by the Committee on the Ethics of Animal Experiments of Consejería de Agricultura,Ganadería,Pesca y Desarrollo Sostenible, Spain (06/03/2018/022 and 16/11/2020/134). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Acknowledgments: S.H.-E. is supported by the program PFIS (Contratos Predoctorales de Formación en Investigación en Salud), Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia e Innovación under grant (FI21/00280). M.E.P.-I. is a “Nicolás Monardes” researcher (RC1-0006-2023, Servicio Andaluz de Salud, Junta de Andalucía, Spain. 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