# Intermittent antibiotic exposure of Escherichia coli biofilms drives resistance in catheter-associated infection models

Yutaka Yoshii, Stanislas Thiriet-Rupert, David Lebeaux, Jean-Marc Ghigo, Christophe Beloin

► To cite this version:

Yutaka Yoshii, Stanislas Thiriet-Rupert, David Lebeaux, Jean-Marc Ghigo, Christophe Beloin. Intermittent antibiotic exposure of Escherichia coli biofilms drives resistance in catheter-associated infection models. npj Biofilms and Microbiomes, 2026, 12 (1), pp.41. 10.1038/s41522-025-00906-4. pasteur-05464676

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Submitted on 19 Jan 2026

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1 Intermittent Antibiotic Exposure of *Escherichia coli* Biofilms Drives Resistance in Catheter-

2 Associated Infection Models

3

4 Yutaka YOSHII¹*†, Stanislas THIRIET-RUPERT¹, David LEBEAUX¹,², Jean-Marc GHIGO¹,

5 Christophe BELOIN¹*

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7 ¹ Institut Pasteur, Université Paris Cité, Genetics of Biofilms Laboratory, CNRS UMR6047,

8 75015, Paris, France.

9 ² Department of Infectious Diseases, Saint Louis-Lariboisière Hospital, Assistance Publique-

10 Hôpitaux de Paris, Université Paris Cité, Paris, France.

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12 *Co-corresponding author: Christophe BELOIN (christophe.beloin@pasteur.fr), Yutaka YOSHII

13 (yutaka.yoshii@inserm.fr)

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15 †Present address: Université Paris Cité, INSERM, IAME, Paris 75018, France.

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17 **Keywords:** Amikacin, Biofilm, catheter-related infection, *Escherichia coli*, Intermittent

18 treatment

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ABSTRACT

The use of antibiotic lock therapy (ALT) to protect catheters from infection is still being debated due to its inconsistent effectiveness and the potential risk of promoting antibiotic resistance. Using *in vitro* approaches and a clinically relevant rat model of pediatric venous access ports, which are commonly used in clinical settings, we demonstrated that 10 days of continuous therapy eradicates *Escherichia coli* biofilms *in vitro* without the emergence of antibiotic resistance. By contrast, an 8-hour intermittent therapy used for infected parenteral nutrition patients rapidly selected low-level amikacin-resistant mutants primarily due to convergent *fusA*, *sbmA*, and *cpxA* mutations. Our findings indicate that intermittent dosing generates pulsed selective pressure, favoring the development of resistance mutants within spatially structured biofilm communities. This suggests that biofilms may act as evolutionary incubators, in which medical interventions could unintentionally influence adaptation outcomes. Furthermore, the low-level resistance developing in treated biofilms may be overlooked in clinical settings and contribute to the selection of high-level resistant mutants.

Our study, therefore, underscores that, in addition to dosing, optimizing the timing of antimicrobial treatment could mitigate the emergence of resistance. These principles are applicable beyond catheters to any biofilm-related infections where short-term antibiotic exposure may impact microbial community adaptation.
## INTRODUCTION

Antimicrobial resistance (AMR) is rapidly emerging in a group of bacterial species collectively known by the acronym ESKAPEE (*Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Enterobacter* species, and *Escherichia coli*), posing a global threat to human and animal health ¹,². Among the ESKAPEE group, the Gram-negative bacterium *E. coli* is a leading cause of AMR-related morbidity and mortality worldwide ¹. Whereas *E. coli* is generally susceptible to many antimicrobial agents, the misuse of antibiotics or ill-adapted treatment regimens can rapidly lead to resistance ³,⁴. Considering the difficulties in developing new antibiotics, understanding how *E. coli* evolves antibiotic resistance during and after antibiotic treatment could mitigate the impact of AMR in this bacterium ⁵.

Pathogenic *E. coli* are well-known agents of acute gastrointestinal infections and chronic urinary tract infections, but also responsible for recalcitrant catheter infections ⁶⁻¹⁰. Among the causative pathogens of central venous catheter-related infections, although *Staphylococcus aureus* and coagulase-negative staphylococci remain the most prevalent microorganisms ¹¹, recent surveillance data reveal a growing proportion of gram-negative bacteria, including *E. coli*, which account for 9.7–20% of these infections ¹¹⁻¹⁶. Therefore, in clinical settings, the significance of *E. coli* as a cause of catheter-related infections, exhibiting high and multifactorial antibiotic tolerance due to biofilms ¹⁷,¹⁸, has gained increased attention ¹⁹.

Laboratory evolution experiments on biofilms under antibiotic treatments with sub- to above-minimum Inhibitory concentration (MICs) for short to long periods showed that biofilm tolerance could serve as a stepping stone for AMR ²⁰⁻²⁶. Using an *in vitro* biofilm model on silicone disks, we recently demonstrated that *E. coli* biofilms evolved resistance faster and at higher levels than planktonic cells after repeated treatments with the aminoglycoside amikacin at above-MIC concentrations ²⁷. However, the evolutionary dynamics of biofilms are highly influenced by environmental and experimental factors, such as bacterial strains, inoculum sizes, nutrient levels, and experimental duration ²⁸. Understanding the emergence of AMR in biofilms exposed to clinically relevant conditions is therefore crucial for developing effective treatments and limiting antibiotic resistance.
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In this study, we used *in vitro* and *in vivo* pre-clinical rat models to assess the efficacy and risks of the emergence of antibiotic resistance after continuous and intermittent treatment for totally implantable venous access ports (TIVAP), a medical device widely used in clinics to administer intravenous drips or nutrients <sup>29,30</sup>. These TIVAPs, which are composed of a subcutaneous injectable chamber connected to the venous circulation by a catheter, are routinely contaminated upon manipulation or anterograde colonization, often leading to biofilm infections <sup>30,31</sup>. The standard treatment to salvage or protect these devices consists of an antibiotic lock therapy (ALT), during which high concentrations of antibiotics, typically 100–1,000 times the MIC, are injected into the catheter lumen. This treatment is performed continuously for 7–14 days or intermittently in parenteral nutrition infusion and hemodialysis cases <sup>30,32-36</sup>, with mixed clinical efficiency outcomes <sup>37-39</sup>. Moreover, concerns are rising about antibiotic resistance observed after intermittent ALT in hemodialysis patients <sup>40,41</sup>.

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Here, we investigated the efficacy and AMR-associated risks of continuous and intermittent ALT amikacin (ALT<sub>AMK</sub>) on biofilm in pediatric TIVAP, both *in vitro* and *in vivo*. We showed that a 10-day continuous ALT<sub>AMK</sub> efficiently eradicated *E. coli* biofilms without favoring the emergence of resistance. In contrast, intermittent ALT<sub>AMK</sub> not only failed to fully eradicate *E. coli* biofilms but also promoted the emergence of antibiotic resistance to amikacin. This study highlights the need to consider potential evolutionary paths toward antibiotic resistance in biofilm-associated infections and provides valuable insights for developing a safer and more efficient management of catheters.

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RESULTS

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Continuous 10- and 7-day ALT treatments of E. coli biofilms with amikacin do not promote
the emergence of antibiotic resistance

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To study the potential emergence of AMR in biofilms in clinical devices, we established an *in vitro* model using pediatric TIVAP, where biofilms of the adherent-invasive *E. coli* strain LF82 ⁴² composed of ca 4.0 × 10⁸ cells/TIVAP were formed after 48-hour continuous flow of LB bacterial growth medium (Figure 1a, Supplementary Figure 1a–d). This model was subjected to amikacin antibiotic lock therapy (ALT<sub>AMK</sub>), demonstrating that 48-hour *E. coli* LF82 biofilms are sensitive to a 1-day ALT<sub>AMK</sub> in a dose-dependent manner (Supplementary Figure 1e and f). The ALT<sub>AMK</sub> at a concentration of 500 times the MIC resulted in approximately a five-log reduction in free-floating and biofilm cells compared to the PBS control (ALT<sub>PBS</sub>), confirming that the ALT<sub>AMK</sub> effectively reduces the survival of biofilm cells in our *in vitro* TIVAP system.

Considering that guidelines for the treatment of TIVAP infections recommend consecutive 7–14 days of ALT using antibiotic concentrations at 100–1,000 times the MIC<sup>33-36</sup>, we compared the effects of a daily-exchanged ALT<sub>AMK</sub> at 500 times the MIC for 10 and 7 days (Figure 1b–f). 10-day continuous ALT<sub>AMK</sub> treatment prevented bacterial regrowth and successfully eradicated biofilm bacteria at the endpoint (Figure 1c and e). By contrast, despite showing undetectable levels of free-floating cells one hour after the recovery phase, the 7-day treatment allowed bacterial regrowth after the 3-day recovery phase in all three tested TIVAPs (Figure 1d and Supplementary Figure 2a). The final bacterial counts in the 7-day ALT<sub>AMK</sub> group were almost equivalent to the ALT<sub>PBS</sub> control group, with approximately 10<sup>8</sup> colony-forming units (CFU)/mL of free-floating cells and 10<sup>9</sup> CFU/TIVAP in biofilm samples (Figure 1d and f).

We also assessed the development of amikacin resistance in the recovered bacterial population during the 7-day continuous ALT<sub>AMK</sub> experiment by measuring growth frequency on amikacin plates with 1 time the MIC. The ALT<sub>AMK</sub>-treated biofilm populations showed growth frequencies of less than 0.1%, like those of the ALT<sub>PBS</sub> control group (Figure 1g and Supplementary Figure 2b). Additionally, the population amikacin MICs for treated and control biofilm bacteria were 16 µg/mL in LB and 4 µg/mL in Mueller-Hinton (MH), matching those of the ancestor (Figure 1h and Supplementary Figure 2c). Consistent with this absence of increase in MICs, no genetic mutation in genes related to aminoglycoside antibiotic resistance
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was identified in endpoint biofilm populations from these ALT<sub>PBS</sub> and ALT<sub>AMK</sub> groups (Supplementary Data 1). This demonstrated that the 7-day continuous ALT<sub>AMK</sub>, while inefficient regarding biofilm eradication, did not cause the emergence of amikacin resistance.

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Intermittent ALT treatment of TIVAP in vitro biofilms leads to low-level amikacin resistance

We then investigated the impact of intermittent ALT<sub>AMK</sub> treatment, which is recommended for patients on total parenteral nutrition (TPN) with infected long-term catheters. For TPN catheter infection treatment in clinics, antibiotics at 100–1,000 times the MIC are installed in the catheter lumen for 8 to 12 hours and alternated with TPN administration during the rest of the day, and treatment/nutrition cycles are repeated over several days <sup>36</sup>, which led to mixed clinical efficiency outcomes in some parenteral nutrition infusion and hemodialysis cases <sup>37-39</sup>. We assessed treatment efficacy and the emergence of amikacin resistance in two biofilm-forming <i>E. coli</i> strains, LF82 and the enteroaggregative 55989 <sup>43</sup>. We conducted 7 cycles of a 1-day cycle: 8 hours of intermittent ALT<sub>AMK</sub> at 500 times the MIC and 16 hours of continuous LB flow without antibiotics. These cycles concluded with a 3-day recovery phase (Figure 2a). The treatment regimen used in our study closely mimics the one used in clinics.

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When 48-hour biofilms were subjected to the intermittent ALT<sub>AMK</sub>, both strains exhibited a reduction in survival during each ALT cycle. However, this reduction diminished during the final cycle. Moreover, after the 3-day recovery phase, the numbers of free-floating and biofilm cells were similar between the ALT<sub>AMK</sub> and ALT<sub>PBS</sub> control groups, indicating treatment failure (Figure 2b–e and Supplementary Figure 3a, b, e, and f).

Growth frequencies on amikacin plates at 1 time the MIC revealed a gradual increase in the number of free-floating cells of the ALT<sub>AMK</sub> groups over the cycles. By the end of the cycles, two out of three LF82 replicates reached 10–100% growth frequencies, and all three 55989 replicates reached 1–10%. In contrast, the ALT<sub>PBS</sub> control groups maintained low growth frequencies of 0.001–0.1% for both free-floating and biofilm cells of both strains (Figure 2f and g, and Supplementary Figure 3c and g). In the ALT<sub>AMK</sub> groups, the population amikacin MICs increased to 32 µg/mL in LB (8 µg/mL in MH) for all three LF82 replicates (Figure 2h and Supplementary Figure 3d) and to 24 µg/mL in LB (6 µg/mL in MH) for all three 55989 replicates, higher than the ancestor strains’ MICs (Figure 2i and Supplementary Figure 3h). According to
CLSI guidelines, the MIC breakpoints for amikacin against E. coli in MH medium are ≤ 4 µg/mL for susceptible isolates and ≥ 16 µg/mL for resistant ones ⁴⁴. Therefore, the observed MICs were indicative of the selection of low-level antibiotic resistance.

To identify the genetic factors associated with the increased MIC and growth frequency observed in the ALT<sub>AMK</sub> group, we sequenced endpoint LF82 and 55989 biofilm populations obtained from each part of ALT<sub>AMK</sub>-treated TIVAPs (silicone septum, port, and catheter parts of TIVAPs n°4–6). Mutations with a frequency of 20% or more for LF82 and 10% or more for 55989, compared to the ancestral population, are shown in Figure 2j and k. We identified convergence in the mutation profiles for the two strains with mutations in the *sbmA* and *fusA* genes (Supplementary Data 1). *sbmA* encodes an inner-membrane peptide transporter while *fusA* encodes the essential elongation factor G. Loss-of-function mutations in *sbmA* and *fusA* have been previously associated with increased aminoglycoside resistance<sup>27,45-50</sup>. The *sbmA* gene was mutated in two out of the three LF82 biofilm populations and deleted in two out of three 55989 biofilm populations, along with surrounding neighboring genes, including *phoA_2*, *iraP*, *ddIA*, *KOPPCICB_04469*, *yaiY*, *yaiW*, *KOPPCICB_04473*, *ampH*, *iprA*, and *KOPPCICB_04476* (Figure 2j and k, Table 1, and Supplementary Data 1). The *fusA* gene was mutated in two out of three 55989 biofilm populations (Figure 2k). Additionally, mutations in the *fusA* gene were also detected at low frequencies in two out of three LF82 biofilm populations (Supplementary Data 1). To validate that these mutations are specific to AMK treatment, we sequenced untreated PBS biofilm populations from one of the two strains, LF 82. *sbmA* and *fusA* mutations were absent in this PBS control group (Supplementary Data 1). Interestingly, however, in this LF82 PBS control group, we detected mutations in the *fimA* gene, which encodes the major subunit of type 1 fimbriae. These mutations were also present in the AMK intermittently treated biofilm LF82 and 55989 populations (Supplementary Data 1). Since these *fimA* mutations were also present in the 7-day continuous control and treated LF82 biofilms, it is likely that they have been selected by the biofilm lifestyle and may affect biofilm-forming capacity in TIVAPs, hence possibly tolerance to AMK.

Overall, these results demonstrate that *in vitro* intermittent ALT<sub>AMK</sub> treatment of TIVAP failed to eradicate biofilms and promoted the emergence of low-level resistance, potentially caused notably by mutations in *sbmA* and *fusA*.
Emergence of resistance occurs when using a clinically relevant total parenteral nutrition

To further explore the clinical relevance of the observed emergence of amikacin resistance in intermittent ALT<sub>AMK</sub> under flow conditions, considering that total TPN in clinical settings is composed of highly controlled compounds capable of bacterial growth but not optimal compared to LB<sup>51,52</sup>, we tested whether using total TPN medium instead of LB in our *in vitro* model of TIVAP infection would influence the effectiveness of ALT<sub>AMK</sub> treatment. First, we verified that the bioluminescent *E. coli* 55989 carrying the pAT881 plasmid<sup>53</sup> formed biofilms under continuous TPN flow (Supplementary Figure 4a and b). We then applied four one-day cycles of an 8-hour lock/16-hour regrowth regimen of intermittent ALT<sub>AMK</sub> at 500 times the MIC, using TPN instead of LB at all stages of the experiment after the inoculation/adhesion step (Figure 3a). In the ALT<sub>AMK</sub> group, CFU counts of free-floating cells exhibited less survival reduction compared to what we observed in LB (Figure 3b and Supplementary Figure 4d), consistent with reduced bacterial load in TPN vs LB. After the 3-day recovery phase, the biofilm cells in the ALT<sub>AMK</sub> group were almost equivalent to those in the control ALT<sub>PBS</sub> group, indicating treatment failure (Figure 3c and Supplementary Figure 4f). Consistent with the CFU counts of free-floating samples over time, bioluminescence imaging revealed that biofilm bioluminescent intensities decreased during ALT<sub>AMK</sub> treatment but rebounded during the recovery phase (Figure 3d, and Supplementary Figure 4c and e). Although growth frequencies on amikacin plates at 1 time the MIC could not be analyzed for the ALT<sub>AMK</sub> group due to low bacterial counts, this group showed *ca.* 10-fold increased frequencies (0.1–1%) in recovery-phase free-floating and endpoint biofilm samples in two out of three ALT<sub>AMK</sub> group replicates (TIVAPs n°5 and 6), compared to the PBS group (0.01–0.1%). In the PBS group, resistance frequencies also increased, to a lesser extent. This result is consistent with the well-documented enhanced mutation rate in biofilms, increasing the likelihood of accumulating emergent antibiotic resistance mutations, even in the absence of antibiotic pressure<sup>27,54-56</sup> (Figure 3e and Supplementary Figure 4g). The two ALT<sub>AMK</sub> group replicates demonstrated low-level resistance at 24 µg/mL in LB (6 µg/mL in MH) of amikacin (Figure 3f and Supplementary Figure 4h).

We then sequenced endpoint biofilm populations in the two TIVAPs with the increased MIC (ALT<sub>AMK</sub> TIVAPs n°5 and 6), using collected samples from amikacin plates at 1 time the MIC.
Consistent with *in vitro* TIVAP experiments performed with LB, we identified convergent mutations in the *fusA* and *cpxA* genes. *fusA* mutations were detected at frequencies ≥10% in all parts of TIVAP n°6 (Figure 3g) and at lower frequencies in TIVAP 5 (Supplementary Data 1). *cpxA* mutations were detected at frequencies ≥10% in all parts of both TIVAPs n°5 and n°6. *CpxA* serves as the sensor kinase of the CpxAR two-component regulatory system. Mutations in the *cpxA* gene can confer aminoglycoside resistance by regulating the *acrD* gene, encoding AcrD transporter belonging to the resistance-nodulation-division family and a component of the AcrAD-TolC efflux pump⁵⁷.

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These results confirmed that *in vitro* amikacin intermittent treatment of biofilms using a clinically relevant parenteral nutrition medium and regimen also fails to eradicate biofilms, leading to the emergence of low-level antibiotic resistance.

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**Intermittently treated biofilm with amikacin leads to the emergence of resistance in a pre-clinical catheter-associated infection model**

To validate our *in vitro* findings *in vivo*, we used a previously described rat model that reproduces most clinically relevant situations associated with TIVAP biofilm infections ⁵³,⁵⁸ (Figure 4a). Following TIVAP implantation, we established a 3-day biofilm of bioluminescent 55989 strain (55989pAT881) under static conditions using TPN medium. We then administered four one-day cycles of 8-hour intermittent ALT<sub>AMK</sub> at 500 times the MIC and 16-hour withdrawal by installing the TPN solution, concluding with a 3-day recovery phase. Throughout this period, we monitored bioluminescent biofilms *in situ* (Figure 4b and c, and Supplementary Figure 5a). In the ALT<sub>PBS</sub> control group, bioluminescence intensities remained stable on either the dorsal or ventral sides of all three catheterized rats (Figure 4b). Conversely, the ALT<sub>AMK</sub> group gradually reduced bioluminescence intensity, which disappeared after the second treatment cycle. However, in one rat (ALT<sub>AMK</sub> Rat 4), bioluminescence intensity resurfaced during the recovery phase. Upon extracting the TIVAPs, bioluminescent bacterial biofilms were present in all rats of the ALT<sub>PBS</sub> group and in ALT<sub>AMK</sub> Rat 4 (Figure 4c). Additionally, the TIVAP of ALT<sub>AMK</sub> Rat 5 also demonstrated very weak bioluminescence intensity (Supplementary Figure 5a). CFU counting revealed that all parts of the ALT<sub>PBS</sub> group and 6 out of 9 parts in the ALT<sub>AMK</sub> group recovered biofilm cells at the endpoint. As a result, the ALT<sub>AMK</sub> group tended to have lower CFU numbers of biofilm bacteria on extracted TIVAPs than the
ALT<sub>PBS</sub> group, although the difference was not statistically significant (Figure 4d and 253 Supplementary Figure 5b). Small amounts of biofilm bacteria were found on TIVAPs from 254 ALT<sub>AMK</sub> Rat 6, although bioluminescence was not detected during the recovery phase 255 (Supplementary Figure 5a and b). This lack of significance may be due to the small size of the 256 sample (n = 3 per group), which could limit the statistical power of the analyses and make it 257 more challenging to identify true differences between the groups. Overall, these results 258 indicate that the *in vivo* intermittent ALT<sub>AMK</sub> treatment failed to eradicate biofilms in all three 259 animals in our preclinical model. 260

More concerning was the observed higher growth frequency of biofilm samples on amikacin 261 plates at 1 time the MIC in the ALT<sub>AMK</sub> group, ranging from 10–100%, compared to the ALT<sub>PBS</sub> 262 control group (Figure 4e and Supplementary Figure 5c), with population amikacin MICs 263 reaching 24–96 µg/mL in LB (6 to 24 µg/mL in MH) (Figure 4f and Supplementary Figure 5d). 264 Sequence analysis of surviving endpoint biofilm populations obtained from three ALT<sub>AMK</sub>- 265 treated TIVAPs confirmed that, *in vivo* as well, the *fusA* mutation is the most common 266 mutation observed in all six parts of the three TIVAPs (Figure 4g). 267

Taken together, our experiment demonstrated that treatment of biofilms formed in a clinically 269 relevant *in vivo* TIVAP model also led to the emergence of amikacin resistance. 270

This study examined the impact of high-concentration amikacin antibiotic lock therapy 272 (ALT<sub>AMK</sub>) recommended by guidelines for treating catheter-related *E. coli* biofilms. Our findings 273 reveal how treatment frequency (timing) modulates eco-evolutionary dynamics in clinically 274 relevant biofilm ecosystems, where pulsed antibiotic exposure creates strong selection 275 windows for resistance emergence. 276

We found that a continuous 10-day lock regimen successfully eradicated *in vitro* LF82 biofilms, 281 whereas a 7-day regimen did not. The favorable results associated with the 10-day continuous 282
lock, compared to the 7-day lock, were likely due to the superior total exposure and possibly
accumulation of AMK within the biofilms over the extended period. These additional 3 days
of AMK locking may have reduced the likelihood of any residual tolerant bacterial regrowth
once the treatment was completed. Despite this, the 7-day lock regimen still prevented the
rise of resistant subpopulations. Although the link between treatment duration and resistance
is debated ⁵⁹, treatment failure from short-term regimens, even if not leading to the
emergence of antibiotic resistance, still requires retreatment and is therefore associated with
a significant health and economic burden ⁶⁰. Mathematical modeling and clinical studies
support the effectiveness of longer treatment durations, recommending at least 10 days of
continuous ALT for successful catheter disinfection ³³,³⁶,⁶¹,⁶². Our data are consistent with
these findings, underscoring the clinical relevance of our *in vitro* model.

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In contrast to continuous treatments, all three intermittent ALT<sub>AMK</sub> treatments conducted *in vitro* failed, leading to the emergence of amikacin resistance with frequencies ranging from 1% to over 10%. This demonstrates how intermittent dosing turns biofilms into evolutionary incubators, where spatial structuring accelerates mutant selection. Analysis of free-floating cells revealed differences in bacterial numbers and resistance rates between LF82 and 55989 biofilms in LB, despite having the same amikacin MIC value of 16 µg/mL. Previous reports demonstrated that, in addition to biofilm formation capacity, the trajectory of antibiotic resistance evolution after antibiotic exposure is strain-specific<sup>63-65</sup>, suggesting that the genetic differences between LF82 and 55989 might affect biofilm formation capacity and, consequently, their tolerance to amikacin. Although the reduction in medium richness (when comparing TPN with LB media) slightly decreased the frequency of resistance observed during intermittent treatments in the *in vitro* TPN experiment, the bacterial load on TIVAP in the experiment ranged from 10<sup>7</sup> to 10<sup>8</sup> CFU/TIVAPs, comparable to what is found in clinical situations where bacterial loads on devices typically range from 10<sup>2</sup> to 10<sup>7</sup> CFUs/device<sup>66-68</sup>. Previous studies show strain-specific genetic diversification in biofilm populations<sup>69</sup>, suggesting that high-level resistance is selectively promoted during antibiotic treatment when nutrient levels are high<sup>70</sup>. Thus, while nutrient heterogeneity influences evolutionary trajectories, intermittent selection pressure remains the primary driver of resistance amplification in these structured communities. While our study demonstrates that
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intermittent ALT induces antibiotic resistance, future studies should consider the nature of
the tested bacterial strains and nutritional conditions to better mimic clinical settings.

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We also showed that intermittent treatment, performed *in vivo* in our pre-clinical rat catheter biofilm model, promoted the development of amikacin resistance, consistent with our *in vitro* observations. Despite varying bacterial counts at the endpoint (10³ to 10⁷ CFU/TIVAP), these counts are comparable with those observed in the clinical studies⁶⁶⁻⁶⁸. Moreover, growth frequencies on amikacin plates at 1 time the MIC were high for all three rats treated with ALT<sub>AMK</sub>, around 10 %, compared to the ALT<sub>PBS</sub> control (0.01%). This increased resistance seemed to develop faster and at higher levels than *in vitro*, which is consistent with another *in vivo* study on ciprofloxacin resistance in *P. aeruginosa* lung infection in mice<sup>71</sup>. The mechanisms behind this rapid and high-level resistance are unclear. However, the host immune environment, failing to cure the infectious disease, could finally synergize with antibiotic selection to accelerate the evolution of resistance. For instance, the host's reactive oxygen species, which can be triggered by infections, are not only bactericides that eliminate infections but also, conversely, promote the development of antibiotic resistance in bacteria<sup>71,72</sup>. In our *in vivo* experiment, since ceftriaxone is provided as a systemic treatment, it can also be considered that the temporary presence of blood containing ceftriaxone during the transition from ALT treatment to TPN infusion influences the *in vivo* dynamics of amikacin resistance selection. Further *in vivo* investigation is needed to clarify these dynamics. For instance, using a continuous drip infusion pig catheter model<sup>73</sup>, in which continuous nutrition infusion is more feasible than in our rat *in vivo* model, could provide more clinically relevant results.

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In both our *in vitro* and *in vivo* intermittent ALT experiments with lethal antibiotic concentrations, endpoint biofilm populations exhibited low levels of resistance. We observed only a 1.5 to 2.0-fold increase in MICs for most conditions, rising from 4 to 6–8 µg/mL when measured in MH, and from 16 to 24–32 µg/mL when measured in LB. These MIC values still fall within the susceptible range based on the CLSI definition⁴⁴. Meanwhile, one specific *in vivo* condition (ALT<sub>AMK</sub> rat 6) exceptionally showed a more significant increase, with a 6-fold rise from 4 to 24 µg/mL in MH and from 16 to 96 µg/mL in LB. Additionally, similar studies have shown that biofilms of other bacteria also develop antibiotic resistance when exposed
to various antibiotics<sup>21,23,74,75</sup>. These stepwise adaptations exemplify how biofilm communities serve as reservoirs for incremental evolution under pulsed selection. In clinical contexts, such low-level resistance increases might be easily overlooked during standard routine MIC testing, leading to repeated administration of the same antibiotic, then causing the selection of high-level resistant mutants. For example, studies have shown that in vancomycin-treated *S. aureus* bloodstream infections, low-level resistant variants—present initially as minor subpopulations that routine susceptibility tests still classify as “susceptible”—can become enriched under drug pressure once treatment begins. This enrichment may contribute to treatment failure and the eventual development of higher-level resistance<sup>76-78</sup>. A similar pattern has also been observed in *in vitro*<sup>79</sup> or *in vivo* experiments<sup>80</sup>. Therefore, as discussed previously<sup>81</sup>, these clinical and experimental data, including our findings, emphasize the importance of low-level resistance mutants as a pathway to clinically relevant high-level resistance.

Genome sequencing revealed that the convergent mutations in the *fusA*, *sbmA*, and *cpxA* genes may have contributed to the increased resistance to amikacin observed in endpoint biofilm populations. The *fusA* gene is essential for mRNA translocation in the ribosome and is conserved in diverse bacteria. A very recent study demonstrated that elongation factor G mutations selectively silence drug-bound ribosomes, thereby preventing error cluster formation and reducing the second self-promoted uptake phase of aminoglycosides into bacteria by preserving proteome and membrane integrity<sup>50</sup>. Our study found that *fusA* gene mutations are observed in all four experimental conditions treated with intermittent ALT<sub>AMK</sub> (*in vitro* LB LF82, *in vitro* LB 55989, *in vitro* TPN 55989pAT881, and *in vivo* 55989pAT881 experiments; Table 1), at frequencies greater than or equal to 5%. Other research also found *fusA* point mutations in laboratory and clinical isolates of different bacteria, *S. aureus*, *Salmonella*, and *P. aeruginosa*, warranting *fusA* mutation risks across species<sup>82-88</sup>.

We also identified nonsense and deletion mutations in the *sbmA* gene, encoding an inner membrane peptide transporter, from *in vitro* LF82 LB and 55989 LB experiments. Deleting the *sbmA* gene has been reported to reduce susceptibility to aminoglycosides, which aligns with our results<sup>27,45-49</sup>. An *Enterobacter cloacae* clinical isolate has exhibited aminoglycoside resistance due to the loss of the *sbmA* gene and approximately 20,000 nucleotides of adjacent
379 genes<sup>89</sup>. This massive deletion is similar to our findings that biofilm populations of TIVAP 4
380 and 5 from the *in vitro* 55989 experiment lost 11,024 nucleotides, including the *sbmA* and
381 neighbor genes (Table 1), suggesting that this region might be a fragile spot where breaks
382 occur in some bacteria.

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384 Point mutations in the *cpxA* gene, encoding CpxA histidine kinase of the CpxRA signal
385 transduction system, were also identified from *in vitro* experiments with LF82 in LB (at low
386 frequencies, less than 20%; see Supplementary Data 1) and 55989pAT881 in TPN. The CpxRA
387 system senses cell envelope stresses, regulating the transcription of downstream genes <sup>90</sup>.
388 Some point mutation mutants of the *cpxA* gene have been demonstrated to emerge from a
389 clinical Klebsiella strain isolated from a patient after antibiotic treatment <sup>91</sup> and to increase
390 resistance to aminoglycosides <sup>45,46,92</sup>, highlighting their clinical significance. These single amino
391 acid substitutions could alter the functionality of the CpxRA system, affecting various regulons,
392 including the *acrD* gene. Similar to the previously reported point mutations, the *cpxA* mutants
393 identified in our study are likely to be linked with amikacin resistance. Collectively, convergent
394 evolution in stress-response pathways underscores how antibiotic pulses reshape regulatory
395 networks in biofilm-adapted populations.

396

397 Although the amikacin MICs of biofilm populations obtained from the TIVAP4 catheter in the
398 *in vitro* LB LF82 intermittent experiment and the TIVAP6 silicone septum in the *in vitro* LB
399 55989 experiment were also slightly elevated at 32 µg/mL and 24 µg/mL, respectively, we
400 detected no mutations in the previously mentioned *sbmA*, *cpxA*, and *fusA* genes (Table 1).
401 Nevertheless, mutations in the major facilitator superfamily transporter genes and the
402 NADH-quinone oxidoreductase subunits *nuoA-C/D* genes were observed in the biofilms from
403 LF82 TIVAP4; mutations in the NADH-quinone oxidoreductase subunits *nuoC/D-N* genes were
404 observed in the biofilms from 55989 TIVAP6, which are linked to aminoglycoside resistance,
405 tolerance, and persistence <sup>93-96</sup> (Supplementary Data 1). These findings suggest that these
406 other mutated genes may also have collectively contributed to the development of low-level
407 resistance.

408

409 Furthermore, mutations in the *fimA* and *fimH* genes, which encode fimbrial and adhesin
410 proteins known to influence biofilm formation <sup>97-99</sup>, were identified in some biofilms during
411 continuous and intermittent *in vitro* LB experiments with LF82, *in vitro* TPN, and *in vivo* studies
412 (Supplementary Data 1). The emergence of mutations in these genes may have increased
413 biofilm formation, leading to a higher frequency of persister cells and overall population
414 tolerance. Consequently, along with the emergence of amikacin-resistant genes, the *fimA* and
415 *fimH* mutations are believed to contribute to enhanced biofilm survival and tolerance to the
416 antibiotic, promoting the emergence of resistance mutations²⁷.

417

418 In conclusion, this study demonstrated that an 8-hour intermittent ALT regimen leads to low-
419 level antibiotic resistance, primarily due to *fusA*, *sbmA* and *cpxA* mutations in two biofilm-
420 forming *E. coli* strains under various nutritional conditions, as observed in both *in vitro* and
421 preclinical *in vivo* catheter models. These findings establish treatment frequency as a critical
422 evolutionary driver in clinically relevant biofilm ecosystems. Additionally, intermittent
423 treatment can also occur in other infections because of low compliance to prescribed
424 medication regimens. Such irregular dosing can also adversely affect treatment efficacy and
425 potentially contribute to antibiotic resistance. Hence, while mathematical models and some
426 promising data suggest the potential of intermittent regimens for bacterial biofilm eradication
427 ¹⁰⁰-¹⁰⁷, further research is needed to optimize the dose and management schedule to enhance
428 their effectiveness and minimize resistance development.
429 METHODS

430 Bacterial strains and growth conditions

431 The bacterial strains used in this study are listed in Supplementary Table 1. Overnight cultures
432 were grown at 37 °C at 180 rpm in Miller's LB (Corning Inc., cat. 46-050-CM, Corning, NY, USA)
433 or Mueller-Hinton broth (Beckton, Dickinson and Company (BD), cat. 212322, Franklin Lakes,
434 NJ, USA), with 50 µg/mL spectinomycin (Sigma, S9007, St Louis, MO, USA) for the
435 55989pAT881 strain.
436

436

437 *In vitro* TIVAP experiments using LB broth or total parenteral nutrition (TPN) infusion

438 System setting up

439 The *in vitro* TIVAP model described in the previous paper 53 was employed, and some
440 modifications were made in this study. Briefly, first, medical-grade pediatric 5 Fr TIVAPs
441 (POLYSITE 2005 Micro kit, Perouse Medical, Ivry-le-Temple, France) were prepared using a 10
442 cm catheter and an accessory connector at the tip of the catheter, as provided with the kit
443 (the internal volume of the TIVAP is approximately 250 µL). TIVAPs were connected to a
444 medium bottle and a waste container via a 22 G Huber needle (VYGON, Inject-site, cat.
445 1057.27, Écouen, France) and connecting tubes (TYGON flexible plastic tube S3 E-3603, Saint-
446 Gobain Performance Plastics, Courbevoie, France). Fresh LB medium or the TPN infusion
447 (Baxter International, PERIOLIMEL N4E, cat. 3865924, Deerfield, IL, USA) was stored in the
448 medium bottle and supplied from the medium bottle to the waste container through the
449 inside of the TIVAPs by a peristaltic pump (Watson MARLOW, cat. 205S, Falmouth, UK) (Figure
450 1a and Supplementary Figure 1a). Several luer plugs were used to connect each part (Fisher
451 Scientific, ARK-PLAS, LAX22-PP0, LHX10-PP0, LNC31-PP0, LNC69-PP0, Pittsburgh, USA). All
452 devices were sterilized before use. This system was placed in a microbiological safety cabinet
453 to maintain sterility. The experimental temperature was maintained at 37°C with a heater
454 (Heidolph Incubator 1000, Heidolph Instruments, Schwabach, Germany) in the safety cabinet.
455

455

456 Bacterial inoculation into the TIVAP

457 After the system was set up, LB or TPN priming was performed for all lumens at a speed of
458 300 µL/min for one hour. Then, after priming was stopped, approximately 10² CFUs (per 100
459 µL) of LF82 or 55989 strains suspended in fresh LB prepared from the overnight culture for *in
460 vitro* LB experiments or 10⁶ CFUs (per 100 µL) of the 55989pAT881 strain suspended in
461

phosphate-buffered saline (PBS, Corning, no. 21-040-CM, USA) for the *in vitro* TPN experiment
were inoculated into the port using a 1-mL syringe (BD Plastipak) with a Huber needle. The
inoculated bacteria were then allowed to adhere to the TIVAP for three hours without flow.

462

463

464

465

*Growth phase*

466

The *in vitro* experiments consisted of three phases: growth, ALT, and recovery. After three
hours of bacterial inoculation, the continuous flow was started, and non-adherent bacteria
were washed out. Biofilm bacteria were grown continuously at a rate of 300 µL/min for 48
hours in the LB and 72 hours in the TPN experiments, according to a clinical study that revealed
a mean time of three days from clinical suspicion of catheter-related infections to
administering antibiotics <sup>108</sup>. During the growth phase, the floating cells were sampled at
designated time points (1, 4, 24, 48, and 72 hours) from the catheter tip under the continuous
flow by placing a hemolysis tube for CFU counting and growth frequency analysis on amikacin
plates with one time the MIC (described below). The remaining was stored at -80°C as a
glycerol stock.

467

468

469

470

471

472

473

474

475

476

477

*ALT phase (continuous or intermittent antibiotic lock)*

478

After the 48- or 72-hour growth phase, the connection tubes upstream and downstream of
the TIVAP were aseptically removed from the TIVAP. The connector at the catheter tip was
then closed with a plug Luer lock (ARK-PLAS, LNC31-PP0, Fisher Scientific). The remaining fluid
in the TIVAP was gently removed from the port using a one-mL syringe with a Huber needle.
Next, 250 µL of amikacin at 8 mg/mL (Sigma; of note, eight mg/mL corresponds to 500 times
the MIC for LF82 and 55989 strains in LB conditions) dissolved in PBS in the ALT<sub>AMK</sub> group or
250 µL of PBS (in the ALT<sub>PBS</sub> control group) were slowly injected into the port (Figure 1a and
Supplementary Figure 1b). The ALT solution was exchanged every 24 hours for 10 or 7 days in
the continuous ALT regimen or installed for 8 hours in the intermittent ALT regimen for 7 or 4
cycles alternating with a 16-hour continuous flow. In the intermittent ALT experiments,
floating cells were sampled at 1 and 16 hours after each ALT cycle.

479

480

481

482

483

484

485

486

487

488

489

490

*Recovery phase*

491

At the end of (each) ALT phase, the new sterilized connection tubes were connected to the
TIVAP upstream and downstream TIVAP to restart the continuous flow for 72 hours. In both

492
continuous and intermittent ALT experiments, the floating cells were sampled at designated time points (1, 24, 48, and 72 hours) in the recovery phase.

495

### Biofilm sample collection from TIVAP at the endpoint

At the endpoint of each experiment, the TIVAP was removed from the system and disinfected externally with 70 % ethanol (VWR Chemicals, cat. 83801.290, Radnor, PA, USA) for 15 min.

The catheter was aseptically cut every two cm long with a sterilized scalpel (hereafter, all preparation was aseptically performed). The silicone septum part was removed from the port and cut into small pieces with a scalpel. For each part, pieces were suspended in one mL LB broth (in a microtube). Biofilm bacteria attaching on/in the port were carefully wiped with a sterile cotton swab (Deltalab, cat. 300201, Barcelona, Spain) moistened with LB broth. The cotton swab tip was then cut at one cm and suspended in one mL LB broth. All microtubes containing TIVAP parts were vortexed for one min at the maximum speed and then sonicated for 10 min in an ultrasonic bath (Branson 5800, Branson Ultrasonics, Danbury, CT, USA). Finally, the biofilm bacterial suspension was used for CFU counting, growth frequency analysis on amikacin plates with one time the MIC, and amikacin MIC testing, and the remaining was stored at -80°C as a glycerol stock.

510

### CFU counting and growth frequency on amikacin plates with 1 time the MIC

A series of 10-fold dilutions was prepared for each sample in 96-well plates, and 10 µL of each dilution (or 100 µL of undiluted samples) was inoculated onto LB agar plates with or without 16 µg/mL amikacin. The plates were incubated at 37°C for 24 hours. The growth frequency on amikacin plates at one time the MIC was calculated as follows:

$$ Growth frequency on one time the amikacin MIC (\%) = \frac{CFU_{amikacin at one time the MIC}}{CFU_{LB}} \times 100 (\%) $$

, where $CFU_{amikacin at one time the MIC}$ is the CFU number of each population grown on LB agar plates containing amikacin at 16 µg/mL (one time the MIC), and $CFU_{LB}$ is the one grown on the LB agar plate corresponding to CFU counting.

520

### Amikacin susceptibility

The amikacin MIC for the obtained samples was determined using the agar dilution method previously described ⁴⁴, with minor modifications. Briefly, the obtained samples were
incubated overnight in LB or MH broth from the glycerol stock. The overnight cultures were diluted to approximately $1.5 \times 10^7$ CFU/mL into fresh LB or MH media. Then, the diluted bacterial culture was inoculated onto LB or MH agar plates containing amikacin at 0, 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, 64, 96, 128, 198, and 256 µg/mL using a multipoint replicating device (Boekel, PA, cat. 140500, USA), which transfers approximately 1 µL of each diluted culture to agar surfaces. The agar plates were incubated at 37°C for 20 hours. The MIC testing was performed with four replicates per sample per agar (two technical replicates prepared from the glycerol stock; this step was repeated twice on different days). The most representative value was adopted as the result.

533

534 *Bioluminescence imaging*

535 In the *in vitro* TPN experiment using the 55989pAT881 strain, bioluminescence images of 536 biofilm formation on the TIVAP were captured using an imaging system (IVIS SpectrumCT; 537 Perkin Elmer, Waltham, USA) each day during the growth and recovery phases, as well as at 1 538 and 16 hours of the withdrawal period during the ALT phase.

539

540 *In vivo TIVAP experiment*

541 *Ethics approval*

542 The French Ministry of Agriculture accredited the animal facility to perform experiments on 543 live rodents (accreditation number A75-15 01, issued on 20 October 2027, and number A75- 544 15 02, issued on 20 October 2027) in compliance with French and European regulations on 545 the care and protection of laboratory animals (EC Directive 2010/63, French Law 2013-118, 546 issued on 1 February 2013). All experiments were approved by the Ethics Committee #89 (le 547 CETEA, Institut Pasteur) and registered under the reference APAFIS #45216- 548 2023101919069048 v1).

549

550 *TIVAP surgery and postoperative care*

551 TIVAP implantation in rats was performed using the same kit as the *in vitro* experiments, 552 following the previous protocol with modifications <sup>58</sup>. Briefly, after one week of 553 acclimatization, TIVAP implantation surgery was performed for anesthetized eight-week male 554 Crl:CD (SD) rats (Charles River, Wilmington, MA, USA) with isoflurane (ISO-VET, Piramal Critical 555 Care, Bethlehem, PA, USA) on Day 0 as follows: the port part was implanted in the
subcutaneous pocket on the back. After the neck skin incision on the ventral side, the catheter was tunnelled subcutaneously from the port on the dorsal side to the neck on the ventral side and exposed through the neck skin incision. The catheter was inserted into the right main jugular vein after a partial microincision. The tip of the inserted catheter was advanced to approximately four cm, where the entrance to the right atrium is located. After fixing the catheter in the jugular vein, the surgical sites were closed with sutures. TIVAP patency was attempted to be maintained with a daily heparin lock (250 µL) containing 500 IU/mL on Day 0 and 250 IU/mL thereafter (Sanofi Aventis, Paris, France) in the TIVAP. An anti-inflammatory drug, meloxicam (1.0 mg/kg, Metacam solution injectable two mg/mL, Boehringer Ingelheim, Germany), was injected subcutaneously on Days 1-3 to reduce postoperative pain. The incision sites on the back and neck, and the behaviour of the rats, were also monitored for three days as postoperative care.

### Bacterial loading into the TIVAP and monitoring of biofilm formations for 3 days

Overnight cultures of E. coli 55989pAT881 grown in LB with 50 µg/mL spectinomycin were diluted to $2 \times 10^8$ CFU/mL with PBS. Then, after removing the previously installed heparin lock solution from the TIVAP, 50 µL of the diluted culture ($1 \times 10^7$ CFU as the final bacterial count) was slowly injected into the TIVAP port of rats anesthetized with isoflurane on Day 4 (hereafter, all injection steps were performed on rats under anesthetized conditions). After 24 hours of bacterial inoculation, floating bacteria were removed by collecting the solution from the port. Then, 250 µL of TPN infusion was installed for 3 days (Days 5–8) into the TIVAP of isoflurane anesthetized rats and exchanged daily by collecting the previous solution from the TIVAP and injecting a new solution. If the previous infusion could not be collected due to reduced catheter patency, a new solution was gently injected without the need for a collection procedure. Ceftriaxone (200 mg/kg, Sigma, C5793) was also administered subcutaneously once daily until the end of the experiment (Day 15) to prevent systemic infections. Bioluminescence images of biofilm formation on the TIVAP were regularly captured on the dorsal and ventral sides of isoflurane anesthetized rats using IVIS Spectrum CT, including the condition before bacterial loading.

### Intermittent ALT and recovery phases
After the three-day biofilm formation, 250 µL of amikacin lock solutions at eight mg/mL dissolved in PBS containing heparin (250 IU/mL of the final conc.) was installed into the TIVAP of isoflurane anesthetized rats for eight hours. 250 µL of PBS with heparin at 250 IU/mL was used in the ALT~PBS~ control group. Then, 250 µL of the TPN infusion was installed into the TIVAP for 16 hours. This intermittent treatment was repeated for four cycles on Days 8–12. Finally, the TPN infusion was installed into the TIVAP for three days (Days 12–15), and the solution was exchanged daily. Bioluminescence images of biofilm formation on the TIVAP of isoflurane anesthetized rats were captured before and after intermittent ALT procedures and each day in the recovery phase. Note that during the biofilm growth, regrowth, and recovery periods, TPN was administered as a 250 µL instillation, not as a continuous flow, due to the impossibility of immobilizing the rat.

## TIVAP extraction from euthanized rats

On Day 15, after the rats were euthanized with carbon dioxide, the TIVAP was aseptically removed from the euthanized rat. Then, the bioluminescence images of the biofilms in the extracted TIVAP were captured. Next, after the surface of the TIVAP was carefully disinfected with 70% ethanol, the TIVAP was disassembled into silicone septum, port, and catheter parts as described above. Biofilm bacteria on each part were analyzed for CFU counting, growth frequency analysis on amikacin plates at 1 time the MIC, and amikacin MIC testing.

## Whole genome sequencing, assembly, and data analyses

Whole-population genome sequencing (WGS) was applied to endpoint biofilm populations treated with continuous or intermittent amikacin in the *in vitro* and *in vivo* experiments to investigate gene mutations. LF82 biofilm samples saved in 15% glycerol (obtained from the continuous and intermittent *in vitro* LB experiments) were directly transferred from the glycerol stock to fresh LB broth and regrown overnight at 37 °C under shaking conditions. Meanwhile, for each sample from 55989 biofilm samples obtained from the *in vitro* LB experiment and 55989pAT881 from the *in vitro* TPN experiments, given the relatively low frequency of mutants growing on amikacin plates at 1 time the MIC in the growth frequency analysis and considering that the corresponding mutations would likely be missed by WGS analysis since Breseq has a detection limit of 5%, colonies growing on amikacin plates at 1 time the MIC were pooled and incubated in LB broth without antibiotics overnight at 37 °C
619 under shaking conditions. This enriched method was also applied to 55989pAT881 biofilm
620 samples from the *in vivo* experiment. Similarly to the previous study 27, the genomic DNA was
621 then extracted from these cultures, prepared for whole genome sequencing using the Nextera
622 XT DNA library preparation kit (Illumina, San Diego, CA, USA), and sequenced on the Hiseq and
623 Miseq platforms (Illumina) with an average depth of 150 ×. Read quality was assessed using
624 FastQC version 0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and
625 trimmed using Trimmomatic version 0.39 109. The trimmed sequence reads were analyzed by
626 BreSeq version 0.35.0 110 to detect genetic variants.

627

628 Statistical analysis

629 Data are presented as the mean ± standard error (SE), with or without individual data points,
630 from three independent biological experiments. Statistical analyses were conducted using
631 Prism 10.2.3 (GraphPad Software Inc.). They corresponded to an unpaired two-tailed t-test
632 with Welch's correction between two groups and to the Welch one-way analysis of variance
633 (ANOVA) with Dunnett post hoc test for comparisons involving more than two groups. P-
634 values <0.05 were deemed statistically significant.

635
636 **Data availability**

637 Supplementary Data 1 corresponds to the Mutations identified using Breseq in end-point
638 biofilm populations at a frequency higher than 5%. It can be downloaded at
639 https://zenodo.org/records/17655296.

640 Supplementary Data 2 file corresponds to the raw data of the different experiments presented
641 in this manuscript. It can be downloaded at https://zenodo.org/records/17655323.

642

643 Acknowledgements

644 We thank the animal facility and UTechS Photonic BioImaging (PBI) facility at Institut Pasteur.
645 This work was supported by grants from the French National Research Agency (ANR), project
646 EvolTolAB (ANR-18-CE13-0010), the French Government's Investissement d'Avenir program,
647 Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (grant no. ANR-
648 10-LABX-62-IBEID), and the Fondation pour la Recherche Médicale (grant no.
649 DEQ20180339185). Y.Y. received support from a Grant-in-Aid for JSPS Fellow (grant no.
650 17J10232). S.T.-R was also supported by the French National Research Agency (ANR), project
651 EvolTolAB (ANR-18-CE13-0010).

652

653 Author contributions

654 Y.Y., D.L., and C.B. designed the experiments. Y.Y. and S.T.-R. performed the experiments.
655 Y.Y., S.T.-R., D.L., J.-M.G., and C.B. analysed the data. Y.Y. and C.B. wrote the manuscript with
656 significant contributions from other authors.

657

658 Competing interests

659 Authors Y.Y, D.L. and S.T.-R. declare no financial or non-financial competing interests. Author
660 J.-M.G. serves as Editor-In-Chief of this journal and had no role in the peer review or decision
661 to publish this manuscript. Author J.-M.G. declares no financial competing interests. Author
662 C.B. serves as Associate Editor of this journal and had no role in the peer review or decision to
663 publish this manuscript. Author C.B. declares no financial competing interests.

664
<table><caption>Table 1. Identified non-synonymous *fusA*, *sbmA*, and *cpxA* mutations from biofilm populations at a frequency higher than 5% in all experiments. Si, po, and ca represent biofilm samples from the silicone septum, port, and catheter parts of each TIVAP. Del. represents deletion of the gene. †The *sbmA* gene was deleted together with neighbor genes of *phoA_2*, *iraP*, *ddlA*, *KOPPCICB_04469*, *yaiY*, *yaiW*, *KOPPCICB_04473*, *ampH*, *iprA*, and *KOPPCICB_04476*.</caption><thead><tr><th>Gene</th><th>Strain</th><th>Experiment</th><th>Sample (TIVAP)</th><th>Mutation</th><th>Gene</th><th>Strain</th><th>Experiment</th><th>Sample (TIVAP)</th><th>Mutation</th></tr></thead><tbody><tr><td rowspan="2">fusA</td><td rowspan="2">LF82</td><td rowspan="2">In vitro LB</td><td>4 si</td><td>K653N G-&gt;T</td><td rowspan="2">sbmA</td><td rowspan="2">LF82</td><td rowspan="2">In vitro LB</td><td>5 si</td><td>W27* G-&gt;A</td></tr><tr><td>5 ca</td><td>K653N G-&gt;T</td><td>Q120H A-&gt;C</td></tr><tr><td rowspan="6">55989</td><td rowspan="6">In vitro LB</td><td>4 si</td><td>F605L T-&gt;C</td><td rowspan="6"></td><td rowspan="6"></td><td>po</td><td>W27* G-&gt;A</td></tr><tr><td>po</td><td>F605L T-&gt;C</td><td>ca</td><td>coding 12/1221 nt</td></tr><tr><td>ca</td><td>F605L T-&gt;C</td><td>W27* G-&gt;A</td></tr><tr><td>6 po</td><td>A678V C-&gt;T</td><td>6 si</td><td>C67* C-&gt;A</td></tr><tr><td>ca</td><td>A678V C-&gt;T</td><td>po</td><td>C67* C-&gt;A</td></tr><tr><td>ca</td><td>A678V C-&gt;T</td><td>ca</td><td>C67* C-&gt;A</td></tr><tr><td rowspan="10">55989pAT881</td><td rowspan="10">In vitro TPN</td><td>5 si</td><td>S672F C-&gt;T</td><td rowspan="10">cpxA</td><td rowspan="10">LF82</td><td rowspan="10">In vitro LB</td><td>4 si</td><td>del. Δ11024 nt†</td></tr><tr><td>po</td><td>S672F C-&gt;T</td><td>po</td><td>del. Δ11024 nt†</td></tr><tr><td>6 si</td><td>A678V C-&gt;T</td><td>ca</td><td>del. Δ11024 nt†</td></tr><tr><td>S672F C-&gt;T</td><td>5 si</td><td>del. Δ11024 nt†</td></tr><tr><td>po</td><td>R671H G-&gt;A</td><td>po</td><td>del. Δ11024 nt†</td></tr><tr><td>ca</td><td>R671H G-&gt;A</td><td>ca</td><td>del. Δ11024 nt†</td></tr><tr><td>S672F C-&gt;T</td><td>E100K G-&gt;A</td><td>ca</td><td>del. Δ11024 nt†</td></tr><tr><td>po</td><td>S672F C-&gt;T</td><td>5 si</td><td>L19R T-&gt;G</td></tr><tr><td>ca</td><td>A592V C-&gt;T</td><td>po</td><td>L19R T-&gt;G</td></tr><tr><td>ca</td><td>A592V C-&gt;T</td><td>6 si</td><td>M22L A-&gt;T</td></tr><tr><td rowspan="10">55989pAT881</td><td rowspan="10">In vivo</td><td>4 si</td><td>R671H G-&gt;A</td><td rowspan="10"></td><td rowspan="10"></td><td>po</td><td>L19R T-&gt;G</td></tr><tr><td>S672F C-&gt;T</td><td>55989pAT881 In vitro TPN</td><td>po</td><td>L19R T-&gt;G</td></tr><tr><td>po</td><td>S672F C-&gt;T</td><td>po</td><td>T6I C-&gt;T</td></tr><tr><td>R671H G-&gt;A</td><td>po</td><td>T6I C-&gt;T</td></tr><tr><td>ca</td><td>A592V C-&gt;T</td><td>ca</td><td>T6I C-&gt;T</td></tr><tr><td>5 si</td><td>S672F C-&gt;T</td><td>6 si</td><td>D162E C-&gt;A</td></tr><tr><td>R671H G-&gt;A</td><td>po</td><td>D162E C-&gt;A</td></tr><tr><td>ca</td><td>S672F C-&gt;T</td><td>ca</td><td>D162E C-&gt;A</td></tr><tr><td>R671H G-&gt;A</td><td>R671C C-&gt;T</td><td>R671C C-&gt;T</td></tr><tr><td>6 ca</td><td>S672F C-&gt;T</td><td>R671H G-&gt;A</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>**Figure 1.** 10- and 7-day continuous amikacin lock therapy at 500 times the MIC for *E. coli* LF82 biofilms formed *in vitro* on TIVAP under continuous LB flow. (a) Schematic illustrations of the *in vitro* TIVAP model, including growth/recovery and antibiotic lock therapy (ALT) phases. (b) Experimental protocol for 10- and 7-day daily-exchanged continuous amikacin lock therapy (ALT<sub>AMK</sub>) at 500 times the MIC. Experiments were conducted with 3-hour bacterial inoculation/adhesion on TIVAPs without continuous flow, followed by 2-day biofilm growth under continuous LB flow, 10- or 7-day ALT without continuous flow, and 3-day biofilm formation recovery with continuous LB flow. PBS was injected into TIVAPs as the control (ALT<sub>PBS</sub>). (c, d) Time course of CFUs of floating cell samples collected from TIVAP catheter tips in the 10- (c) or 7-day (d) experiments. (e, f) CFUs of biofilm samples at the endpoint in the 10-(e) and 7-day (f) experiments. (g) Growth frequencies of floating cell and biofilm samples in the 7-day experiment on amikacin plates with 1 time the MIC (ALT<sub>PBS</sub> control group on the left panel and ALT<sub>AMK</sub> group on the right panel). (h) Amikacin MIC for biofilm population samples at the endpoint on LB and Mueller-Hinton (MH) media. The tested biofilm samples were the same as (f). The data used in (c and e) were extracted from the same 10-day experiment, and the data in (e, f, and g) were extracted from the same 7-day experiment. The limit of detection (LOD) indicates the lowest CFU that can be measured. Values below the limit of detection (LOD) were included and plotted at the LOD threshold. Means, individual values, and standard errors from three biological replicates (TIVAPs) are shown (Welch's t-test, ***p < 0.001). Figure 1a was generated using BioRender.</figcaption></figure>
a

<figure><img src="image_3.png" /></figure>

d

<figure><img src="image_4.png" /></figure>

f

<figure><img src="image_5.png" /></figure>

LF82 AMK

<figure><img src="image_6.png" /></figure>

b

<figure><img src="image_7.png" /></figure>

e

<figure><img src="image_8.png" /></figure>

g

<figure><img src="image_9.png" /></figure>

55989 AMK

<figure><img src="image_10.png" /></figure>

c

<figure><img src="image_11.png" /></figure>

h

<table><caption>LF82 biofilm samples at 72 h
of the recovery period</caption><thead><tr><th rowspan="2"></th><th colspan="4">Amikacin MIC (µg/mL)</th></tr><tr><th>LB</th><th>MH</th><th>LB</th><th>MH</th></tr></thead><tbody><tr><td>LF82 ancestor</td><td>16</td><td>4</td><td></td><td></td></tr><tr><td>TIVAP 1 ○</td><td>16</td><td>4</td><td>TIVAP 4 ◁</td><td>32<sup>†</sup> 8<sup>†</sup></td></tr><tr><td>TIVAP 2 □</td><td>16</td><td>4</td><td>TIVAP 5 ◁</td><td>32 8</td></tr><tr><td>TIVAP 3 △</td><td>16</td><td>4</td><td>TIVAP 6 ▽</td><td>32 8</td></tr></tbody></table>

i

<table><caption>55989 biofilm samples at 72 h
of the recovery period</caption><thead><tr><th rowspan="2"></th><th colspan="4">Amikacin MIC (µg/mL)</th></tr><tr><th>LB</th><th>MH</th><th>LB</th><th>MH</th></tr></thead><tbody><tr><td>55989 ancestor</td><td>16</td><td>4</td><td></td><td></td></tr><tr><td>TIVAP 1 ○</td><td>16</td><td>4</td><td>TIVAP 4 ◁</td><td>24 6</td></tr><tr><td>TIVAP 2 □</td><td>16</td><td>4</td><td>TIVAP 5 ◁</td><td>24 6</td></tr><tr><td>TIVAP 3 △</td><td>16</td><td>4</td><td>TIVAP 6 ▽</td><td>24 6</td></tr></tbody></table>

j

<figure><img src="image_12.png" /><figcaption>LF82</figcaption></figure>

k

<figure><img src="image_13.png" /><figcaption>55989</figcaption></figure>
<caption>**Figure 2. 7 cycles of intermittent amikacin lock therapy at 500 times the MIC for *in vitro* E. coli LF82 and 55989 TIVAP biofilms under continuous LB flow.** ((a) Experimental Protocol: Seven cycles of 8-hour intermittent amikacin lock therapy at 500-fold MIC (ALT<sub>AMK</sub>) were conducted on biofilms formed by E. coli LF82 and 55989 in TIVAPs. The control group received PBS (ALT<sub>PBS</sub>). The experiment included 3-hour bacterial inoculation and adhesion without continuous flow, followed by 2 days of biofilm growth under continuous LB flow, and subsequent alternating 8-hour ALT and 16-hour recovery phases, culminating in a 3-day biofilm recovery with continuous flow. (b, c) CFU time course: Time course data of CFUs from floating cell samples collected from the catheter tips of TIVAPs exhibiting LF82 (b) and 55989 (c) biofilms were analyzed. Both panels compared the floating cell dynamics in response to ALT<sub>AMK</sub> and ALT<sub>PBS</sub>. (d, e) Endpoint biofilm CFU: The CFUs at the endpoint for LF82 (d) and 55989 (e) biofilm samples on TIVAPs were measured. (f, g) Growth frequencies on amikacin plates: Growth frequencies of LF82 (f) and 55989 (g) floating cell and biofilm samples on amikacin plates with 1 time the MIC were measured (ALT<sub>PBS</sub> group on the left panel and ALT<sub>AMK</sub> on the right panel in each). (h, i) Amikacin MIC determination for biofilm populations: The amikacin MIC values for LF82 (h) and 55989 (i) biofilm populations were assessed on LB and MH media. (j, k) Whole-population genome sequencing (WGS)-based mutational analysis: Heatmaps identifying mutations from endpoint biofilm populations, determined by WGS, are displayed in panels (j) for LF82 and (k) for 55989. Of note, mutations at frequencies of ≥20% for LF82 and ≥10% for 55989 are demonstrated (see Supplementary Data 1 for all identified mutations). Si, Po, and ca represent biofilm samples from the silicone septum, port, and catheter parts of each TIVAP. The data used in (b, d, f, h, and j) were extracted from the same LF82 intermittent amikacin lock experiment, and the data in (c, e, g, i, and k) were extracted from the same 55989 intermittent amikacin lock experiment. The limit of detection (LOD), which indicates the lowest CFU that can be measured, was established as 10 CFU/mL. Values below LOD were included and plotted at the LOD threshold. Means, individual values, and standard errors from three biological replicates (TIVAPs) are shown (Welch's t-test, *p* <0.05). For †, 32 and 8 µg/mL in LB and MH were obtained from the catheter biofilm and were demonstrated as the highest amikacin MIC value compared to silicone and port biofilms (see Supplementary Figure 3d).</caption>
<figure><img src="image_14.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>55989pAT881 AMK</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_21.png" /></figure>
**Figure 3. 4 cycles of intermittent amikacin lock therapy at 500 times the MIC for bioluminescent 55989pAT881 in vitro TIVAP biofilms under continuous TPN flow.** (a) Experimental protocol for four cycles of 8-hour intermittent amikacin lock therapy (ALT<sub>AMK</sub>). PBS was injected into TIVAPs as the control (ALT<sub>PBS</sub>). Experiments were conducted with 3-hour inoculation/adhesion of the PBS-suspended bacteria on TIVAPs without continuous flow, followed by 3-day biofilm growth under continuous total parenteral nutrition (TPN) flow, 4-cycle sets of 8-hour ALT phase (without flow) and 16-hour intermittent recovery phase (with continuous TPN flow), and 3-day biofilm formation recovery with continuous TPN flow. (b) Time course of CFUs of floating cell samples collected from the catheter tips of TIVAPs, where 55989pAT881 biofilms were formed and treated with four cycles of 8-hour intermittent ALT<sub>AMK</sub> at 500-fold MIC or ALT<sub>PBS</sub>. (c) CFUs of bioluminescent 55989pAT881 biofilm samples at the endpoint. (d) Representative time course of bioluminescence images of 55989pAT881 biofilms formed on TIVAP treated with or without amikacin (ALT<sub>PBS</sub> 3 and ALT<sub>AMK</sub> 6). (e) Growth frequencies of 55989pAT881 floating cell and biofilm samples on amikacin plates at 1 time MIC (ALT<sub>PBS</sub> control group on the upper panel and ALT<sub>AMK</sub> group on the lower panel). (f) Amikacin MIC for 55989pAT881 biofilm population samples on LB and MH media. (g) Heatmap for identified mutations from endpoint biofilm populations by whole-population genome sequencing (WGS). Endpoint 55989pAT881 biofilm populations obtained from TIVAPs 5 and 6, and grown on amikacin plates at 1 time the MIC, were subjected to WGS. Mutations at a frequency greater than or equal to 10% are highlighted in orange (see Supplementary Data 1 for all identified mutations). Si, Po, and ca represent biofilm samples from the silicone septum, port, and catheter parts of each TIVAP. Means, individual values, and standard errors from three biological replicates (TIVAPs) are shown (Welch's t-test, *p* <0.05). For †, 24 and 6 µg/mL in LB and MH were obtained from the port biofilm and were demonstrated as the highest MICs compared to silicone and catheter biofilms.
<figure><img src="image_22.png" /></figure>

Surgery

Heparin exchange

Bacterial loading

Daily TPN injection

AMK lock
for 8 h

Daily TPN injection

TIVAP
sampling

CFU counts

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

Anti-inflammatory
drug injection

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

TPN injection
for 16 h

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

Bioluminescent
imaging

<figure><img src="image_30.png" /></figure>

In situ monitoring

Periodic imaging

<figure><img src="image_31.png" /></figure>

Extracted TIVAP

Systemic
antibiotic

<figure><img src="image_32.png" /></figure>

Daily ceftriaxone

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

c

PBS 3 Δ

AMK 4 ◀

(p/s/cm²/sr)

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

PBS rat 3 Δ

Dorsal
Ventral

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

g

Population MIC (µg/mL)

TIVAP 4

Si 24

Po 24

Ca 24

TIVAP 5

Si 24

Po –

Ca 24

TIVAP 6

Si –

Po 96

Ca 96

◇

AMK rat 4

Dorsal
Ventral

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

(p/s/cm²/sr)

7.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

4.5

abgT

atpG

cirA_2

elaD

fusA

gadE

glnH

gspE_1

Inter mgtS/dgcZ

ndh

nuoC

nuoJ

repA

sbcB

secM

ubiF

Inter wcaK/wzxC

ydeA

Inter yojl/alkB

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

Amikacin MIC (µg/mL)

55989pAT881 ancestor

Rat 1 ○ 16 4

Rat 4 ◁ 24 6

PBS Rat 2 ☐ 16 4

AMK Rat 5 ◁ 24 6

Rat 3 △ 16 4

Rat 6 ▽ 96 24
**Figure 4.** 4 cycles of intermittent amikacin lock at 500 times the MIC for bioluminescent 55989 *in vivo* TIVAP biofilms. (a) Experimental Protocol: Four cycles of 8-hour intermittent amikacin lock therapy (ALT<sub>AMK</sub>) at 500-fold MIC were performed on biofilms formed by the bioluminescent 55989 strain in TIVAPs, with a control group receiving PBS (ALT<sub>PBS</sub>). Medical-grade 5 Fr TIVAPs were implanted in rats after a 1-week acclimatization. On day 4, the bioluminescent strain was inoculated into the TIVAP port. Biofilm formation was established over 3 days with daily TPN on days 5-8. The intermittent ALT and 16-hour recovery phases were conducted from days 8-12, followed by a biofilm recovery phase with TPN on days 12-15. TIVAPs were collected from euthanized rats on day 15 for analysis. (b) Bioluminescence time course: Examples of bioluminescent imaging of biofilms treated with ALT<sub>AMK</sub> versus ALT<sub>PBS</sub> are presented (ALT<sub>PBS</sub> rat 3 and ALT<sub>AMK</sub> rat 4). (c) Endpoint TIVAP bioluminescence images: Extracted TIVAP images illustrating biofilm presence in each treatment group are shown. (d) Endpoint biofilm CFU: CFUs for biofilm samples were measured at the endpoint. (e) Growth frequencies on amikacin plates: Growth frequencies of biofilm cell samples on amikacin plates at 1 time the MIC are reported. (f) Amikacin MIC determination: The amikacin MIC values for biofilm cell samples grown on LB and MH media were assessed. (g) Whole-Population Genome Sequencing (WGS)-based mutational analysis: Heatmaps showcasing mutations from endpoint biofilm populations, identified through WGS, are presented, with mutations at frequencies of ≥10% highlighted in orange (see Supplementary Data 1). Biofilms were not recovered from the ALT<sub>AMK</sub> TIVAP 5 port and TIVAP 6 silicone and port (see Supplementary Figure 5b). Si, Po, and ca represent biofilm samples from the silicone septum, port, and catheter parts of each TIVAP. Means, individual values, and standard errors from three biological replicates (TIVAPs) are shown (Welch's t-test, **p < 0.001). Figure 4a was generated using BioRender.

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Supplementary information

Intermittent Antibiotic Exposure of *Escherichia coli* Biofilms Drives Resistance in Catheter-Associated Infection Models

Yutaka YOSHII, Stanislas THIRIET-RUPERT, David LEBEAUX, Jean-Marc GHIGO, Christophe BELOIN

Supplementary information includes:

Supplementary Data 1: Mutations identified using Breseq in end-point biofilm populations at frequencies ≥5%.

It can be downloaded at [https://zenodo.org/records/17655296](https://zenodo.org/records/17655296).

Supplementary Data 2: contains the raw data of the different experiments.

It can be downloaded at [https://zenodo.org/records/17655323](https://zenodo.org/records/17655323).

Supplementary Table 1

Supplementary Figures 1 to 5
<table><caption>Supplementary Table 1. Bacterial strains and plasmids used in this study</caption><thead><tr><td><i>E. coli</i> strains</td><td>Description</td><td>Reference</td></tr></thead><tbody><tr><td>LF82 (LF82Δ<i>ampC</i>::mars)</td><td>A derivative of adherent invasive <i>E. coli</i> LF82<sup>1)</sup> isolated from an ileal biopsy of a Crohn's disease patient. LF82Δ<i>ampC</i>::mars-FRT. Deletion of <i>ampC</i> by mars-KmFRT and removal of the kanamycin resistance.</td><td>2)</td></tr><tr><td>55989</td><td>Enteroaggregative <i>E. coli</i></td><td>3)</td></tr><tr><td>55989pAT881</td><td>A 55989 strain transformed with stable plasmid pAT881 providing production of bioluminescence</td><td>4)</td></tr></tbody></table>

Supplementary References

1) Glasser AL, Boudeau J, Barnich N, Perruchot MH, Colombel JF, Darfeuille-Michaud A. Adherent invasive *Escherichia coli* strains from patients with Crohn's disease survive and replicate within macrophages without inducing host cell death. *Infect Immun*. 2001; 69:5529-37. DOI: 10.1128/IAI.69.9.5529-5537.2001

2) Usui M, Yoshii Y, Thiriet-Rupert S, Ghigo J-M, Beloin C. Intermittent antibiotic treatment of bacterial biofilms favors the rapid evolution of resistance. *Communications Biology* 2023; 6:275. DOI: 10.1038/s42003-023-04601-y

3) Bernier C, Gounon P, Le Bouguénec C. Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative *Escherichia coli* as a sensitive probe for detecting the AAF-encoding operon family. *Infect Immun*. 2002; 70:4302-11. DOI: 10.1128/IAI.70.8.4302-4311.2002

4) Chauhan A, Ghigo J-M, Beloin C. Study of in vivo catheter biofilm infections using pediatric central venous catheter implanted in rat. *Nature Protocols*. 2016;11(3):525-541. DOI: 10.1038/nprot.2016.033
Supplementary Figures

<figure><img src="image_68.png" /><figcaption>**Supplementary Figure 1.** LF82 biofilm formation on *in vitro* TIVAP under continuous LB flow. (a) Representative photographs showing the *in vitro* TIVAP model combined with continuous LB flow. (b) The actual lock therapy step after removing the continuous flow system. (c) Time course of CFUs of floating cell samples collected from the tips of the TIVAP catheters after 48-hour continuous LB flow incubation. (d) CFUs of biofilm samples formed on each TIVAP part (silicone septum, port, and catheter) after 48-hour continuous LB flow in the same experiment with (c). (e) Time course of CFUs of floating cell samples collected from TIVAPs treated with a 24-hour amikacin lock at different concentrations (PBS control, 5 times, 80 times, and 500 times MIC), followed by 1-hour recovery continuous LB flow. (f) CFUs of biofilm samples at the end-point in the same experiment with (c). Three TIVAPs were used for each condition in each experiment. LOD indicates the lowest CFU that can be measured. Means, individual values, and standard errors from three biological replicates (TIVAPs) are shown (Welch ANOVA with Dunnett post hoc test, ****p <0.0001).</figcaption></figure>
<figure><img src="image_69.png" /><figcaption>Fig. S2</figcaption></figure>

c

<table><thead><tr><th rowspan="2"></th><th colspan="2">PBS 1 ○</th><th colspan="2">PBS 2 □</th><th colspan="2">PBS 3 △</th></tr><tr><th>LB</th><th>MH</th><th>LB</th><th>MH</th><th>LB</th><th>MH</th></tr></thead><tbody><tr><td>Silicone</td><td>16</td><td>4</td><td>16</td><td>4</td><td>16</td><td>4</td></tr><tr><td>Port</td><td>16</td><td>4</td><td>16</td><td>4</td><td>16</td><td>4</td></tr><tr><td>Catheter</td><td>-</td><td>-</td><td>16</td><td>4</td><td>-</td><td>-</td></tr></tbody></table>

<table><thead><tr><th rowspan="2"></th><th colspan="2">AMK 4 ◁</th><th colspan="2">AMK 5 ◁</th><th colspan="2">AMK 6 ▽</th></tr><tr><th>LB</th><th>MH</th><th>LB</th><th>MH</th><th>LB</th><th>MH</th></tr></thead><tbody><tr><td>Silicone</td><td>16</td><td>4</td><td>16</td><td>4</td><td>16</td><td>4</td></tr><tr><td>Port</td><td>16</td><td>4</td><td>16</td><td>4</td><td>16</td><td>4</td></tr><tr><td>Catheter</td><td>16</td><td>4</td><td>-</td><td>-</td><td>16</td><td>4</td></tr></tbody></table>

**Supplementary Figure 2. Individual TIVAP sample data in the 7-day continuous *in vitro* experiment.** (a) CFUs of biofilm samples of each TIVAP part in individual TIVAPs. Using the total CFU of individual TIVAPs, Figure 1f was generated. (b) Growth frequencies of biofilm cell samples of each TIVAP part on amikacin plates at 1 time the MIC. Total growth frequencies are also shown as part of Figure 1g. (c) Amikacin MIC for biofilm population samples obtained from each TIVAP part of individual TIVAPs on LB and Mueller-Hinton (MH) media.
<figure><img src="image_70.png" /><figcaption>Fig. S3</figcaption></figure>

**Supplementary Figure 3.** Individual TIVAP sample data in the *in vitro* seven-cycle intermittent amikacin lock experiment. The data encompasses the following key parameters: CFUs of biofilm samples, growth frequency, and amikacin MIC in the seven cycles of the intermittent amikacin lock experiment with LB medium. (a–d) Individual LF82 TIVAP sample data: CFU time course (a), endpoint biofilm CFU (b), growth frequencies on amikacin plates with 1 time the MIC (c), and population amikacin MIC for biofilm samples (d). (e–h) Individual 55989 TIVAP sample data: CFU time course (e), endpoint biofilm CFU (f), growth frequencies on amikacin plates with 1 time the MIC (g), and population amikacin MIC for biofilm samples (h). Using the data in (a, e), Figure 2b and c were generated. Using total CFUs of
individual TIVAPs in (b, f), Figure 2d and e were generated. Total growth frequencies in (c, g) are shown as part of Figure 2f and g. The highest MIC values in each condition (d, h) are shown in Figure 2h and i.
<figure><img src="image_71.png" /><figcaption>Fig. S4</figcaption></figure>

**Supplementary Figure 4.** *In vitro* TIVAP biofilms formed by bioluminescent 55989pAT881 under continuous TPN flow and individual TIVAP sample data for four cycles of intermittent amikacin lock. (a) Time course of CFUs of floating cell samples obtained from the catheter tips of TIVAPs after 72-hour continuous TPN flow. (b) CFUs of endpoint biofilm samples formed on each TIVAP part (silicone septum, port, and catheter) in the same experiment as (a). (c) Bioluminescence image of TIVAP before bacterial loading. (d) Individual data for the time course
of floating cell samples obtained from each TIVAP. Using the data in (d), Figure 3b was generated. (e) Time course bioluminescence images of biofilms formed on TIVAPs. (f) CFUs of biofilm samples of each TIVAP part. Using the total CFUs of each TIVAP, Figure 3c was generated. (g) Growth frequencies of biofilm samples of each TIVAP part on amikacin plates at 1 time the MIC. Total growth frequencies are shown as part of Figure 3e. (h) Amikacin MIC for biofilm population samples obtained from each TIVAP part (silicone septum, port, and catheter) on LB and MH media. The highest MIC values in each condition in (h) are listed in Figure 3f.
<figure><img src="image_72.png" /><figcaption>Fig. S5</figcaption></figure>
**Supplementary Figure 5. Individual *in vivo* TIVAP sample data.** The following are presented: the time course of bioluminescence images, CFUs of biofilm samples, growth frequency and amikacin MIC. (a) Individual time course of bioluminescence images of 55989pAT881 biofilms and TIVAPs at the endpoint treated with or without amikacin lock. (b) CFUs of biofilm samples of each TIVAP part in individual TIVAPs. Using the total CFUs of each TIVAP, Figure 4d was generated. (c) Growth frequencies of biofilm samples of each TIVAP part on amikacin plates at 1 time the MIC. Using the total growth frequencies of each TIVAP, Figure 4e was generated. (d) Population amikacin MIC for biofilm samples obtained from each TIVAP part (silicone septum, port, and catheter) on LB and MH media. The highest and most frequent MIC values in each condition in (d) were listed in Figure 4f.