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. 2025 Dec 21;56(1):315–325. doi: 10.55730/1300-0144.6165

Fitness cost and biofilm formation in fosfomycin-resistant clinical Escherichia coli and Klebsiella pneumoniae isolates

Mustafa ÖKEER 1, Sabire Şöhret AYDEMİR 2, Bayrı ERAÇ 1,*
PMCID: PMC12974299  PMID: 41816737

Abstract

Background/aim

Fosfomycin has regained importance owing to its unique mechanism of action and effectiveness against extended-spectrum β-lactamase-producing Gram-negative bacteria. This study aimed to evaluate the biological fitness cost associated with fosfomycin resistance and its impact on biofilm formation in clinical Enterobacteriaceae isolates.

Materials and methods

A total of 78 Escherichia coli and 34 Klebsiella pneumoniae strains isolated from urine samples at Ege University Hospital were analyzed. Fosfomycin minimum inhibitory concentrations (MICs) were determined using the reference agar dilution method. Resistance was induced by exposing two K. pneumoniae strains with a fosfomycin MIC of 4 μg/mL and two E. coli strains susceptible to fosfomycin (MIC ≤ 8 μg/mL) to gradually increasing concentrations of the antibiotic. Biofilm-forming capacities, growth rates, and expression levels of selected virulence genes (fimH and papC in E. coli; entB, mrkD, uge, wabG, and ycfM in K. pneumoniae) were compared between variants with low and high fosfomycin MICs.

Results

Of the 78 E. coli isolates, 13 (16.6%) were resistant to fosfomycin. Additionally, eight (23.5%) of 34 K. pneumoniae isolates exhibited high fosfomycin MICs (MIC > 32 μg/mL). No significant differences in biofilm formation were observed between the variants. However, the expression of the fimH gene decreased in one E. coli resistant variant compared with its susceptible counterpart. While the expression of the uge gene decreased in one K. pneumoniae isolate with a high MIC, the expression of the wabG gene increased. Slower growth rates were observed in two fosfomycin-resistant E. coli strains and one K. pneumoniae strain with a high fosfomycin MIC than in their counterparts.

Conclusion

These findings suggest that, in the examined isolates, decreased susceptibility to fosfomycin was associated with slower growth, whereas biofilm formation ability remained largely unaffected. Continued surveillance of fosfomycin resistance is essential owing to its potential implications for bacterial fitness and pathogenicity.

Keywords: Fosfomycin, fitness cost, virulence, biofilm, Escherichia coli, Klebsiella pneumoniae

1. Introduction

Antibiotic resistance has emerged as one of the most critical public health challenges of the modern era. The excessive and inappropriate use of antibiotics has led to the emergence of infections that are increasingly difficult or even impossible to treat. Infections caused by antibiotic-resistant microorganisms are currently responsible for approximately 700,000 deaths each year. By 2050, it is estimated that antibiotic-resistant infections could cause up to 10 million deaths and impose a global economic burden exceeding 1 trillion USD annually [1]. Fosfomycin has become a preferred therapeutic option for the treatment of uncomplicated urinary tract infections owing to its unique mechanism of action and relatively low rate of resistance development [2,3]. Moreover, the convenience of single-dose administration, which improves patient compliance, further underscores its therapeutic importance. Given these advantages, the use of fosfomycin in treating infections caused by multidrug-resistant (MDR) bacteria is expected to increase. Consequently, the risk of rising fosfomycin resistance rates is anticipated to become more pronounced in the coming years [3]. Fosfomycin exerts its antibacterial effect by inactivating the MurA enzyme, which catalyzes an essential step in bacterial cell wall synthesis, thereby inhibiting peptidoglycan formation. The uptake of fosfomycin into the bacterial cell is mediated by the GlpT and UhpT transport systems. Structural alterations in the MurA enzyme, its overproduction, or changes in the GlpT and UhpT transport systems are major factors contributing to the development of fosfomycin resistance in bacteria [4,5]. The acquisition of fosfomycin-modifying enzymes such as the metalloenzymes FosA, FosB, and FosX or the kinases FomA and FomB also contributes to fosfomycin resistance [6].

The development of antibiotic resistance in bacteria imposes a biological burden known as the fitness cost, which generally results in reduced growth rates and decreased expression of virulence-related genes and proteins [7–10]. However, conflicting results have been reported regarding biofilm formation; some researchers have shown that the fitness cost decreases biofilm formation [11,12], whereas others have reported the opposite effect or no significant difference [9,10,13]. Resistant bacteria must transcribe resistance-related genes in addition to the essential genes required for their metabolic activities. This may lead to a decrease in bacterial growth rate, thereby potentially reducing bacterial virulence and infectivity [14]. Clinically, the fitness cost is important because it influences how long resistant strains can persist once antibiotic pressure is reduced. When the cost is high, the number of resistant strains tends to decline once antibiotic use is discontinued. However, compensatory mutations may emerge to mitigate these costs, allowing resistant strains to remain stable even in the absence of antibiotic pressure [9,15,16]. Thus, a comprehensive understanding of both fitness costs and compensatory adaptive mechanisms is crucial for the rational design of antibiotic rotation and deescalation strategies aimed at effectively managing the long-term dynamics of antimicrobial resistance in clinical settings.

Although fosfomycin is widely used, the development of resistance in clinical strains remains relatively rare. This phenomenon is thought to result from the biological burden (fitness cost) that resistance imposes on bacteria. The unique mechanism of action of fosfomycin makes the development of cross-resistance unlikely [5]. Fosfomycin is primarily recommended for the treatment of uncomplicated urinary tract infections, including cystitis caused by antimicrobial-resistant or extended-spectrum β-lactamase (ESBL)-producing E. coli. It also serves as an alternative treatment option for prostatitis caused by ESBL-producing E. coli [17]. The Infectious Diseases Society of America does not recommend fosfomycin for the treatment of infections caused by K. pneumoniae isolates, which frequently carry fosA or other fosfomycin-hydrolyzing enzymes [18]. However, some studies have reported that multidrug-resistant and NDM-producing clinical K. pneumoniae isolates remain susceptible to fosfomycin [19,20]. Furthermore, fosfomycin has the potential to serve as a component of combination therapy in the treatment of infections caused by Enterobacterales.

To date, numerous studies have investigated fosfomycin resistance and its effects on virulence factors such as biofilm formation [21–23]. Nevertheless, our understanding of the effects of fosfomycin resistance on fitness, biofilm formation, and virulence-related genes in clinical K. pneumoniae strains remains limited. In this study, we aimed to investigate the potential biological burden (fitness cost) associated with fosfomycin resistance in E. coli and K. pneumoniae strains.

2. Materials and methods

2.1. Bacterial strains

A total of 112 bacterial isolates, including 78 E. coli and 34 K. pneumoniae strains obtained from urine samples collected from various departments at Ege University Faculty of Medicine Hospital, were examined in this study. Bacterial strains were identified using the MALDI-TOF MS system (Biotyper; Bruker Daltonics GmbH and Co. KG, Bremen, Germany), and antibiotic susceptibility testing was performed using the VITEK-2 automated system (bioMérieux SA, Marcy-l’Étoile, France). E. coli ATCC 25922 was used as the quality control strain. All bacterial strains were stored at −80 °C in 10% glycerol–brain heart infusion broth until use.

2.2. Determination of fosfomycin resistance

The minimum inhibitory concentrations (MICs) of fosfomycin against the collected E. coli and K. pneumoniae isolates were determined using the reference agar dilution method in accordance with the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Agar plates containing nine different concentrations of fosfomycin (0.5–128 μg/mL) supplemented with a fixed concentration (25 μg/mL) of glucose-6-phosphate were prepared. The lowest concentration of fosfomycin that inhibited visible bacterial growth after 16–18 h of incubation at 37 °C was recorded as the MIC. While the current EUCAST breakpoint (8 μg/mL) was used for interpreting the results of E. coli isolates, no accepted breakpoint exists for K. pneumoniae isolates in the current EUCAST documents. Therefore, K. pneumoniae isolates were not classified as resistant or susceptible. Instead, strains with MIC > 32 μg/mL were described as having a “high fosfomycin MIC” and those with MIC ≤ 32 μg/mL as having a “low fosfomycin MIC”.

2.3. Induction of fosfomycin resistance by serial passaging

To investigate the potential effects of fosfomycin resistance on bacterial fitness and virulence factors, two K. pneumoniae strains with low fosfomycin MICs (no. 4 and 24) and two fosfomycin-susceptible E. coli strains (no. 27 and ATCC 25922) were selected. Among the K. pneumoniae isolates, strain no. 4 was resistant to seven antibiotics—nitrofurantoin, amoxicillin/clavulanic acid, imipenem, gentamicin, ciprofloxacin, trimethoprim/sulfamethoxazole, and cefoxitin—whereas strain no. 24 was resistant only to nitrofurantoin. The E. coli strain (no. 27) exhibited resistance to four antibiotics—amoxicillin/clavulanic acid, gentamicin, ciprofloxacin, and trimethoprim/sulfamethoxazole. Fosfomycin resistance was induced through serial passaging following the method described by Li et al., with minor modifications [24]. Bacterial suspensions and culture media were prepared as described in the agar dilution method. Each strain was inoculated into culture media containing fosfomycin beginning at half of the MIC (MIC/2) and at twofold increasing concentrations up to 2048 μg/mL. After incubation at 37 °C for 48 h, bacterial suspensions were adjusted to 0.5 McFarland turbidity in sterile saline, and 50 μL of each suspension was transferred onto plates containing the next fosfomycin concentration. Passaging was continued until no visible bacterial growth was observed after 48 h of incubation. The fosfomycin MICs of the resistant variants growing on media containing the highest fosfomycin concentration were confirmed using the agar dilution method and then preserved in 10% glycerol–brain heart infusion broth at −80 °C. Fosfomycin-resistant variants were designated by adding the letter “R” to their original strain numbers.

2.4. Comparison of virulence gene expression levels between fosfomycin-susceptible and -resistant variants

The expression levels of the fimH and papC genes, encoding adhesion molecules, were compared among E. coli variants using quantitative real-time polymerase chain reaction (RT-qPCR) [25,26]. In K. pneumoniae variants, the expression levels of the uge, ycfM, and wabG genes associated with capsule biosynthesis, the mrkD gene encoding an adhesion molecule, and the entB gene encoding siderophore biosynthetic enzymes were also analyzed by RT-qPCR [27]. The recA and infB genes were used as reference genes for E. coli and K. pneumoniae, respectively. Total RNA was isolated from bacterial cultures, and complementary DNA (cDNA) was synthesized for RT-qPCR analysis. For each reaction, 2 μL of the obtained cDNA was transferred into 96-well RT-qPCR microplates, followed by the addition of 18 μL of reaction mixture to each well. Amplification was performed using the LightCycler 480 II system (Roche Diagnostics GmbH, Mannheim, Germany) under the following cycling conditions: one cycle at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 15 s, and extension at 72 °C for 20 s. Relative gene expression was calculated using the delta–delta CT method based on the threshold cycle (CT) values. All experiments were performed in triplicate to ensure reproducibility. A minimum twofold change in expression level between the variants was regarded as biologically significant.

2.5. Determination of biofilm-forming capacities of fosfomycin-susceptible and -resistant variants

The biofilm-forming capacities of E. coli and K. pneumoniae variants were determined using the spectrophotometric crystal violet (CV) staining method [28]. The resistant or high-MIC variants were incubated at 37 °C for 16–18 h on Mueller–Hinton agar (MHA) containing half the MIC concentration of fosfomycin (MIC/2) and 25 μg/mL glucose-6-phosphate. The susceptible or low-MIC variants were maintained under identical conditions on MHA plates without antibiotics. After incubation, several colonies were collected, and bacterial suspensions were prepared in 3 mL of sterile saline adjusted to a turbidity equivalent to 0.5 McFarland standard. Biofilm formation was assessed using the CV method in sterile 96-well flat-bottomed microplates [29]. The absorbance values were measured at 570 nm using a microplate reader (Varioskan Flash; Thermo Fisher Scientific Inc., Waltham, MA, USA). All experiments and measurements were performed in triplicate to ensure reproducibility. Tryptic soy broth was used as the negative control, and Enterococcus faecalis ATCC 29212 served as the positive control. The optical density (OD) values with standard deviations (SD) were calculated separately for each strain. To determine the biofilm-forming capacities of the variants, the OD values of each variant were compared with that of the negative control, as described in Table 1 [28,29]. The tested strains and their variants were classified as nonbiofilm, weak (low), moderate, or strong biofilm formers. Statistical analyses were performed using the Student’s t-test in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). A p < 0.05 was considered statistically significant.

Table 1.

Classification of biofilm-forming capacities of the examined strains.

OD ≤ Negative control (NC) No biofilm formation (−)
NC < OD ≤ 2 × NC Low biofilm formation (+)
2 × NC < OD < 4 × NC Moderate biofilm formation (++)
4 × NC ≤ OD Strong biofilm formation (+++)
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2.6. Determination of growth rates of fosfomycin-susceptible and -resistant variants

The growth rates of E. coli and K. pneumoniae variants were determined using a spectrophotometric method [30]. For this purpose, several colonies were collected from fosfomycin-resistant or high-MIC variants grown on MHA containing fosfomycin, and from fosfomycin-susceptible or low-MIC variants grown on MHA without fosfomycin. These colonies were used to prepare bacterial suspensions in sterile saline adjusted to a turbidity of 0.5 McFarland standard (approximately 1.5 × 108 colony-forming units/mL). Each suspension (5 μL) was inoculated into tubes containing 5 mL of Luria–Bertani broth. The inoculated tubes were incubated at 37 °C in a shaking incubator at 200 rpm for 10 h to ensure optimal aeration. The time point at which bacteria were inoculated into the broth was designated as time 0 h, and samples were collected at 0, 2, 4, 6, 8, and 10 h of incubation. Each sample (100 μL) was transferred in triplicate into sterile 96-well microplates. The OD was measured at 600 nm using a spectrophotometer (Varioskan Flash; Thermo Fisher Scientific Inc., Waltham, MA, USA), and absorbance values were recorded. Differences in bacterial growth rates were assessed based on the obtained OD values. For each sampling time point, the OD values of resistant and susceptible variants were compared using the Student’s t-test in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). A p < 0.05 was considered statistically significant. Statistical significance levels were denoted as p < 0.05*, p < 0.01**, and p < 0.001***.

3. Results

3.1. Fosfomycin resistance

The MICs of a total of 112 isolates, including 78 E. coli and 34 K. pneumoniae strains, were determined using the reference agar dilution method. According to EUCAST guidelines, E. coli strains with MIC > 8 μg/mL were considered resistant to fosfomycin. For K. pneumoniae isolates, those with MIC > 32 μg/mL were designated as having high fosfomycin MICs. Based on these criteria, 13 (16.6%) of 78 E. coli isolates were resistant to fosfomycin, whereas eight (23.5%) of 34 K. pneumoniae isolates exhibited high fosfomycin MIC values. The distribution of fosfomycin MIC values among the tested strains is presented in Table 2.

Table 2.

Fosfomycin MICs of E. coli and K. pneumoniae strains determined by the agar dilution method. Underlined values indicate the number of fosfomycin-resistant isolates.

Fosfomycin minimum inhibitory concentrations (MICs) (μg/mL)
<0.5 1 2 4 8 16 32 64 128
Escherichia coli (n = 78) 40 19 2 2 2 4 2 6 1
Klebsiella pneumoniae (n = 34) 8 1 3 6 3 2 3 0 8
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3.2. Fosfomycin-susceptible and-resistant bacterial variants

Fosfomycin-resistant and high-MIC variants were generated from two fosfomycin-susceptible E. coli strains (no. 27 and ATCC 25922) and two K. pneumoniae strains with low fosfomycin MICs (no. 4 and 24). Serial passaging on agar plates containing increasing concentrations of fosfomycin (from MIC/2 to 2048 μg/mL) was continued until no visible growth was observed after 48 h of incubation. The fosfomycin MICs for K. pneumoniae variants increased from 4 μg/mL to >2048 μg/mL. The fosfomycin MIC for E. coli strain no. 27 increased from <0.5 μg/mL to 1024 μg/mL, while that for the E. coli ATCC 25922 reference strain increased from <0.5 μg/mL to 512 μg/mL.

3.3. Differences in virulence gene expression levels between fosfomycin-susceptible and-resistant variants

A significant decrease in the expression level of the fimH gene was observed in the E. coli 25922R variant compared to its parental E. coli 25922 strain, whereas no significant change was detected between the susceptible and resistant variants of E. coli strain no. 27 (Figure 1a). No significant difference was observed in the expression levels of the papC gene between fosfomycin-resistant and susceptible E. coli variants (Figure 1b).

Figure 1.

Figure 1

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Relative expression levels of the fimH (a) and papC (b) genes in fosfomycin-resistant and -susceptible E. coli variants. * indicates a minimum twofold change in expression level.

The expression levels of the entB (Figure 2a), mrkD (Figure 2b), and ycfM (Figure 2c) genes did not differ significantly among the K. pneumoniae variants. A significant decrease in the expression of the uge gene was observed in the K. pneumoniae 4R variant compared to its parental 4 variant. Conversely, no significant difference in the expression level of the same gene was detected between K. pneumoniae 24 and 24R variants (Figure 3a). Additionally, the wabG gene was significantly overexpressed in the K. pneumoniae 4R variant compared with its parental 4 variant. In contrast, no significant difference in the expression level of the wabG gene was observed between K. pneumoniae 24 and 24R variants (Figure 3b).

Figure 2.

Figure 2

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Relative expression levels of the entB (a), mrkD (b), and ycfM (c) genes in K. pneumoniae variants. Low fosfomycin MIC value: 4 μg/mL; high fosfomycin MIC value: 2048 μg/mL. * indicates a minimum twofold change in expression level (not observed in this figure).

Figure 3.

Figure 3

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Relative expression levels of the uge (a) and wabG (b) genes in K. pneumoniae variants. Low fosfomycin MIC value: 4 μg/mL, high fosfomycin MIC value: 2048 μg/mL. * indicates a minimum twofold change in expression level.

3.4. Biofilm-forming capacities of fosfomycin-susceptible and -resistant variants

The tested strains and their variants were classified as nonbiofilm, weak (low), moderate, or strong biofilm formers according to the CV staining method. It was observed that K. pneumoniae strain no. 4 and its resistant variant (4R), as well as the two E. coli strains (no. 27 and ATCC 25922) and their resistant variants (27R and 25922R), were classified as weak biofilm formers. Moreover, K. pneumoniae strain no. 24 and its resistant variant (24R) exhibited a moderate level of biofilm formation. No significant difference was detected in the biofilm-forming capacities between the E. coli and K. pneumoniae variants examined (Figure 4).

Figure 4.

Figure 4

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Optical density (OD570) values of the examined E. coli and K. pneumoniae variants. Low fosfomycin MIC value: 4 μg/mL, high fosfomycin MIC value: 2048 μg/mL. NC: negative control; PC: positive control.

3.5. Comparison of growth rates between fosfomycin-susceptible and -resistant variants

The growth rates of the variants were determined by measuring the OD of samples collected at fixed time intervals from the bacterial suspensions. K. pneumoniae strain no. 4 exhibited a significantly higher absorbance value only at the 6th h compared with its resistant variant (4R). No significant differences were observed in the absorbance values between the variants of the same strain at the other time points (Figure 5a). However, K. pneumoniae strain no. 24 exhibited significantly higher absorbance values than its resistant variant (24R) at 2, 4, 6, 8, and 10 h (Figure 5b). Similarly, at 4, 6, 8, and 10 h, E. coli strains no. 27 and ATCC 25922 exhibited higher absorbance values than their resistant variants (27R and 25922R) (Figures 5c and 5d).

Figure 5.

Figure 5

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Growth curves showing the absorbance (OD600) values of the examined E. coli and K. pneumoniae variants labeled as 4/4R (a), 24/24R (b), 27/27R (c), 25922/25922R (d) over time. * p < 0.05, ** p < 0.01, *** p < 0.001.

4. Discussion

Because of the prolonged discovery process and the high costs associated with developing new antibiotics, the therapeutic options available for treating antibiotic-resistant bacteria are steadily decreasing [31]. Consequently, there has been renewed interest in so-called “old” antibiotics, such as colistin and fosfomycin [32]. Since its discovery, fosfomycin has been used primarily for the treatment of uncomplicated urinary tract infections, with limited application to other types of infections [5,33]. Given the limited number of antibiotics available for treating infections caused by antibiotic-resistant bacteria and its proven effectiveness against ESBL-producing strains, fosfomycin is increasingly being considered for therapeutic use beyond urinary tract infections. Furthermore, several studies have demonstrated that fosfomycin is effective against MDR K. pneumoniae isolates, thereby expanding its potential clinical applications [19,20]. However, the expanded clinical use of fosfomycin may accelerate the emergence of resistance [34,35]. In the present study, the MICs of fosfomycin were determined using the reference agar dilution method. Accordingly, the resistance rate to fosfomycin was 16.6% (13/78) among E. coli isolates. In a study conducted by Soydan et al. (2021), fosfomycin resistance was reported in 4.9% (4/89) of E. coli isolates [36]. In a 2022 study by Öcal and colleagues, fosfomycin resistance was detected in only six (3%) of 175 E. coli strains using the agar dilution method (MIC > 32 μg/mL indicating resistance) [37]. The fosfomycin resistance rate (approximately 17%) observed among E. coli isolates in this study was higher than those reported in previous studies. However, this discrepancy may be explained by the updated EUCAST breakpoints for fosfomycin, which lowered the susceptibility threshold for E. coli from 32 μg/mL to 8 μg/mL. Globally, numerous studies have addressed this issue; however, some did not employ the reference agar dilution method, and in certain cases, fosfomycin resistance was interpreted according to the Clinical and Laboratory Standards Institute breakpoints [38–41].

In this study, fosfomycin-resistant variants were developed from two fosfomycin-susceptible E. coli and two K. pneumoniae strains, and the expression levels of several virulence-associated genes were analyzed in these variants. The underlying mechanisms of fosfomycin resistance were not investigated in these selected strains, which represents a limitation of the present study. The analysis focused on variations in the fimH and papC genes in E. coli strains, and in the entB, mrkD, uge, ycfM, and wabG genes in K. pneumoniae strains. The fimH gene, responsible for adhesion to host epithelial cells, has been reported as one of the most frequently detected virulence factors in uropathogenic E. coli isolates. Its prevalence has been reported to range from 68% to 100% in various studies [25,42–45]. The pyelonephritis-associated pili (pap), also known as P fimbriae, are adhesins found in E. coli strains associated with pyelonephritis and significantly contribute to the virulence of uropathogenic E. coli [46]. In this study, the expression level of the fimH gene did not differ significantly between E. coli variants 27 and 27R. However, its expression was significantly reduced in the fosfomycin-resistant E. coli 25922R variant compared with the susceptible E. coli 25922 strain. No significant difference was observed in the expression levels of the papC gene between fosfomycin-resistant and susceptible E. coli variants. Harwalkar and colleagues reported that the papC gene was detected in 72.9% of ciprofloxacin-susceptible uropathogenic E. coli strains, whereas it was present in only 40.2% of ciprofloxacin-resistant strains [47]. Moreover, Pira et al. demonstrated that fosfomycin-resistant E. coli strains exhibited reduced adhesive capacity [48]. These findings suggest that resistant bacteria may prioritize other essential metabolic processes over the synthesis of P fimbriae, thereby reallocating their cellular energy and resources. Campos et al. reported that several resistance-associated genes were significantly downregulated or upregulated in fosfomycin-resistant E. coli subpopulations compared with their parental heteroresistant isolates [22]. However, they also reported that these alterations in gene expression did not impose a detectable fitness cost. Bermudez et al. observed that fosfomycin resistance did not impair virulence in uropathogenic E. coli strains [21]. Collectively, these studies suggest that fosfomycin-resistant isolates may display transcriptional alterations in virulence-related genes, although such molecular changes are not always manifested phenotypically.

K. pneumoniae regulates capsule and polysaccharide biosynthesis through genes such as uge, wabG, and ycfM. The absence of these genes impairs capsule formation in the bacteria, resulting in a marked reduction in virulence [49,50]. Some genes, including fimH and ycfM, are responsible for the synthesis of adhesion molecules that facilitate bacterial attachment to host epithelial cells [51]. Enterobactin, encoded by the entB gene, is one of the major siderophores produced by K. pneumoniae [52]. In this study, no significant differences were observed in the expression levels of the entB, mrkD, and ycfM genes between the high- and low-MIC K. pneumoniae variants. However, in the high-MIC K. pneumoniae variant 4R, the expression of the uge gene was significantly reduced, whereas the wabG gene was upregulated compared with its parental 4 variant. In contrast, no significant differences in the expression levels of these genes were observed between K. pneumoniae 24 and 24R variants. Since both the uge and wabG genes are involved in bacterial polysaccharide biosynthesis, it is plausible that the decreased expression of uge in the K. pneumoniae 4R variant was compensated by the overexpression of wabG. This study presents, for the first time, data demonstrating how fosfomycin resistance influences the expression of virulence-associated genes in clinical K. pneumoniae isolates. Although fosfomycin resistance did not induce alterations in most of the virulence-associated genes examined in K. pneumoniae variants, compensatory regulation may occur among genes with overlapping functions.

Biofilm formation plays a crucial role in Enterobacteriaceae infections and is frequently observed among K. pneumoniae isolates [53,54]. Consistent with our biofilm formation results, no significant differences were detected between the E. coli and K. pneumoniae variants examined. These findings suggest that the acquisition of fosfomycin resistance did not alter the biofilm-forming capacity of the strains examined.

The development of urinary tract infections requires the presence of a sufficient bacterial load within the urinary tract, and these bacteria must possess the ability to adhere to host tissues. The continuous flushing of the urinary tract by urine creates a challenging environment for bacterial adherence. Moreover, the biological disadvantage associated with the acquisition of antibiotic resistance reduces bacterial growth rates, thereby hindering the attainment of the critical inoculum required for infection establishment. The observation that fosfomycin resistance can be easily acquired in vitro, whereas clinical isolates from infections often remain susceptible to fosfomycin, supports this hypothesis [55]. In the present study, the growth rates of fosfomycin-susceptible and resistant bacterial variants were determined, and the results indicated that fosfomycin resistance generally tended to reduce the growth rates of the examined strains, except for the K. pneumoniae variant 4R. Pira et al. compared the growth rates of fosfomycin-resistant E. coli (two strains) and K. pneumoniae (one strain) with those of their isogenic susceptible counterparts. They found that the growth rates of resistant E. coli variants were lower than those of the susceptible variants. However, no significant difference in growth rate was observed between the K. pneumoniae variants [48]. Additionally, other studies have suggested that the acquisition of resistance to certain antibiotics may not impose a measurable fitness cost among members of the order Enterobacterales [56].

The results of this study revealed that, although some differences were detected in the expression of virulence-associated genes between fosfomycin-susceptible and -resistant E. coli variants, as well as between low- and high-MIC K. pneumoniae variants, these differences were not reflected in their biofilm-forming capacities. Furthermore, it was determined that fosfomycin resistance generally imposes a fitness cost on the examined strains. Although the inclusion of only four strains represents a limitation of this study, it nevertheless provides valuable insights into the biological profiles of the selected clinical isolates.

In light of the increasing rates of antibiotic resistance, expanding the use of fosfomycin against MDR pathogens, including K. pneumoniae isolates, is being considered. In this context, the expression levels of virulence-associated genes in fosfomycin-susceptible and -resistant K. pneumoniae variants were assessed for the first time in this study. The findings of this study may contribute to the rational evaluation of fosfomycin use in K. pneumoniae infections. These findings also suggest that the clinical use of fosfomycin should be carefully monitored to prevent the emergence of resistance.

Acknowledgments

This study was supported by the Ege University Scientific Research Projects Coordination Unit (project no: TDK-2020-21813). All biological activity measurements were performed at the Pharmaceutical Sciences Research Center (FABAL), Faculty of Pharmacy, Ege University.

Funding Statement

This study was supported by the Ege University Scientific Research Projects Coordination Unit (project no: TDK-2020-21813).

Footnotes

Ethical approval: This study was approved by the Ege University Medical Research Ethics Committee (approval date: 05 February 2020; approval no: 20-2T/26).

Author contributions: Mustafa Ökeer: Conceptualization: equal; data curation: equal; formal analysis: equal; investigation: equal; methodology: equal; software: lead; visualization: lead; writing–original draft: equal; writing–review and editing: equal.

Sabire Şöhret Aydemir: Conceptualization: equal; data curation: equal; resources: lead; writing–review and editing: supporting.

Bayrı Eraç: Conceptualization: lead; data curation: equal; formal analysis: lead; funding acquisition: lead; investigation: equal; methodology: lead; project administration: lead; supervision: lead; writing–original draft: equal; writing – review and editing: equal.

Conflicts of interest: On behalf of all authors, the corresponding author declares that there are no conflicts of interest.

Availability of data and material

All data generated or analyzed during this study are included in this published article.

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