Diabetic foot (DF) is a serious late complication of diabetes that dramatically increases the risk of lower limb amputations (1). More importantly, it increases patient morbidity and mortality (2). Infection is one of the key components of DF, contributing to unfavorable patient prognosis and poor podiatric outcomes (3). Early diagnosis of diabetic foot infection (DFI), followed by prompt and aggressive therapy, has the potential not only to slow disease progression, but also to delay or even reverse the above complications (4).

For DFI management, podiatrists have several treatment modalities at their disposal. In mild forms of DFI, antiseptics or local devices with anti-infective substances can be effective in certain cases (5). For mild and moderate stages of DFI, antibiotics (ATBs) administered in various oral or intravenous regimens are recommended (6). For severe forms of DFI accompanied by sepsis, parenteral ATB therapy is strictly indicated (7).

ATBs are selected based on causative microbial agents and microbial sensitivity. To ensure an adequate antibacterial response, it is essential to administer ATBs at levels conducive to optimal bactericidal activity in both serum and peripheral tissues (8). However, the efficacy of ATBs, particularly in peripheral tissues, can be affected by several factors. We hypothesize that macro- and microangiopathy are the most influential of these. However, researchers have yet to comprehensively address the serum and tissue concentrations of time-dependent ATBs, that are not routinely monitored in patients with DFI, especially those suffering from peripheral arterial disease (PAD). Therefore, the aim of our study was to assess the availability and bactericidal effect of ATBs in patients with infected diabetic foot ulcers (IDFUs) in the context of micro- and macrocirculation status.

The study cohorts did not differ significantly in basal characteristics, including circulation parameters ( Table 1 ). DFUs were infected by 2 to 3 pathogens on average (2.5 ± 0.9 microorganisms per DFU in the AMC group vs. 2.5 ± 1.4 bacteria per DFU in the CTZ group; NS). However, there were variations in the MIC of the causative agents (0.54 ± 0.54 vs. 4.68 ± 3.2; P = 0.002; (PH=0.008); Table 2 ).

Concerning the assessment of pharmacokinetic parameters for AMC, the maximum serum levels of AMC (C_max) after bolus administration peaked at around 91.8 ± 52.5 μg mL^-1. AMC concentrations in peripheral tissues (C_max-tissue) reached approximately 9 – 31% of serum levels (ranging from 0.78 ± 0.97 to 7.3 ± 4.5 μg mL^-1; P = 0.0001 - 0.036; (PH=0.0004 - >0.1; Figure 2A ).

When examining the area under the concentration–time curve for plasma or tissue, these microbicidal parameters proved relatively satisfactory. Similarly, when comparing the same parameters with MIC values, their ratios were within an acceptable range ( Table 2 ). To treat DFI effectively, more than half of the daily tissue ATB concentrations must be greater than the MIC of the causative microbial agent. Among AMC-treated patients, only 67% had tissue concentrations that were above the MIC for at least 50% of the time (≥ 50% _tissue fT > MIC; Figure 3A ). In contrast, only 33% of patients with IDFUs maintained tissue concentrations above the MIC for at least 100% of the time (≥ 100% _tissue fT > MIC).

Patients treated with CTZ reached maximum serum CTZ levels of almost 187 μg mL^-1. In patients with IDFUs treated with CTZ, the maximum peripheral tissue concentration was 18.6 ± 7.4 μg mL^-1. Compared to serum levels, tissue CTZ levels ranged between 4.8 ± 2.1 and 18.6 ± 7.4 μg mL^-1; P = 0.0001; (PH=0.0004); Figure 2B ), reaching approximately 9 – 22% of serum CTZ concentrations. Other pharmacological parameters detected in patients from the CTZ group are given in Table 2 . Similar to the study participants treated with AMC, only 55% patients achieved more than half of the daily tissue CTZ concentrations higher than the MIC of pathogens ( Figure 3B ). Of the CTZ-treated patients, 45% had ≥ 100% _tissue fT > MIC.

We identified positive and negative correlations of ATB bactericidal activity markers with some patient characteristics, particularly renal functions and body weight ( Table 3 ). Regarding circulation parameters, only arterial flow correlated significantly with tissue ATB concentrations. The correlation coefficient for the first arterial phase was r = 0.42 (P = 0.045; (PH - >0.1)), and for the second phase, it was r = 0.44 – 0.55 (P = 0.0463 - 0.01; PH>0.1 - >0.1). Other macro- and microcirculation determinants (including plethysmographic interarm difference with cut-point of 25 mm, Figures 4A, B ) did not correlate significantly with pharmacokinetic parameters.

The maximum effect of ATB therapy in iDFUs depends on achieving serum and tissue concentrations that provide effective bactericidal action. The efficacy of ATB therapy is typically assessed based on clinical evaluation during podiatric care, rarely using diagnostic methods. Serum ATB levels are usually only monitored for dose-dependent ATBs such as amikacin and gentamicin. However, time-dependent ATBs used to treat DFIs, including beta-lactams and cephalosporins (32), are not routinely assessed in the laboratory. In fact, there are almost no data on the pharmacokinetic and pharmacodynamic effects of these ATBs on the serum and peripheral tissues of DF patients (33).

The DFIATIM study aims not only to fill this knowledge gap, but also to test a number of clinical hypotheses. One of these is that PAD or microcirculatory impairment significantly affects the bactericidal activity and tissue availability of administered ATBs, especially in the DF. The potential influence of PAD on the accessibility of time-dependent ATBs in the lower limbs has yet to be addressed in the literature. Microdialysis is the most suitable method for clearly determining tissue concentrations of ATBs, monitoring their pharmacokinetic and pharmacodynamic properties, and ensuring the efficacy of intravenous ATB administration in patients with infected DF and PAD (29, 34). Before the introduction of microdialysis into clinical research, ATBs were detectable only in samples obtained from surgical resections or amputations, including even bone material (34, 35). Microdialysis facilitates real-time in vivo monitoring of ATBs (34). This is largely due to the use of a porous catheter membrane, which allows ATBs to diffuse across the membrane into the catheter according to the concentration gradient and the molecular weight of ATBs. We have previously validated this technique using our own in vitro and in vivo experiments (19).

Several methods, each with its own advantages and disadvantages, can be used to determine the concentrations of ATBs in serum and tissue. For our ongoing DFIATIM trial, we use a modern analytical method based on CE–C⁴D (23) to sensitively determine ATBs in microdialysis samples (35). Compared to conventional HPLC techniques, CE approaches to ATB monitoring in microdialysis offer greater insights for several reasons (36). The amount of the sample injected into the separation capillary varies in nanoliter volumes, forming a complementary technique intended for tissue sampling by microdialysis. CE–C⁴D enables direct measurement of a wide range of ATBs in their native states, bypassing the need for complex derivatization reactions (37) or sample treatments that are difficult to perform in microliter amounts. The short migration times of electrophoretic analysis (38) are essential for continuous monitoring of microdialysates, particularly in pharmacological and physiological studies (39). Our previous study clearly demonstrated that the electrophoretic stacking technique is suitable for accurate determination of cephalosporin CTZ and beta-lactam AMC in the blood plasma and microdialysate of foot tissue after ATB administration (19). The concentrations we obtained were in close agreement with a conventional HPLC technique (40).

After incorporating new microdialysis and CE methods within our podiatric practice, we decided to carry out the prospective DFIATIM trial to elucidate the time-dependent concentrations of ATBs in serum and tissue as well as their bactericidal activities. We were particularly interested to discover in one substudy of mentioned trial how the vascular status of the study participants could influence these parameters.

Our results revealed that bolus intravenous administration of both AMC and CTZ yielded satisfactory serum ATB levels. Similarly, Gariani et al. confirmed the satisfactory clinical impact of oral AMC on DFI, similar to clinical outcomes for other antimicrobial regimens (41). However, data on CTZ are practically non-existent in the literature. The bactericidal activity of ATBs are determined by the time interval over which ATB concentrations in tissue exceed the MIC. For AMC, tissue concentration must be ≥ 50% fT > MIC, and for CTZ, the tissue concentration must be ≥ 60% fT > MIC. In our study, these criteria were met in only 67% of patients treated with AMC and 55% of patients with CTZ. However, it was very difficult from the point of macro- and microcirculation parameters to subanalyse patients achieving or non-achieving appropriate MIC of ATBs in peripheral tissue. The subgroups were very small ((8 patients treated by AMC achieved MIC for more than 50% of evaluation time vs. 4 patients non-achieving these levels/similarly 6 patients treated by CTZ achieved MIC for more than 60% of evaluation time vs. 5 patients without sufficient tissue concentrations), and it was impossible to perform appropriate statistical power analyses. Similar situation is when we would like to compare patients achieving or not appropriate plethysmographic parameters including interarm distance.

In our study, ATB levels in serum and tissue, particularly in the AMC group, positively correlated with serum creatinine levels and negatively with weight, BMI, albumin, and glomerular filtration. Similar correlations in connection to AMC treatment were recently documented by Brazil experts (42). Therefore, it is imperative to highlight the importance, during DFI therapy, of tailoring ATB dosage schemes based on factors such as patient weight, BMI, drug distribution, nutrition, and renal functions.

Regarding the relationship between PAD, microcirculatory impairment, ATB availability, and bactericidal activity, we noted significant positive correlations solely between maximum ATB tissue levels and their AUC with arterial flow, as detected in the treated lower limb by OP. Interestingly, neither ABI, TBI, nor TcPO₂ correlated significantly with measured pharmacokinetic and pharmacodynamic parameters. These original findings on the interconnection between vascular status, ATB availability, and bactericidal activity have yet to be reported in the literature. A study by Raymakers et al. underscored the importance of tissue perfusion (TcPO₂) in the penetration of CTZ into the skin, muscles, and bones. However, this study did not follow ATB concentrations in real time, and results were based on immediate ATB levels detected from tissue obtained during amputation procedures (33).

In our study, TcPO2, like ABI and TBI, did not correlate with real-time pharmacology data in either AMC or CTZ. This indicates that the macrocirculation status represented by ABI and TBI and the microcirculation status represented by TcPO₂, are not as important as the volume of blood that reaches the treated foot through the native arterial network and pre-existing capillaries. Our results clearly demonstrate the important role of plethysmography in the diagnosis of vascular changes (exhibiting sensitivity of 73% to 90% and specificity of 77% to 96% for PAD diagnosis) in patients with IDFUs, reinforcing the need for comprehensive vascular examination in podiatric patients at all times (43).

One of the limitations of this study is the small number of patients enrolled. However, microdialysis studies are technically and logistically demanding to perform and generally involve small numbers of participants. Additionally, patients with critical limb-threatening ischemia were excluded from the study, since these patients typically require urgent revascularization procedures or even major amputations.

In summary, ATB treatment of patients with IDFUs proved satisfactory in only half to two-thirds of study participants. The availability and bactericidal activity of ATBs appear to be impaired by vascular changes, particularly by the volume of arterial flow in the treated lower limbs.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by The ethics committees of the Institute for Clinical and Experimental Medicine and Thomayer University Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

VF: Writing – original draft. RJ: Investigation, Writing – review & editing. SA: Methodology, Writing – review & editing. JH: Investigation, Writing – review & editing. VW: Investigation, Writing – review & editing. PB: Methodology, Writing – review & editing. JM: Supervision, Writing – review & editing. PT: Investigation, Methodology, Supervision, Writing – original draft. JP: Methodology, Supervision, Writing – review & editing. MD: Supervision, Writing – review & editing. DS: Supervision, Writing – review & editing. VL: Investigation, Writing – review & editing. MP: Supervision, Writing – review & editing.