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. 2024 Sep 1;13(9):829.
doi: 10.3390/antibiotics13090829.

Combinatory Effect of Nitroxoline and Gentamicin in the Control of Uropathogenic Enterococci Infections

Davorka Repac Antić  1   2 , Bruno Kovač  3 , Marko Kolenc  4 , Irena Brčić Karačonji  5   6 , Ivana Gobin  1 , Mirna Petković Didović  7
Affiliations

Combinatory Effect of Nitroxoline and Gentamicin in the Control of Uropathogenic Enterococci Infections

Davorka Repac Antić et al. Antibiotics (Basel). .

Abstract

Enterococcus faecalis, responsible for a majority of human and nosocomial enterococcal infections, is intrinsically resistant to aminoglycoside antibiotics (such as gentamicin, GEN), which must be used in a combined therapy to be effective. Nitroxoline (NTX) is an old antibiotic, underused for decades, but rediscovered now in an era of growing antibiotic resistance. In this in vitro study, the types of interactions between NTX and GEN on 29 E. faecalis strains were analyzed with an aim to find synergistic antimicrobial and antiadhesion combinations. Transmission electron microscopy (TEM) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to analyze changes in cell morphology and bacterial proteome after monotreatments and combined treatments. The results showed the synergistic effect for six combinations on eight strains, including the ATCC29212, and an additive effect for most strains. Combinations causing a complete inhibition of adhesion were established. Cell membrane integrity was affected by NTX, while combined NTX/GEN treatment caused dramatic changes in cell morphology. Upregulation of the expression of many proteins was established, with some emerging only after combined treatment. The results strongly imply that NTX has the potential for use in combined therapy with GEN against enterococci and it could further provide a substantial contribution to an ongoing fight against antimicrobial resistance and nosocomial infections.

Keywords: E. faecalis; antiadhesion; antimicrobial resistance; enterococci; proteome; synergism.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Susceptibility of Enterococcus faecalis isolates to nitroxoline and gentamicin. MIC–minimum inhibitory concentration.
Figure 2
Figure 2
Number of adhered bacteria of (a) E15; (b) ATCC29212; (c) E55; and (d) E1 Enterococcus faecalis strains on polystyrene after monotreatments with nitroxoline (NTX), gentamicin (GEN), and combined treatment with selected concentrations. The result is presented as mean ± SD. Uppercase letters (A–F) mark statistically significant differences (p < 0.05) between combined treatment and individual treatments contained in the combined treatment. For example, the uppercase letter A in (a) marks a statistically significant difference between NTX1/GEN16 (combined treatment) and NTX1 and GEN16 (individual treatments). Also, uppercase letters BCDE above NTX2 mark a statistically significant difference between NTX2 and combined treatments NTX2/GEN8, NTX2/GEN16, NTX2/GEN32, and NTX2/GEN64.
Figure 3
Figure 3
Transmission electron microscopy (TEM) micrographs showing morphological changes of Enterococcus faecalis ATCC29212 strain: (a) control; after 12 h of exposure to (b) nitroxoline (NTX) 8 μg/mL, (c) gentamicin (GEN) 8 μg/mL, (d) synergistic NTX/GEN combination 2/0.5 μg/mL, (e) additive NTX/GEN combination 2/4 μg/mL, and (f) additive NTX/GEN combination 4/0.5 μg/mL. Red arrows indicate cell wall disruption; blue arrows indicate cellular debris (decayed cells).
Figure 4
Figure 4
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra of the Enterococcus faecalis control strain ATCC29212 without treatment, after monotreatments with nitroxoline and gentamicin, and after the combined treatment. The spectra are moved up the y-axis for easier comparison. Complete spectra are given in (a) and designated sections are zoomed in in images (b–e). The full and empty stars designate genus-specific and E. faecalis-specific biomarkers. Full black triangles designate species-characteristic peaks (found in E. faecalis strains, but also in the strains of other species); full red triangles designate peaks not found within the fingerprinting data of E. faecalis strains [62]; empty triangles designate peaks found only in certain E. faecalis strains.
Figure 5
Figure 5
Venn diagrams of four tested Enterococcus faecalis ATCC29212 mutants (untreated; treated with nitroxoline, NTX; treated with gentamicin, GEN; treated with NTX/GEN combination) and their significantly (a) upregulated and (b) downregulated peaks. The four-digit numbers represent the m/z positions of peaks extracted from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra shown in Figure 4. Bolded values represent peaks not present in the untreated strain. Single- or double-digit numbers represent the total peak count in the particular category. Red number designates the peak found in vanB-positive isolate of Enterococcus faecium.
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