Synergistic effect of repurposed mitomycin C in combination with antibiotics against Aeromonas infection: in vitro and in vivo studies.

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Cheng-Fa Yeh, Chi-Chung Chen, Chih-Cheng Lai, Jin-Wei Liu, Hung-Jen Tang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4317037/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Aeromonas infections pose a significant threat, presenting a potentially life-threatening condition associated with high mortality rates. This study investigates the potential of mitomycin C (MMC), an anticancer drug, as a novel antimicrobial agent against Aeromonas infections. Methods The minimum inhibitory concentrations (MICs) of MMC and antibiotics against clinical isolates of Aeromonas species were assessed, using broth microdilution. Synergistic effects of MMC with antibiotics were assessed through time-kill studies. Additionally, we analyzed MMC's intracellular killing effects using a representative Aeromonas isolate. Efficacy of combined therapies was evaluated in neutropenic mice model. Results We found that only three antibiotics-ciprofloxacin (CIP), levofloxacin (LVX), and tigecycline (TGC) maintained susceptibility rates exceeding 90% under high inoculum conditions. Significantly, in vitro investigations demonstrated a synergistic effect over a 24-hour period when combining the 1/8x MIC of MMC with the 1x or 1/2x MICs of LVX, in a time–kill study. Furthermore, in vivo in neutropenic mice, the combination of 2 mg/kg and 1 mg/kg MMC with LVX resulted in 100% and 80% survival, respectively, whereas treatment with either drug alone resulted in 0% survival rate. Conclusions These findings suggest that MMC can be repurposed as an antimicrobial agent, particularly with a minimized dose and in combination with antibiotics. Aeromonas infections antimicrobial combination therapy mitomycin C synergistic effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Aeromonas , a member of the Aeromonadaceae family, poses a notable threat to human health due to the Capacity to induce severe illnesses, including septic arthritis, diarrheal enteritis, skin and soft tissue infections, meningitis, and bacteremia [ 1 – 5 ]. A recent study indicated that the average annual incidence of bacteremia caused by Aeromonas species in Southern Taiwan was 76 cases per million inhabitants from 2008 to 2010, surpassing rates in Western countries [ 6 ]. The three most common etiological agents are A. hydrophila , A. caviae , and A. veronii biovar sobria , and the possible virulence factors include cytotoxic toxins, proteases, hemolysins, lipases, adhesins, agglutinins, enterotoxins, and VacB [ 7 – 8 ]. Early and aggressive surgical debridement and the administration of broad-spectrum antibiotics, including the combination of doxycycline (DOX) with either ciprofloxacin (CIP) or ceftriaxone, is crucial [ 9 ]. Due to the slow pace of antimicrobial agent development, there is an urgent need to develop novel strategies to augment the diminishing efficacy of antibiotics in mitigating the mortality risk associated with Aeromonas infections. MMC, a naturally occurring entity originally isolated from the Gram-negative bacterium treptomyces caespitosus , exerts its effects by attacking DNA through a reduction cascade, leading to cross-linking of DNA double helix strands and inhibition of DNA synthesis [ 10 ]. A previous study reported a synergistic effect of co-administering MMC, an anticancer agent, and the tobramycin-ciprofloxacin antibiotic hybrid against multidrug-resistant (MDR) Gram-negative bacteria, including Pseudomonas aeruginosa , Acinetobacter baumannii , Escherichia coli , Klebsiella pneumoniae , and Enterobacter cloacae [ 11 ]. Combining antibiotics with nonantibiotic compounds that enhance their activity represents an effective approach to combat severe infectious diseases is crucial. In this study, our primary objective was the systematic exploration of the repurposing of MMC in combination with antimicrobial agents to against Aeromonas species. Concurrently, we underscore the importance of utilizing minimal effective dosages, with a focus on mitigating MMC-associated side effects through combination with antimicrobial agents. Methods Bacterial Isolates The thirty-one Aeromonas isolates, including 11 isolates of Aeromonas hydrophila , 10 isolates of A. caviae , and 10 isolates of A. sobria , were identified by MALDI-TOF MS (Bruker Daltonik, Bremen, Germany) and rpoB sequencing [ 12 – 13 ]. The species were identified by DNA sequence matching of rpoD and gyrB (or rpoB if necessary). The sequences of amplified DNA products were compared with reference sequences available in the GenBank database using BLAST ( http://www.ncbi.nlm.nih.gov/BLAST/ ). Isolates with a dissimilarity value of ≤ 1% were considered the same species. These isolates were stored at − 80°C in Protect Bacterial Preservers (Technical Service Consultants Limited, Heywood, UK) before investigation. Antimicrobial Susceptibility Testing Standard amikacin (AMK), aztreonam (ATM), cefazolin (CZ), cefmetazole (CMZ), cefotaxime (CTX), cefpirome (CPO), chloramphenicol (CHL), ciprofloxacin (CIP), doxycycline (DOX), fosfomycin (FOS), gentamicin (GM), imipenem (IPM), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), tigecycline (TGC), and mitomycin C (MMC) were used for antimicrobial susceptibility testing. Antibiotic MICs were determined by the broth microdilution method, and the interpretation criteria were based on the recommendations of the Clinical and Laboratory Standards Institutes (CLSI, 2023) or the US Food and Drug Administration (FDA). The normal inoculum 5 x 10 5 CFU/mL and high inoculum 5 x 10 7 CFU/mL were detected by this method. Quality control testing was performed using Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, and Pseudomonas aeruginosa ATCC 27853 [ 14 ]. Combination Killing Method Eight A. hydrophila isolates were randomly selected for in vitro measurement of the inhibitory effect of combination regimens, modified as reported by Tang et al, 2017. In brief, bacterial suspensions were diluted to 5 × 10 7 CFU/mL in fresh Mueller–Hinton broth. The drug concentrations of CIP, LVX, or TGC were adjusted to 1x MIC, 1/2x MIC, 1/4x MIC, and 1/8x MIC combined with 1x MIC, 1/2x MIC, 1/4x MIC, and 1/8x MIC of MMC. Bacterial counts were measured at 24 h by enumerating the colonies in 10-fold serially diluted specimens of 100-µL aliquots plated on the nutrient agar ((Difco™ Laboratories, Sparks, MD, USA) at 37°C. All experiments were performed in duplicate. The statistical significances were compared with that of each drug alone and no drug control: * P < 0.05, ** P < 0.01, and *** P < 0.001 [ 15 ]. Assessment of the Intracellular Antibacterial Activity of Antibiotics Raw 264.7 cells diluted to 5 x 10 5 CFU/mL cells in 24-well culture plates. The cells were incubated with 5 x 10 6 CFU/mL well of Aeromonas strain Aero01-243. Thus, the ratio of viable bacteria to cells was 10:1. One hour later, the culture plates were incubated with 8 µg/mL gentamicin for 2 h at 37°C to kill the extracellular bacteria and then washed twice with phosphate-buffered saline (PBS). The drug concentration of mitomycin C was adjusted to 1x, 1/2x, and 1/4x MIC and then added to the culture plates. At selected time intervals (0, 2, 4, and 24 h), the bacterial loads in the wells were counted. The cells were washed with ice-cold PBS. The bacteria were resuspended in 0.5 mL of 1% Triton X-100 in PBS, and the lysates with released bacteria were serially diluted (1:10 in PBS), plated on MH agar plates, and cultured overnight prior to bacterial counting. All experiments were performed in triplicate [ 16 – 17 ]. Murine Infection Model Female inbred BALB/c mice (Animal Center, National Science Council, Taipei, Taiwan) weighing 18–20 g (6–8 weeks old) were used in this study. This study was reviewed and approved by the Chi-Mei Medical Center Institutional Animal Care and Use Committee. The animals were maintained and used in accordance with the National Research Council recommendations and were provided food and water ad libitum. The mice were rendered neutropenic by intraperitoneal injections with 100 and 250 mg/kg of body weight of cyclophosphamide (Sigma-Aldrich, St. Louis, MO, USA) given one and four days prior to inoculation, respectively [ 18 ]. The Aeromonas strain was cultured in the respective broth medium at 37°C for 3 h, and the pellet obtained after centrifugation was diluted to the anticipated turbidity for the mouse experiments. Each dose was injected intramuscularly with 0.05 mL of bacterial suspension into ten mice. Antimicrobials were initiated two hours after bacterial inoculation and administered for 48 hours. The dosage of MMC for the mice was 2 mg/kg every 24 hours and levofloxacin 20 mg/kg every 12 hours. The selection of the 2 mg/kg MMC dosage in vivo was based on converting the clinically administered human dose (10 mg/m 2 ) to the mouse-equivalent dose [ 19 ]. Low doses of MMC (1 and 0.5 mg/kg) were also used in this study. The number of surviving mice was recorded at 8-hour intervals for 7 days. Results MIC results for Various Antibiotics and Mitomycin C Table 1 demonstrate the overall susceptibility rates exceeding 90% under normal inoculum 5 × 10 5 CFU/mL conditions for AMK, GM, ATM, CTX, CPO, CIP, LVX, MIN, TGC, FOS, and MEM, and less than 90% were susceptible to CZ, CMZ, CHL, DOX, and IPM. Finally, there are three antibiotics CIP, LVX, and TGC maintained the overall susceptibility rates exceeding 90% under high inoculum 5 x 10 7 CFU/mL conditions. Table 1 The MICs values were determined using broth microdilution for sixteen antimicrobial agents against 31 isolates of the Aeromonas species. Normal inoculum High inoculum MIC 50, mg/L MIC 90, mg/L susceptible MIC 50, mg/L MIC 90, mg/L susceptible AMK 4 8 100 16 32 67.86 GM 1 2 96.42 4 8 71.43 ATM ≦ 0.03 0.25 100 1 16 85.71 CZ > 128 > 128 0 > 128 > 128 0 CMZ 16 > 64 53.57 > 64 > 64 35.71 CTX 0.25 1 92.86 2 16 64.29 CPO 0.06 0.5 92.86 16 > 32 39.29 CHL 1 4 89.29 2 8 89.29 CIP 0.12 0.5 96.42 0.25 1 92.86 LVX 0.12 0.25 100 0.25 0.5 100 DOX 4 8 71.43 8 16 25 MIN 2 4 100 4 8 75 TGC 1 2 100 1 2 96.42 FOS 4 16 100 16 64 89.29 MEM 0.12 1 96.42 32 > 32 35.71 IPM 2 8 79.31 > 64 > 64 21.43 MMC 0.12 0.5 - 0.25 0.5 - AMK, amikacin; GM, gentamicin; ATM, aztreonam; CZ, cefazolin; CMZ, cefmetazole; CTX, cefotaxime; CPO, cefpirome; CHL, chloramphenicol; CIP, ciprofloxacin; LVX, levofloxacin; DOX, doxycycline; MIN, minocycline; TGC, tigecycline; FOS, fosfomycin; MEM, meropenem; IPM, imipenem; MMC, mitomycin C Table 2. The 24- killing effect of the antibiotic combination on the 8 isolates of Aeromonas species with a high inoculum (5 × 10 7 CFU/mL) using broth methods with 1x, 1/2x, 1/4x, 1/8x and 0x mitomycin C (MMC) in 1x, 1/2x, 1/4x, 1/8x and 0x MIC of ciprofloxacin (CIP) (A), levofloxacin (LVX) (B), and tigecycline (TGC) (C). A 24hr 1x CIP 1/2x CIP 1/4x CIP 1/8x CIP 0x CIP 1x MMC 0.83 ± 1.49 *** 3.14 ± 2.61 *** 6.46 ± 1.59 * 7.16 ± 0.81 7.51 ± 0.11 1/2x MMC 2.24 ± 2.37 *** 5.00 ± 2.41 *** 7.14 ± 1.39 * 8.00 ± 0.40 8.11 ± 0.45 1/4x MMC 3.70 ± 2.35 *** 5.68 ± 2.49 *** 7.78 ± 0.35 *** 8.14 ± 0.41 8.31 ± 0.35 1/8x MMC 5.06 ± 2.45 ** 7.42 ± 0.57 *** 7.86 ± 0.37 *** 8.12 ± 0.41 8.36 ± 0.31 No MMC 7.18 ± 0.81 8.09 ± 0.39 8.40 ± 0.28 7.46 ± 2.91 8.59 ± 0.08 B 24hr 1x LVX 1/2x LVX 1/4x LVX 1/8x LVX 0x LVX 1x MMC 0.00 ± 0.00 *** 3.13 ± 2.25 *** 5.68 ± 2.40 * 5.87 ± 2.46 * 7.25 ± 0.43 1/2x MMC 1.62 ± 1.69 *** 5.25 ± 2.25 *** 6.31 ± 2.50 * 7.45 ± 0.35 ** 7.86 ± 0.37 1/4x MMC 3.09 ± 2.09 *** 6.46 ± 1.28 *** 7.51 ± 0.50 *** 7.90 ± 0.42 *** 8.45 ± 0.18 1/8x MMC 3.83 ± 2.48 *** 7.43 ± 0.37 *** 8.15 ± 0.47 * 8.25 ± 0.47 8.57 ± 0.11 No MMC 6.31 ± 1.07 8.01 ± 0.45 8.34 ± 0.34 8.49 ± 0.27 8.68 ± 0.17 C 24hr 1x TGC 1/2x TGC 1/4x TGC 1/8x TGC 0x TGC 1x MMC 4.10 ± 2.67 *** 6.84 ± 0.81** 7.42 ± 0.56 7.69 ± 0.71 7.57 ± 0.51 1/2x MMC 5.34 ± 2.36 ** 7.44 ± 0.70 ** 7.82 ± 0.41 7.90 ± 0.72 8.10 ± 0.46 1/4x MMC 5.88 ± 2.49 * 7.89 ± 0.65 8.19 ± 0.46 8.09 ± 0.73 8.33 ± 0.40 1/8x MMC 7.24 ± 0.71 8.17 ± 0.49 8.34 ± 0.46 8.25 ± 0.52 * 8.54 ± 0.16 No MMC 7.44 ± 0.59 8.27 ± 0.48 8.44 ± 0.34 8.63 ± 0.22 8.75 ± 0.16 Colony counts are shown as the log means ± standard deviations. P value was compared with that of each drug alone and no drug control. *: P < 0.05. **: P < 0.01. ***: P < 0.001. Time-Kill Studies The synergistic effects of MMC in combination with antibiotics were investigated against eight Aeromonas hydrophila isolates with a high inoculum 5 × 10 7 CFU/mL. Broth microdilution methods were employed for a 24-hour duration, using MMC concentrations at 1x, 1/2x, 1/4x, 1/8x, and 0x MICs in combination with CIP, LVX, and TGC at 1x, 1/2x, 1/4x, 1/8x, and 0x MICs (Table. 2). The findings revealed a synergistic effect when employing MMC at a concentration of 1x, 1/2x, and 1/4x MICs in combination with CIP at a concentration of 1x MIC at 24-hour intervals compared with that of each drug alone and no drug control (p < 0.001; Table 2 A). Significantly, the data exhibited better activity when employing MMC at a concentration of 1x, 1/2x, 1/4x, and 1/8x MICs in combination with 1x MIC LVX at 24-hour intervals compared with that of each drug alone and no drug control (p < 0.001; Table 2 B). However, TGC exhibited a less effect at a concentration of 1x MIC, in combination with 1x MIC of MMC compared with that of each drug alone and no drug control (p < 0.001; Table 2 C). Consequently, LVX was selected as the primary agent for further investigations in combination with MMC in animal studies. Intracellular Antibacterial Activity of Mitomycin C The intracellular killing effect of MMC against Aeromonas Aero01-243 is depicted in Fig. 1 . The results indicate the absence of detectable Aeromonas Aero01-243 when subjected to 1x and 1/2x MICs of MMC at 24-hour intervals. Conversely, treatment with 1/4x MIC of MMC or the control group demonstrated no inhibition of intracellular colony counts, leading to the proliferation of Aeromonas Aero01-243 to over log 6 within the same timeframe. Notably, the application of 1x and 1/2x MIC of MMC exerted a substantial inhibitory effect on intracellular colony counts at 24-hour intervals compared with the 1/4x MIC of MMC or the control group. Animal Study Neutropenic mice were intramuscularly administered Aeromonas Aero01-243 at concentrations of 1.8 × 10 2 , 1.8 × 10 3 , 1.8 × 10 4 , and 1.8 × 10 5 CFU/mL (Figure. 2). The group with inoculum of 1.8 × 10 2 CFU/mL exhibited a 100% survival rate (10/10) on day 1, 70% (7/10) on day 2, 60% (6/10) on day 3, and 0% (0/10) after day 4. The group inoculated with 1.8 × 10 3 CFU/mL exhibited a 100% survival rate (10/10) on day 1 and 0% (0/10) after day 2. The group inoculated with 1.8 × 10 4 CFU/mL exhibited a 0% survival rate (0/10) on day 1. Remarkably, the group inoculated with 1.8 × 10 4 CFU/mL and treated with 2 mg/kg MMC maintained a 100% survival rate (10/10) until day 7. However, the group inoculated with 1.8 × 10 5 CFU/mL and treated with 2 mg/kg MMC exhibited a 30% survival rate (3/10) on day 1 and 0% (0/10) after day 2. The group inoculated with 1.8 × 10 4 CFU/mL exhibited a significantly higher survival rate compared to the other groups. Synergy of Mitomycin C with Levofloxacin In neutropenic mice, Aeromonas Aero01-243 was administered intramuscularly with inoculated of 0.99 × 10 5 CFU/mL (Figure. 3A) or 0.99 × 10 6 CFU/mL (Figure. 3B) treated with MMC, LVX, or a combination of MMC and LVX. In Fig. 3A, the control group without MMC or LVX exhibited a 0% survival rate (0/10) on day1. The MMC group exhibited a 100% survival rate (10/10) on day 1 but declined to 0% (0/10) after day 2. The LVX group exhibited a 100% survival rate (10/10) on day 2, 80% (8/10) on day 3, and 60% (6/10) after day 4. The combination group maintained a 100% survival rate (10/10) until day 7. As shown in Fig. 3B, the control and MMC groups exhibited a 0% survival rate at 0.99 × 10 6 CFU/mL. The LVX group displayed a 100% survival rate (10/10) on day 1, 80% (8/10) on day 2, 40% (4/10) on day 3, and 0% (0/10) after day 4. The combination group maintained a 100% survival rate (10/10) until day 7. A significant synergistic effect was observed, with a 100% survival rate when administering MMC in combination with LVX at 0.99 × 10 5 CFU/mL or 0.99 × 10 6 CFU/mL. In contrast, MMC monotherapy resulted in a 0% survival rate, and LVX monotherapy exhibited a 60% survival rate at 0.99 × 10 5 CFU/mL or a 0% survival rate at 0.99 × 10 6 CFU/mL. Efficacy of Lower Dosages of Mitomycin C in Combination with Levofloxacin We also evaluated survival rates employing a reduced dosage of MMC in combination with LVX, using a higher inoculum (1.89 × 10 6 CFU/mL) of Aeromonas Aero01-243 (Figure. 4). The results reveal a 0% survival rate (0/10) for the control group on day 1. In contrast, the LVX group exhibited a 100% survival rate (10/10) on day 1, 20% (2/10) on day 2, and 0% (0/10) post day 3. The MMC group exhibited survival rates of 20% (2/10), 80% (8/10), and 100% (10/10) on day 7 at MMC doses of 0.5, 1, and 2 mg/kg, respectively, when combined with LVX. Discussion In this study, we observed a remarkably low MIC of MMC against Aeromonas species, with MIC 50 /MIC 90 values of 0.12/0.5 mg/L at normal inoculum 5 x 10 5 CFU/ mL and 0.25/0.5 mg/L at high inoculum 5 x 10 7 CFU/ mL. The inoculum effect for MMC was less obvious than that of other antibiotics. This study contributes valuable insights into the complexities associated with severe Aeromonas species infections and underscores the potential of combination therapy involving MMC and selected antibiotics in clinical practice. Infections characterized by a high bacterial burden, such as septic bloodstream infections, necrotizing fasciitis, and abscesses, are frequently encountered and pose a challenge due to the limited efficacy of the currently available therapies [ 20 ]. To address this issue, adjustments to the in vitro testing protocol require an increase in the inoculum quantity. From our data, most antibiotics exhibited susceptibility rates exceeding 90% under the normal inoculum 5 × 10 5 CFU/mL condition. However, only CIP, LVX, TGC, and MMC maintained susceptibility rates exceeding 90% under the high inoculum 5 × 10 7 CFU/mL condition. The findings underscore the significant susceptibility of CIP, LVX and TGC compared to antibiotics that are effective against A eromonas species, such as CTX and MIN. These results may have implications for the selection of optimal therapeutic agents against severe and high inoculum Aeromonas infections. Consequently, we conducted investigations employing MMC in combination with three antimicrobial agents under high inoculum 5 × 10 7 CFU/mL conditions in a time-kill assay according to the above results. The results demonstrated a noteworthy synergistic effect when applying 1/8x MIC of MMC with 1x MIC of LVX compared with that of each drug alone and no drug control (p < 0.001). This significant synergistic effect remained consistent over a 24-hour period, distinguishing it from combinations involving CIP and TGC. The intricate biochemical mechanisms underlying phagocytosis and intracellular survival of Aeromonas within host macrophages represent complex processes critical to the outcome of infection [ 21 ]. Intracellular bacteria, protected from high extracellular antibiotic levels, heighten the risk of drug resistance. Antibiotic efflux pumps in eukaryotic cells reduce intracellular drug levels, diminishing their effectiveness against intracellular bacteria [ 22 – 23 ]. According to a previous study, LVX exhibited a 300-fold reduction in intracellular bacterial load at clinically relevant concentrations, while DOX and CIP exhibited 40-fold and 4-fold reductions, respectively [ 24 ]. Our investigation provides compelling evidence that the application of 1x and 1/2x MICs of MMC exerted a substantial inhibitory effect on intracellular colony counts at 24-hour intervals compared with the 1/4x MIC of MMC or the control group in vitro. LVX was then chosen as the primary agent for further exploration in combination with MMC in an animal study. In individuals with compromised immune function, such as those affected by conditions such as diabetes, malignancies, liver cirrhosis, or HIV infection, Aeromonas infections can present a life-threatening and invasive risk. Among this cohort, bloodstream infections are highly prevalent, with associated mortality rates reaching as high as 68% [ 25 – 27 ]. To replicate the immunocompromised state, a well-established neutropenic mouse model was employed. In the group treated with MMC at an initial inoculum of 1.8 × 10 4 CFU/mL, a notable 100% survival rate was observed; much above that of groups exposed to a higher initial inoculum of 1.8 × 10 5 CFU/mL, which had 0% survival rates. The effectiveness of monotherapy using MMC in treating mild infections with low inoculum levels is underscored, although its impact is less significant when considering the inoculum effect. Nevertheless, its efficacy declines notably when the inoculum reaches 1.8 × 10 5 CFU/mL, indicating that combining MMC with antibiotics is advisable for managing severe Aeromonas infections, such as septicemia or necrotizing fasciitis with abscess formation. Our objective then was to investigate the combined efficacy of MMC and the antimicrobial agent LVX in enhancing synergistic effects against Aeromonas species in an animal model. We conducted experiments to evaluate the synergistic impact of MMC in combination with LVX utilizing a neutropenic mouse model. Our study focused on a higher bacterial inoculum (0.99 × 10 5 CFU/mL and 0.99 × 10 6 CFU/mL) of Aeromonas Aero01-243. The results revealed significant synergistic effects, with a 100% survival rate observed in mice treated with 2 mg/kg MMC in combination with 20 mg/kg LVX across all levels of high bacterial inoculum. This is in stark contrast to the outcomes of MMC and LVX monotherapy, where both exhibited 0% survival rates. Our findings illustrate that the in vivo synergistic efficacy of MMC combined with antibiotics yields promising results, warranting consideration for potential clinical applications in patient care. Given the considerable adverse effects linked with MMC, there is an imperative to investigate lower-dose MMC combination regimens that adequately alleviate these effects. We evaluated survival rates by adjusting MMC dosages in combination with LVX in neutropenic mice challenged with a higher inoculum 1.89 × 10 6 CFU/mL of Aeromonas Aero01-243. The results revealed survival rates of 80% and 100% at doses of 1 and 2 mg/kg, respectively. These findings establish a notable survival rate of 80% in immunocompromised individuals infected with a higher inoculum, who were treated with 1 mg/kg of MMC in combination with LVX. This approach may have clinical implications for reducing MMC-related adverse effects. Conclusion Our findings highlight the absence of an inoculum effect in MMC and underscore its substantial inhibitory impact on intracellular colony counts. Similarly, LVX also demonstrated exceptional intracellular bactericidal activity. Notably, neutropenic mice subjected to a combined regimen of MMC, and LVX exhibited a 100% survival rate, with even a half-dose combination sustaining an impressive 80% survival rate. These outcomes provide compelling evidence for the pivotal synergy between MMC and LVX, showcasing significant efficacy against Aeromonas species both in vitro and in vivo. Our forthcoming investigations aim to delineate an optimally effective lower dose of MMC to mitigate potential adverse effects. Abbreviations MMC Mitomycin C MICs Minimum inhibitory concentrations CIP Ciprofloxacin LVX Levofloxacin TGC Tigecycline AMK Amikacin ATM Aztreonam CZ Cefazolin CMZ Cefmetazole CTX Cefotaxime CPO Cefpirome CHL Chloramphenicol DOX Doxycycline FOS Fosfomycin GM Gentamicin IPM Imipenem MEM Meropenem MIN Minocycline CLSI Clinical and Laboratory Standards Institutes FDA Food and Drug Administration PBS Phosphate-buffered saline IRB Institutional Review Board Declarations Acknowledgements The authors would like to thank Laboratory of Medical Research, Chi Mei Medical Center for the technical assistance in operating time - kill studies, intracellular antibacterial activity, and animal studies. Author contributions Chi-Chung Chen and Chih-Cheng Lai performed the experiments. Cheng-Fa Yeh, Chi-Chung Chen, and Hung-Jen Tang analyzed the experimental data. Chi-Chung Chen, Chih-Cheng Lai, and Jin-Wei Liu provided experimental resources. Cheng-Fa Yeh wrote the manuscript. Wen-Pin Su and Hung-Jen Tang revised the manuscript. Cheng-Fa Yeh and Hung-Jen Tang provided financial support. All authors reviewed the manuscript. Funding This work was supported by the Chi-Mei Medical Center Research Foundation-(CMFHR11069, to Cheng-Fa Yeh), ( CMFHT11202, to Hung-Jen Tang ), (CMFHR11311, to Cheng-Fa Yeh ). Data availability All data generated or analyzed during this study are included in this published article. Raw data is available from the corresponding author upon request. Ethics approval and consent to participate The study and waiver from the informed consent process were approved by the Institutional Review Board (IRB) of the Chi Mei Medical Center, Tainan City, Taiwan (IRB Serial number LAC. SOP102-104). Consent for publication This manuscript is approved by all authors for publication. Competing interests The authors declare no competing interests. References Abdel-Latif HM, Khafaga AF. Natural co-infection of cultured Nile tilapia Oreochromis niloticus with Aeromonas hydrophila and Gyrodactylus cichlidarum experiencing high mortality during summer. Aquac Res 2020;51:1880-1892. Stratev D, Odeyemi OA. An overview of motile Aeromonas septicaemia management. Aquac Int 2017;25:1095-1105. Lee, CY, Kuo LT, Peng KT, et al. Prognostic factors and monomicrobial necrotizing fasciitis: gram-positive versus gram-negative pathogens. BMC Infect Dis 2011;11:5-12. Huang KF, Huang MH, Lin YS, et al. 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J Basic Clin Pharm 2016;7:27-31. Pletzer D, Hancock RE. Is synergy the key to treating high-density infections? Future Microbiol 2018;13:1629–1632. Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 1999;17: 593-623. Thwaites G, Gant EV. Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus? Nat Rev Microbiol 2011;9:215–22. Seral C, Carryn S, Tulkens PM, Van Bambeke F. Influence of P-glycoprotein and MRP efflux pump inhibitors on the intracellular activity of azithromycin and ciprofloxacin in macrophages infected by Listeria monocytogenes or Staphylococcus aureus. J Antimicrob Chemother 2003;51:1167–73. Peyrusson F, Whelan AO, Hartley MG, et al. Intracellular Activity of Antibiotics against Coxiella burnetii in a Model of Activated Human THP-1 Cells. Antimicrob Agents Chemother 2021;65(12):e01061-21. Nolla-Salas J, Codina-Calero J, Valles-Angulo S, et al. Clinical significance and outcome of Aeromonas spp. infections among 204 adult patients. Eur J Clin Microbiol Infect Dis 2017;36(8):1393-403. Ko WC, Chuang YC. Aeromonas bacteremia: review of 59 episodes. Clin Infect Dis 1995;20(5): 1298-304. Lau SM, Peng MY, Chang FY. Outcomes of Aeromonas bacteremia in patients with different types of underlying disease. J Microbiol Immunol Infect 2000;33(4):241-7. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4317037","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":296523327,"identity":"145b2093-5ea9-4bfd-af61-082a3ec5fcbf","order_by":0,"name":"Cheng-Fa Yeh","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Fa","middleName":"","lastName":"Yeh","suffix":""},{"id":296523331,"identity":"bfeee0ad-2758-419e-a5d4-e1530a1b2042","order_by":1,"name":"Chi-Chung Chen","email":"","orcid":"","institution":"Chi Mei Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Chi-Chung","middleName":"","lastName":"Chen","suffix":""},{"id":296523335,"identity":"933df800-813b-49d8-a3d0-fe170b1325ef","order_by":2,"name":"Chih-Cheng Lai","email":"","orcid":"","institution":"Chi Mei Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Chih-Cheng","middleName":"","lastName":"Lai","suffix":""},{"id":296523339,"identity":"2a85b0be-d23e-48b4-b13e-902cab254901","order_by":3,"name":"Jin-Wei Liu","email":"","orcid":"","institution":"Chi Mei Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Jin-Wei","middleName":"","lastName":"Liu","suffix":""},{"id":296523343,"identity":"a9061b0f-dcba-491f-a6e5-0d1e413ae337","order_by":4,"name":"Hung-Jen Tang","email":"","orcid":"","institution":"Chi Mei Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hung-Jen","middleName":"","lastName":"Tang","suffix":""},{"id":296523347,"identity":"c10e1b16-fc85-4998-8095-7f5b79c033ca","order_by":5,"name":"Wen-Pin Su","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYLCCDwwMPHxAmrEBxDtAhA7GGUAtbEhaIDQ+wMwDJIjXYnC7/Zm0bY6dDBsD78GPM9sY5PhuJLA/5sGn5c4ZM+ncbclAh/ElS25sYzCWvJHA2IxPi9mNHDagFmagFh4zxodtDIkbQFpy8GpJfyZtua0erqWeCC0JZtKM2w5DtAAdlmBASIv9nTPGlr3bjvOwMfMYS844J2E488zDxtl/8GiRnN3+8MbPbdX2/Ow9hh97ymzk+Y4nH/g4A48WBgkYgxnOJRSTEvilR8EoGAWjYBQwMAAAK3dGxrJ7m2EAAAAASUVORK5CYII=","orcid":"","institution":"National Cheng Kung University Hospital, National Cheng Kung University","correspondingAuthor":true,"prefix":"","firstName":"Wen-Pin","middleName":"","lastName":"Su","suffix":""}],"badges":[],"createdAt":"2024-04-24 09:22:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4317037/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4317037/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55796960,"identity":"0ededc69-c9ac-4594-aa5a-c1dd53f44df0","added_by":"auto","created_at":"2024-05-03 11:15:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52536,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure1Aeromonas.png","url":"https://assets-eu.researchsquare.com/files/rs-4317037/v1/bad674f5cd949baa6b1ecf15.png"},{"id":55797179,"identity":"ca8763e7-363c-4a6f-829a-4663b75db0a3","added_by":"auto","created_at":"2024-05-03 11:23:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":232265,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure2Aeromonas.png","url":"https://assets-eu.researchsquare.com/files/rs-4317037/v1/7f1bc8af41fbd016c57f95bc.png"},{"id":55796962,"identity":"bc82dfc2-544a-455b-baba-db338cbdd540","added_by":"auto","created_at":"2024-05-03 11:15:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":230195,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure3Aeromonas.png","url":"https://assets-eu.researchsquare.com/files/rs-4317037/v1/a64516cefbdd639f53d8a13c.png"},{"id":55796964,"identity":"9e33def8-ddfe-4d58-8e4c-281aaa8cc515","added_by":"auto","created_at":"2024-05-03 11:15:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136494,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure4Aeromonas.png","url":"https://assets-eu.researchsquare.com/files/rs-4317037/v1/56a870583b99af6360abb33f.png"},{"id":57261764,"identity":"f4737052-6918-4484-8673-b1548c9c455f","added_by":"auto","created_at":"2024-05-28 09:52:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1242541,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4317037/v1/a7479cb2-1ad0-4c65-ad3f-920c4902cb50.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic effect of repurposed mitomycin C in combination with antibiotics against Aeromonas infection: in vitro and in vivo studies.","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAeromonas\u003c/em\u003e, a member of the \u003cem\u003eAeromonadaceae\u003c/em\u003e family, poses a notable threat to human health due to the Capacity to induce severe illnesses, including septic arthritis, diarrheal enteritis, skin and soft tissue infections, meningitis, and bacteremia [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. A recent study indicated that the average annual incidence of bacteremia caused by \u003cem\u003eAeromonas\u003c/em\u003e species in Southern Taiwan was 76 cases per million inhabitants from 2008 to 2010, surpassing rates in Western countries [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The three most common etiological agents are \u003cem\u003eA. hydrophila\u003c/em\u003e, \u003cem\u003eA. caviae\u003c/em\u003e, and \u003cem\u003eA. veronii biovar sobria\u003c/em\u003e, and the possible virulence factors include cytotoxic toxins, proteases, hemolysins, lipases, adhesins, agglutinins, enterotoxins, and VacB [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Early and aggressive surgical debridement and the administration of broad-spectrum antibiotics, including the combination of doxycycline (DOX) with either ciprofloxacin (CIP) or ceftriaxone, is crucial [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to the slow pace of antimicrobial agent development, there is an urgent need to develop novel strategies to augment the diminishing efficacy of antibiotics in mitigating the mortality risk associated with \u003cem\u003eAeromonas\u003c/em\u003e infections. MMC, a naturally occurring entity originally isolated from the Gram-negative bacterium \u003cem\u003etreptomyces caespitosus\u003c/em\u003e, exerts its effects by attacking DNA through a reduction cascade, leading to cross-linking of DNA double helix strands and inhibition of DNA synthesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A previous study reported a synergistic effect of co-administering MMC, an anticancer agent, and the tobramycin-ciprofloxacin antibiotic hybrid against multidrug-resistant (MDR) Gram-negative bacteria, including \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, and \u003cem\u003eEnterobacter cloacae\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Combining antibiotics with nonantibiotic compounds that enhance their activity represents an effective approach to combat severe infectious diseases is crucial.\u003c/p\u003e \u003cp\u003eIn this study, our primary objective was the systematic exploration of the repurposing of MMC in combination with antimicrobial agents to against \u003cem\u003eAeromonas\u003c/em\u003e species. Concurrently, we underscore the importance of utilizing minimal effective dosages, with a focus on mitigating MMC-associated side effects through combination with antimicrobial agents.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Isolates\u003c/h2\u003e \u003cp\u003eThe thirty-one \u003cem\u003eAeromonas\u003c/em\u003e isolates, including 11 isolates of \u003cem\u003eAeromonas hydrophila\u003c/em\u003e, 10 isolates of \u003cem\u003eA. caviae\u003c/em\u003e, and 10 isolates of \u003cem\u003eA. sobria\u003c/em\u003e, were identified by MALDI-TOF MS (Bruker Daltonik, Bremen, Germany) and rpoB sequencing [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The species were identified by DNA sequence matching of \u003cem\u003erpoD\u003c/em\u003e and \u003cem\u003egyrB\u003c/em\u003e (or \u003cem\u003erpoB\u003c/em\u003e if necessary). The sequences of amplified DNA products were compared with reference sequences available in the GenBank database using BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/BLAST/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/BLAST/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Isolates with a dissimilarity value of \u0026le;\u0026thinsp;1% were considered the same species. These isolates were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in Protect Bacterial Preservers (Technical Service Consultants Limited, Heywood, UK) before investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial Susceptibility Testing\u003c/h2\u003e \u003cp\u003eStandard amikacin (AMK), aztreonam (ATM), cefazolin (CZ), cefmetazole (CMZ), cefotaxime (CTX), cefpirome (CPO), chloramphenicol (CHL), ciprofloxacin (CIP), doxycycline (DOX), fosfomycin (FOS), gentamicin (GM), imipenem (IPM), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), tigecycline (TGC), and mitomycin C (MMC) were used for antimicrobial susceptibility testing.\u003c/p\u003e \u003cp\u003eAntibiotic MICs were determined by the broth microdilution method, and the interpretation criteria were based on the recommendations of the Clinical and Laboratory Standards Institutes (CLSI, 2023) or the US Food and Drug Administration (FDA). The normal inoculum 5 x 10\u003csup\u003e5\u003c/sup\u003e CFU/mL and high inoculum 5 x 10\u003csup\u003e7\u003c/sup\u003e CFU/mL were detected by this method. Quality control testing was performed using \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ATCC 700603, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCombination Killing Method\u003c/h2\u003e \u003cp\u003eEight \u003cem\u003eA. hydrophila\u003c/em\u003e isolates were randomly selected for in vitro measurement of the inhibitory effect of combination regimens, modified as reported by Tang et al, 2017. In brief, bacterial suspensions were diluted to 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/mL in fresh Mueller\u0026ndash;Hinton broth. The drug concentrations of CIP, LVX, or TGC were adjusted to 1x MIC, 1/2x MIC, 1/4x MIC, and 1/8x MIC combined with 1x MIC, 1/2x MIC, 1/4x MIC, and 1/8x MIC of MMC. Bacterial counts were measured at 24 h by enumerating the colonies in 10-fold serially diluted specimens of 100-\u0026micro;L aliquots plated on the nutrient agar ((Difco\u0026trade; Laboratories, Sparks, MD, USA) at 37\u0026deg;C. All experiments were performed in duplicate. The statistical significances were compared with that of each drug alone and no drug control: * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and *** P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of the Intracellular Antibacterial Activity of Antibiotics\u003c/h2\u003e \u003cp\u003eRaw 264.7 cells diluted to 5 x 10\u003csup\u003e5\u003c/sup\u003e CFU/mL cells in 24-well culture plates. The cells were incubated with 5 x 10\u003csup\u003e6\u003c/sup\u003e CFU/mL well of \u003cem\u003eAeromonas\u003c/em\u003e strain Aero01-243. Thus, the ratio of viable bacteria to cells was 10:1. One hour later, the culture plates were incubated with 8 \u0026micro;g/mL gentamicin for 2 h at 37\u0026deg;C to kill the extracellular bacteria and then washed twice with phosphate-buffered saline (PBS). The drug concentration of mitomycin C was adjusted to 1x, 1/2x, and 1/4x MIC and then added to the culture plates. At selected time intervals (0, 2, 4, and 24 h), the bacterial loads in the wells were counted. The cells were washed with ice-cold PBS. The bacteria were resuspended in 0.5 mL of 1% Triton X-100 in PBS, and the lysates with released bacteria were serially diluted (1:10 in PBS), plated on MH agar plates, and cultured overnight prior to bacterial counting. All experiments were performed in triplicate [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMurine Infection Model\u003c/h2\u003e \u003cp\u003eFemale inbred BALB/c mice (Animal Center, National Science Council, Taipei, Taiwan) weighing 18\u0026ndash;20 g (6\u0026ndash;8 weeks old) were used in this study. This study was reviewed and approved by the Chi-Mei Medical Center Institutional Animal Care and Use Committee. The animals were maintained and used in accordance with the National Research Council recommendations and were provided food and water ad libitum. The mice were rendered neutropenic by intraperitoneal injections with 100 and 250 mg/kg of body weight of cyclophosphamide (Sigma-Aldrich, St. Louis, MO, USA) given one and four days prior to inoculation, respectively [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The \u003cem\u003eAeromonas\u003c/em\u003e strain was cultured in the respective broth medium at 37\u0026deg;C for 3 h, and the pellet obtained after centrifugation was diluted to the anticipated turbidity for the mouse experiments. Each dose was injected intramuscularly with 0.05 mL of bacterial suspension into ten mice. Antimicrobials were initiated two hours after bacterial inoculation and administered for 48 hours.\u003c/p\u003e \u003cp\u003eThe dosage of MMC for the mice was 2 mg/kg every 24 hours and levofloxacin 20 mg/kg every 12 hours. The selection of the 2 mg/kg MMC dosage in vivo was based on converting the clinically administered human dose (10 mg/m\u003csup\u003e2\u003c/sup\u003e) to the mouse-equivalent dose [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Low doses of MMC (1 and 0.5 mg/kg) were also used in this study. The number of surviving mice was recorded at 8-hour intervals for 7 days.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMIC results for Various Antibiotics and Mitomycin C\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrate the overall susceptibility rates exceeding 90% under normal inoculum 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL conditions for AMK, GM, ATM, CTX, CPO, CIP, LVX, MIN, TGC, FOS, and MEM, and less than 90% were susceptible to CZ, CMZ, CHL, DOX, and IPM. Finally, there are three antibiotics CIP, LVX, and TGC maintained the overall susceptibility rates exceeding 90% under high inoculum 5 x 10\u003csup\u003e7\u003c/sup\u003e CFU/mL conditions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe MICs values were determined using broth microdilution for sixteen antimicrobial agents against 31 isolates of the \u003cem\u003eAeromonas\u003c/em\u003e species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eNormal inoculum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eHigh inoculum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC \u003csub\u003e50, mg/L\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMIC \u003csub\u003e90, mg/L\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esusceptible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMIC \u003csub\u003e50, mg/L\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMIC \u003csub\u003e90, mg/L\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esusceptible\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e67.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e71.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e≦\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e85.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCMZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e64.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e39.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e92.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLVX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMIN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e96.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIPM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eAMK, amikacin; GM, gentamicin; ATM, aztreonam; CZ, cefazolin; CMZ, cefmetazole; CTX, cefotaxime;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eCPO, cefpirome; CHL, chloramphenicol; CIP, ciprofloxacin; LVX, levofloxacin; DOX, doxycycline; MIN,\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eminocycline; TGC, tigecycline; FOS, fosfomycin; MEM, meropenem; IPM, imipenem; MMC, mitomycin C\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003cp\u003eTable 2. The 24- killing effect of the antibiotic combination on the\u0026nbsp;8 isolates of \u003cem\u003eAeromonas\u0026nbsp;\u003c/em\u003especies with a high inoculum (5\u0026nbsp;\u0026times;\u0026nbsp;10\u003csup\u003e7\u003c/sup\u003e CFU/mL)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e using broth methods with 1x, 1/2x, 1/4x, 1/8x and \u0026nbsp;0x mitomycin C (MMC) in 1x, 1/2x, 1/4x, 1/8x and \u0026nbsp;0x MIC of ciprofloxacin (CIP) (A), levofloxacin (LVX) (B), and tigecycline (TGC) (C).\u003c/p\u003e\n\u003cp\u003eA\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e\u003cstrong\u003e24hr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e1x CIP \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e1/2x CIP \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e1/4x CIP \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e1/8x CIP \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e0x CIP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e1x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e0.83\u0026nbsp;\u0026plusmn;\u0026nbsp;1.49 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e3.14\u0026nbsp;\u0026plusmn;\u0026nbsp;2.61 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e6.46\u0026nbsp;\u0026plusmn;\u0026nbsp;1.59 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e7.16 \u0026plusmn; 0.81\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e7.51 \u0026plusmn; 0.11\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e1/2x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e2.24\u0026nbsp;\u0026plusmn;\u0026nbsp;2.37 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e5.00\u0026nbsp;\u0026plusmn;\u0026nbsp;2.41 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e7.14\u0026nbsp;\u0026plusmn;\u0026nbsp;1.39 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e8.00 \u0026plusmn; 0.40\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e8.11 \u0026plusmn; 0.45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e1/4x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e3.70\u0026nbsp;\u0026plusmn;\u0026nbsp;2.35 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e5.68\u0026nbsp;\u0026plusmn;\u0026nbsp;2.49 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e7.78\u0026nbsp;\u0026plusmn;\u0026nbsp;0.35 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e8.14 \u0026plusmn; 0.41\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e8.31 \u0026plusmn; 0.35\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e1/8x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e5.06\u0026nbsp;\u0026plusmn;\u0026nbsp;2.45 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e7.42\u0026nbsp;\u0026plusmn;\u0026nbsp;0.57 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e7.86\u0026nbsp;\u0026plusmn;\u0026nbsp;0.37 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e8.12 \u0026plusmn; 0.41\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e8.36 \u0026plusmn; 0.31\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003eNo MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e7.18 \u0026plusmn; 0.81\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e8.09 \u0026plusmn; 0.39\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e8.40 \u0026plusmn; 0.28\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e7.46 \u0026plusmn; 2.91\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e8.59 \u0026plusmn; 0.08\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eB\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e\u003cstrong\u003e24hr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1x LVX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1/2x LVX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1/4x LVX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1/8x LVX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e0x LVX\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e1x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e0.00\u0026nbsp;\u0026plusmn;\u0026nbsp;0.00 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e3.13\u0026nbsp;\u0026plusmn;\u0026nbsp;2.25 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5.68\u0026nbsp;\u0026plusmn;\u0026nbsp;2.40 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5.87\u0026nbsp;\u0026plusmn;\u0026nbsp;2.46 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e7.25 \u0026plusmn; 0.43\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e1/2x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e1.62\u0026nbsp;\u0026plusmn;\u0026nbsp;1.69 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e5.25\u0026nbsp;\u0026plusmn;\u0026nbsp;2.25 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e6.31\u0026nbsp;\u0026plusmn;\u0026nbsp;2.50 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e7.45\u0026nbsp;\u0026plusmn;\u0026nbsp;0.35 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e7.86 \u0026plusmn; 0.37\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e1/4x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e3.09\u0026nbsp;\u0026plusmn;\u0026nbsp;2.09 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e6.46\u0026nbsp;\u0026plusmn;\u0026nbsp;1.28 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e7.51\u0026nbsp;\u0026plusmn;\u0026nbsp;0.50 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e7.90\u0026nbsp;\u0026plusmn;\u0026nbsp;0.42 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e8.45 \u0026plusmn; 0.18\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e1/8x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e3.83\u0026nbsp;\u0026plusmn;\u0026nbsp;2.48 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e7.43\u0026nbsp;\u0026plusmn;\u0026nbsp;0.37 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e8.15\u0026nbsp;\u0026plusmn;\u0026nbsp;0.47 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e8.25 \u0026plusmn; 0.47\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e8.57 \u0026plusmn; 0.11\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003eNo MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e6.31 \u0026plusmn; 1.07\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e8.01 \u0026plusmn; 0.45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e8.34 \u0026plusmn; 0.34\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e8.49 \u0026plusmn; 0.27\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\"\u003e\n \u003cp\u003e8.68 \u0026plusmn; 0.17\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eC\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e\u003cstrong\u003e24hr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\"\u003e\n \u003cp\u003e1x TGC \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\"\u003e\n \u003cp\u003e1/2x TGC \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e1/4x TGC \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\"\u003e\n \u003cp\u003e1/8x TGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.463917525773196%\"\u003e\n \u003cp\u003e0x TGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e1x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\"\u003e\n \u003cp\u003e4.10\u0026nbsp;\u0026plusmn;\u0026nbsp;2.67 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\"\u003e\n \u003cp\u003e6.84 \u0026plusmn; 0.81**\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e7.42 \u0026plusmn; 0.56\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\"\u003e\n \u003cp\u003e7.69 \u0026plusmn; 0.71\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.463917525773196%\"\u003e\n \u003cp\u003e7.57 \u0026plusmn; 0.51\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e1/2x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\"\u003e\n \u003cp\u003e5.34\u0026nbsp;\u0026plusmn;\u0026nbsp;2.36 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\"\u003e\n \u003cp\u003e7.44\u0026nbsp;\u0026plusmn;\u0026nbsp;0.70 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e7.82 \u0026plusmn; 0.41\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\"\u003e\n \u003cp\u003e7.90 \u0026plusmn; 0.72\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.463917525773196%\"\u003e\n \u003cp\u003e8.10 \u0026plusmn; 0.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e1/4x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\"\u003e\n \u003cp\u003e5.88\u0026nbsp;\u0026plusmn;\u0026nbsp;2.49 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\"\u003e\n \u003cp\u003e7.89 \u0026plusmn; 0.65\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e8.19 \u0026plusmn; 0.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\"\u003e\n \u003cp\u003e8.09 \u0026plusmn; 0.73\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.463917525773196%\"\u003e\n \u003cp\u003e8.33 \u0026plusmn; 0.40\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e1/8x MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\"\u003e\n \u003cp\u003e7.24 \u0026plusmn; 0.71\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\"\u003e\n \u003cp\u003e8.17 \u0026plusmn; 0.49\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e8.34 \u0026plusmn; 0.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\"\u003e\n \u003cp\u003e8.25\u0026nbsp;\u0026plusmn;\u0026nbsp;0.52 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.463917525773196%\"\u003e\n \u003cp\u003e8.54 \u0026plusmn; 0.16\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003eNo MMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\"\u003e\n \u003cp\u003e7.44 \u0026plusmn; 0.59\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\"\u003e\n \u003cp\u003e8.27 \u0026plusmn; 0.48\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e8.44 \u0026plusmn; 0.34\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\"\u003e\n \u003cp\u003e8.63 \u0026plusmn; 0.22\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.463917525773196%\"\u003e\n \u003cp\u003e8.75 \u0026plusmn; 0.16\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eColony counts are shown as the log means \u0026plusmn; standard deviations. P value was\u0026nbsp;compared with that of each drug alone and no drug control. *: P \u0026lt; 0.05. **: P \u0026lt; 0.01. ***: P \u0026lt; 0.001.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTime-Kill Studies\u003c/h2\u003e \u003cp\u003eThe synergistic effects of MMC in combination with antibiotics were investigated against eight \u003cem\u003eAeromonas hydrophila\u003c/em\u003e isolates with a high inoculum 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/mL. Broth microdilution methods were employed for a 24-hour duration, using MMC concentrations at 1x, 1/2x, 1/4x, 1/8x, and 0x MICs in combination with CIP, LVX, and TGC at 1x, 1/2x, 1/4x, 1/8x, and 0x MICs (Table. 2). The findings revealed a synergistic effect when employing MMC at a concentration of 1x, 1/2x, and 1/4x MICs in combination with CIP at a concentration of 1x MIC at 24-hour intervals compared with that of each drug alone and no drug control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Significantly, the data exhibited better activity when employing MMC at a concentration of 1x, 1/2x, 1/4x, and 1/8x MICs in combination with 1x MIC LVX at 24-hour intervals compared with that of each drug alone and no drug control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, TGC exhibited a less effect at a concentration of 1x MIC, in combination with 1x MIC of MMC compared with that of each drug alone and no drug control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Consequently, LVX was selected as the primary agent for further investigations in combination with MMC in animal studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular Antibacterial Activity of Mitomycin C\u003c/h2\u003e \u003cp\u003eThe intracellular killing effect of MMC against \u003cem\u003eAeromonas\u003c/em\u003e Aero01-243 is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results indicate the absence of detectable \u003cem\u003eAeromonas\u003c/em\u003e Aero01-243 when subjected to 1x and 1/2x MICs of MMC at 24-hour intervals. Conversely, treatment with 1/4x MIC of MMC or the control group demonstrated no inhibition of intracellular colony counts, leading to the proliferation of \u003cem\u003eAeromonas\u003c/em\u003e Aero01-243 to over log 6 within the same timeframe. Notably, the application of 1x and 1/2x MIC of MMC exerted a substantial inhibitory effect on intracellular colony counts at 24-hour intervals compared with the 1/4x MIC of MMC or the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Study\u003c/h2\u003e \u003cp\u003eNeutropenic mice were intramuscularly administered \u003cem\u003eAeromonas\u003c/em\u003e Aero01-243 at concentrations of 1.8 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e, 1.8 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e, 1.8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, and 1.8 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL (Figure. 2). The group with inoculum of 1.8 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e CFU/mL exhibited a 100% survival rate (10/10) on day 1, 70% (7/10) on day 2, 60% (6/10) on day 3, and 0% (0/10) after day 4. The group inoculated with 1.8 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e CFU/mL exhibited a 100% survival rate (10/10) on day 1 and 0% (0/10) after day 2. The group inoculated with 1.8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e CFU/mL exhibited a 0% survival rate (0/10) on day 1. Remarkably, the group inoculated with 1.8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e CFU/mL and treated with 2 mg/kg MMC maintained a 100% survival rate (10/10) until day 7. However, the group inoculated with 1.8 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL and treated with 2 mg/kg MMC exhibited a 30% survival rate (3/10) on day 1 and 0% (0/10) after day 2. The group inoculated with 1.8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e CFU/mL exhibited a significantly higher survival rate compared to the other groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSynergy of Mitomycin C with Levofloxacin\u003c/h2\u003e \u003cp\u003eIn neutropenic mice, \u003cem\u003eAeromonas\u003c/em\u003e Aero01-243 was administered intramuscularly with inoculated of 0.99 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL (Figure. 3A) or 0.99 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL (Figure. 3B) treated with MMC, LVX, or a combination of MMC and LVX. In Fig.\u0026nbsp;3A, the control group without MMC or LVX exhibited a 0% survival rate (0/10) on day1. The MMC group exhibited a 100% survival rate (10/10) on day 1 but declined to 0% (0/10) after day 2. The LVX group exhibited a 100% survival rate (10/10) on day 2, 80% (8/10) on day 3, and 60% (6/10) after day 4. The combination group maintained a 100% survival rate (10/10) until day 7. As shown in Fig.\u0026nbsp;3B, the control and MMC groups exhibited a 0% survival rate at 0.99 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL. The LVX group displayed a 100% survival rate (10/10) on day 1, 80% (8/10) on day 2, 40% (4/10) on day 3, and 0% (0/10) after day 4. The combination group maintained a 100% survival rate (10/10) until day 7. A significant synergistic effect was observed, with a 100% survival rate when administering MMC in combination with LVX at 0.99 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL or 0.99 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL. In contrast, MMC monotherapy resulted in a 0% survival rate, and LVX monotherapy exhibited a 60% survival rate at 0.99 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL or a 0% survival rate at 0.99 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy of Lower Dosages of Mitomycin C in Combination with Levofloxacin\u003c/h2\u003e \u003cp\u003eWe also evaluated survival rates employing a reduced dosage of MMC in combination with LVX, using a higher inoculum (1.89 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL) of \u003cem\u003eAeromonas\u003c/em\u003e Aero01-243 (Figure. 4). The results reveal a 0% survival rate (0/10) for the control group on day 1. In contrast, the LVX group exhibited a 100% survival rate (10/10) on day 1, 20% (2/10) on day 2, and 0% (0/10) post day 3. The MMC group exhibited survival rates of 20% (2/10), 80% (8/10), and 100% (10/10) on day 7 at MMC doses of 0.5, 1, and 2 mg/kg, respectively, when combined with LVX.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we observed a remarkably low MIC of MMC against \u003cem\u003eAeromonas\u003c/em\u003e species, with MIC\u003csub\u003e50\u003c/sub\u003e/MIC\u003csub\u003e90\u003c/sub\u003e values of 0.12/0.5 mg/L at normal inoculum 5 x 10\u003csup\u003e5\u003c/sup\u003e CFU/ mL and 0.25/0.5 mg/L at high inoculum 5 x 10\u003csup\u003e7\u003c/sup\u003e CFU/ mL. The inoculum effect for MMC was less obvious than that of other antibiotics. This study contributes valuable insights into the complexities associated with severe \u003cem\u003eAeromonas\u003c/em\u003e species infections and underscores the potential of combination therapy involving MMC and selected antibiotics in clinical practice.\u003c/p\u003e \u003cp\u003eInfections characterized by a high bacterial burden, such as septic bloodstream infections, necrotizing fasciitis, and abscesses, are frequently encountered and pose a challenge due to the limited efficacy of the currently available therapies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To address this issue, adjustments to the in vitro testing protocol require an increase in the inoculum quantity. From our data, most antibiotics exhibited susceptibility rates exceeding 90% under the normal inoculum 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL condition. However, only CIP, LVX, TGC, and MMC maintained susceptibility rates exceeding 90% under the high inoculum 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/mL condition. The findings underscore the significant susceptibility of CIP, LVX and TGC compared to antibiotics that are effective against A\u003cem\u003eeromonas\u003c/em\u003e species, such as CTX and MIN. These results may have implications for the selection of optimal therapeutic agents against severe and high inoculum \u003cem\u003eAeromonas\u003c/em\u003e infections.\u003c/p\u003e \u003cp\u003eConsequently, we conducted investigations employing MMC in combination with three antimicrobial agents under high inoculum 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/mL conditions in a time-kill assay according to the above results. The results demonstrated a noteworthy synergistic effect when applying 1/8x MIC of MMC with 1x MIC of LVX compared with that of each drug alone and no drug control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This significant synergistic effect remained consistent over a 24-hour period, distinguishing it from combinations involving CIP and TGC.\u003c/p\u003e \u003cp\u003eThe intricate biochemical mechanisms underlying phagocytosis and intracellular survival of \u003cem\u003eAeromonas\u003c/em\u003e within host macrophages represent complex processes critical to the outcome of infection [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Intracellular bacteria, protected from high extracellular antibiotic levels, heighten the risk of drug resistance. Antibiotic efflux pumps in eukaryotic cells reduce intracellular drug levels, diminishing their effectiveness against intracellular bacteria [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. According to a previous study, LVX exhibited a 300-fold reduction in intracellular bacterial load at clinically relevant concentrations, while DOX and CIP exhibited 40-fold and 4-fold reductions, respectively [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our investigation provides compelling evidence that the application of 1x and 1/2x MICs of MMC exerted a substantial inhibitory effect on intracellular colony counts at 24-hour intervals compared with the 1/4x MIC of MMC or the control group in vitro. LVX was then chosen as the primary agent for further exploration in combination with MMC in an animal study.\u003c/p\u003e \u003cp\u003eIn individuals with compromised immune function, such as those affected by conditions such as diabetes, malignancies, liver cirrhosis, or HIV infection, \u003cem\u003eAeromonas\u003c/em\u003e infections can present a life-threatening and invasive risk. Among this cohort, bloodstream infections are highly prevalent, with associated mortality rates reaching as high as 68% [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To replicate the immunocompromised state, a well-established neutropenic mouse model was employed. In the group treated with MMC at an initial inoculum of 1.8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e CFU/mL, a notable 100% survival rate was observed; much above that of groups exposed to a higher initial inoculum of 1.8 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL, which had 0% survival rates. The effectiveness of monotherapy using MMC in treating mild infections with low inoculum levels is underscored, although its impact is less significant when considering the inoculum effect. Nevertheless, its efficacy declines notably when the inoculum reaches 1.8 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL, indicating that combining MMC with antibiotics is advisable for managing severe \u003cem\u003eAeromonas\u003c/em\u003e infections, such as septicemia or necrotizing fasciitis with abscess formation.\u003c/p\u003e \u003cp\u003eOur objective then was to investigate the combined efficacy of MMC and the antimicrobial agent LVX in enhancing synergistic effects against \u003cem\u003eAeromonas\u003c/em\u003e species in an animal model. We conducted experiments to evaluate the synergistic impact of MMC in combination with LVX utilizing a neutropenic mouse model. Our study focused on a higher bacterial inoculum (0.99 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL and 0.99 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL) of \u003cem\u003eAeromonas\u003c/em\u003e Aero01-243. The results revealed significant synergistic effects, with a 100% survival rate observed in mice treated with 2 mg/kg MMC in combination with 20 mg/kg LVX across all levels of high bacterial inoculum. This is in stark contrast to the outcomes of MMC and LVX monotherapy, where both exhibited 0% survival rates. Our findings illustrate that the in vivo synergistic efficacy of MMC combined with antibiotics yields promising results, warranting consideration for potential clinical applications in patient care. Given the considerable adverse effects linked with MMC, there is an imperative to investigate lower-dose MMC combination regimens that adequately alleviate these effects. We evaluated survival rates by adjusting MMC dosages in combination with LVX in neutropenic mice challenged with a higher inoculum 1.89 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL of \u003cem\u003eAeromonas\u003c/em\u003e Aero01-243. The results revealed survival rates of 80% and 100% at doses of 1 and 2 mg/kg, respectively. These findings establish a notable survival rate of 80% in immunocompromised individuals infected with a higher inoculum, who were treated with 1 mg/kg of MMC in combination with LVX. This approach may have clinical implications for reducing MMC-related adverse effects.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings highlight the absence of an inoculum effect in MMC and underscore its substantial inhibitory impact on intracellular colony counts. Similarly, LVX also demonstrated exceptional intracellular bactericidal activity. Notably, neutropenic mice subjected to a combined regimen of MMC, and LVX exhibited a 100% survival rate, with even a half-dose combination sustaining an impressive 80% survival rate. These outcomes provide compelling evidence for the pivotal synergy between MMC and LVX, showcasing significant efficacy against \u003cem\u003eAeromonas\u003c/em\u003e species both in vitro and in vivo. Our forthcoming investigations aim to delineate an optimally effective lower dose of MMC to mitigate potential adverse effects.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMMC Mitomycin C\u003c/p\u003e\n\u003cp\u003eMICs Minimum inhibitory concentrations\u003c/p\u003e\n\u003cp\u003eCIP \u0026nbsp; \u0026nbsp;Ciprofloxacin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLVX \u0026nbsp;Levofloxacin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTGC \u0026nbsp;Tigecycline\u003c/p\u003e\n\u003cp\u003eAMK \u0026nbsp; Amikacin\u003c/p\u003e\n\u003cp\u003eATM \u0026nbsp; Aztreonam\u003c/p\u003e\n\u003cp\u003eCZ \u0026nbsp; \u0026nbsp; Cefazolin\u003c/p\u003e\n\u003cp\u003eCMZ \u0026nbsp; Cefmetazole\u003c/p\u003e\n\u003cp\u003eCTX \u0026nbsp; \u0026nbsp;Cefotaxime\u003c/p\u003e\n\u003cp\u003eCPO Cefpirome\u003c/p\u003e\n\u003cp\u003eCHL Chloramphenicol\u003c/p\u003e\n\u003cp\u003eDOX Doxycycline\u003c/p\u003e\n\u003cp\u003eFOS \u0026nbsp; \u0026nbsp;Fosfomycin \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGM \u0026nbsp; \u0026nbsp;Gentamicin\u003c/p\u003e\n\u003cp\u003eIPM \u0026nbsp; \u0026nbsp;Imipenem\u003c/p\u003e\n\u003cp\u003eMEM Meropenem\u003c/p\u003e\n\u003cp\u003eMIN \u0026nbsp; Minocycline\u003c/p\u003e\n\u003cp\u003eCLSI Clinical and Laboratory Standards Institutes \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFDA \u0026nbsp; Food and Drug Administration\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003eIRB \u0026nbsp; \u0026nbsp;Institutional Review Board\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Laboratory of Medical Research, Chi Mei Medical Center for the technical assistance in operating time - kill studies, intracellular antibacterial activity, and animal studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChi-Chung Chen and Chih-Cheng Lai performed the experiments. Cheng-Fa Yeh, Chi-Chung Chen, and Hung-Jen Tang analyzed the experimental data. Chi-Chung Chen, Chih-Cheng Lai, and Jin-Wei Liu provided experimental resources. Cheng-Fa Yeh wrote the manuscript. Wen-Pin Su and Hung-Jen Tang revised the manuscript. Cheng-Fa Yeh and Hung-Jen Tang provided financial support. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Chi-Mei Medical Center Research Foundation-(CMFHR11069, to \u003cstrong\u003eCheng-Fa Yeh), (\u003c/strong\u003eCMFHT11202, to \u003cstrong\u003eHung-Jen Tang\u003c/strong\u003e), (CMFHR11311, to \u003cstrong\u003eCheng-Fa Yeh\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. Raw data is available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study and waiver from the informed consent process were approved by the Institutional Review Board (IRB) of the Chi Mei Medical Center, Tainan City, Taiwan (IRB Serial number LAC. SOP102-104).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript is approved by all authors for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Latif HM, Khafaga AF. Natural co-infection of cultured Nile tilapia \u003cem\u003eOreochromis niloticus\u003c/em\u003e with \u003cem\u003eAeromonas hydrophila\u003c/em\u003e and \u003cem\u003eGyrodactylus cichlidarum\u003c/em\u003e experiencing high mortality during summer. Aquac Res 2020;51:1880-1892.\u003c/li\u003e\n\u003cli\u003eStratev D, Odeyemi OA. An overview of motile \u003cem\u003eAeromonas\u003c/em\u003e septicaemia management. Aquac Int 2017;25:1095-1105.\u003c/li\u003e\n\u003cli\u003eLee, CY, Kuo LT, Peng KT, et al. Prognostic factors and monomicrobial necrotizing fasciitis: gram-positive versus gram-negative pathogens. BMC Infect Dis 2011;11:5-12.\u003c/li\u003e\n\u003cli\u003eHuang KF, Huang MH, Lin YS, et al. Independent predictors of mortality for necrotizing fasciitis: A retrospective analysis in a single institution. J Trauma 2011;71:467-73.\u003c/li\u003e\n\u003cli\u003eTsai YH, Huang KC, Shen SH, et al. Microbiology and surgical indicators of necrotizing fasciitis in a tertiary hospital of southwest Taiwan. Int J Infect Dis 2012;16:159\u0026ndash;65.\u003c/li\u003e\n\u003cli\u003eTang HJ, Lai CC, Lin HL, et al. Clinical manifestations of bacteremia caused by \u003cem\u003eAeromonas\u003c/em\u003e species in southern Taiwan. PLoS One 2014;9(3):e91642.\u003c/li\u003e\n\u003cli\u003eJanda, JM, Abbott, SL. The genus \u003cem\u003eAeromonas\u003c/em\u003e: taxonomy, pathogenicity, and infection. Clin. Microbiol Rev 2010;23:35-73.\u003c/li\u003e\n\u003cli\u003eErova TE, Kosykh VG, Fadl AA, et al. Cold shock exoribonuclease R (VacB) is involved in \u003cem\u003eAeromonas \u003c/em\u003e\u003cem\u003ehydrophilia\u003c/em\u003e pathogenesis. J Bacteriol 2008;190:3467-74.\u003c/li\u003e\n\u003cli\u003eStevens DL, Bisno AL, Chambers HF, et al. Practice guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the Infectious Diseases Society of America. Clin Infect Dis\u003cem\u003e \u003c/em\u003e2014;15;59(2):147-59.\u003c/li\u003e\n\u003cli\u003eVerweij J, Pinedo HM. Mitomycin C: mechanism of action, usefulness and limitations. Anti-Cancer Drugs 1990;1(1): 5-13.\u003c/li\u003e\n\u003cli\u003eDomalaon R, Ammeter D, Brizuela M, et al. Repurposed Antimicrobial Combination Therapy: Tobramycin-Ciprofloxacin Hybrid Augments Activity of the Anticancer Drug Mitomycin C Against Multidrug-Resistant Gram-Negative Bacteria. Front Microbiol\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e2019;10:1556.\u003c/li\u003e\n\u003cli\u003eKitagawa D, Suzuki Y, Abe N., et al. Comparison of MALDI-TOF mass spectrometry and rpoB gene sequencing for the identification of clinical isolates of \u003cem\u003eAeromonas\u003c/em\u003e spp. Heliyon 2022;8(11):e11585.\u003c/li\u003e\n\u003cli\u003eChen PL, Wu CJ, Chen CS, et al. A comparative study of clinical \u003cem\u003eAeromonas\u003c/em\u003e dhakensis and \u003cem\u003eAeromonas\u003c/em\u003e hydrophila isolates in southern Taiwan: A. dhakensis is more predominant and virulent. Clin Microbiol Infect 2014;20(7): 428-34.\u003c/li\u003e\n\u003cli\u003eClinical and Laboratory Standards Institute. Approved Standard M100-S33 Twenty-Second Informational Supplement. Wayne, PA: Clinical and Laboratory Standards Institute, 2023.\u003c/li\u003e\n\u003cli\u003eTang HJ, Lai CC, Chen CC, et al. Cephalosporin-Glycopeptide Combinations for Use against Clinical Methicillin-Resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e Isolates: Enhanced \u003cem\u003eIn vitro\u003c/em\u003e Antibacterial Activity. Front Microbiol 2017;8:884.\u003c/li\u003e\n\u003cli\u003e Tang HJ, Ko WC, Chen CC, et al. In Vitro and In Vivo Intracellular Killing Effects of Tigecycline against Clinical Nontyphoid Salmonella Isolates Using Ceftriaxone as a Comparator. Antimicrob Agents Chemother 2011;55(6): 2755\u0026ndash;2759.\u003c/li\u003e\n\u003cli\u003ePonnusamy D, Kozlova EV, Sha J, et al. Cross-talk among flesh-eating \u003cem\u003eAeromonas hydrophilia\u003c/em\u003e strains in mixed infection leading to necrotizing fasciitis. Proc Natl Acad Sci U S A 2016;113(3):722\u0026ndash;727.\u003c/li\u003e\n\u003cli\u003eCrandon JL, Kuti JL, Nicolau DP. Comparative efficacies of human simulated exposures of telavancin and vancomycin against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e with a range of vancomycin MICs in a murine pneumonia model. Antimicrob Agents Chemother 2010;54(12):5115\u0026ndash;5119.\u003c/li\u003e\n\u003cli\u003eNair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 2016;7:27-31.\u003c/li\u003e\n\u003cli\u003ePletzer D, Hancock RE. Is synergy the key to treating high-density infections? Future Microbiol 2018;13:1629\u0026ndash;1632.\u003c/li\u003e\n\u003cli\u003eAderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 1999;17: 593-623.\u003c/li\u003e\n\u003cli\u003eThwaites G, Gant EV. Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus? Nat Rev Microbiol 2011;9:215\u0026ndash;22.\u003c/li\u003e\n\u003cli\u003eSeral C, Carryn S, Tulkens PM, Van Bambeke F. Influence of P-glycoprotein and MRP efflux pump inhibitors on the intracellular activity of azithromycin and ciprofloxacin in macrophages infected by Listeria monocytogenes or Staphylococcus aureus. J Antimicrob Chemother 2003;51:1167\u0026ndash;73.\u003c/li\u003e\n\u003cli\u003ePeyrusson F, Whelan AO, Hartley MG, et al. Intracellular Activity of Antibiotics against \u003cem\u003eCoxiella \u003c/em\u003e\u003cem\u003eburnetii\u003c/em\u003e in a Model of Activated Human THP-1 Cells. Antimicrob Agents Chemother 2021;65(12):e01061-21.\u003c/li\u003e\n\u003cli\u003eNolla-Salas J, Codina-Calero J, Valles-Angulo S, et al. Clinical significance and outcome of Aeromonas spp. infections among 204 adult patients. Eur J Clin Microbiol Infect Dis 2017;36(8):1393-403.\u003c/li\u003e\n\u003cli\u003eKo WC, Chuang YC. \u003cem\u003eAeromonas\u003c/em\u003e bacteremia: review of 59 episodes. Clin Infect Dis 1995;20(5): 1298-304.\u003c/li\u003e\n\u003cli\u003eLau SM, Peng MY, Chang FY. Outcomes of \u003cem\u003eAeromonas\u003c/em\u003e bacteremia in patients with different types of underlying disease. J Microbiol Immunol Infect 2000;33(4):241-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aeromonas infections, antimicrobial combination therapy, mitomycin C, synergistic effect","lastPublishedDoi":"10.21203/rs.3.rs-4317037/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4317037/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAeromonas\u003c/em\u003e infections pose a significant threat, presenting a potentially life-threatening condition associated with high mortality rates. This study investigates the potential of mitomycin C (MMC), an anticancer drug, as a novel antimicrobial agent against \u003cem\u003eAeromonas\u003c/em\u003e infections.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe minimum inhibitory concentrations (MICs) of MMC and antibiotics against clinical isolates of \u003cem\u003eAeromonas\u003c/em\u003e species were assessed, using broth microdilution. Synergistic effects of MMC with antibiotics were assessed through time-kill studies. Additionally, we analyzed MMC's intracellular killing effects using a representative \u003cem\u003eAeromonas\u003c/em\u003e isolate. Efficacy of combined therapies was evaluated in neutropenic mice model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that only three antibiotics-ciprofloxacin (CIP), levofloxacin (LVX), and tigecycline (TGC) maintained susceptibility rates exceeding 90% under high inoculum conditions. Significantly, in vitro investigations demonstrated a synergistic effect over a 24-hour period when combining the 1/8x MIC of MMC with the 1x or 1/2x MICs of LVX, in a time\u0026ndash;kill study. Furthermore, in vivo in neutropenic mice, the combination of 2 mg/kg and 1 mg/kg MMC with LVX resulted in 100% and 80% survival, respectively, whereas treatment with either drug alone resulted in 0% survival rate.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that MMC can be repurposed as an antimicrobial agent, particularly with a minimized dose and in combination with antibiotics.\u003c/p\u003e","manuscriptTitle":"Synergistic effect of repurposed mitomycin C in combination with antibiotics against Aeromonas infection: in vitro and in vivo studies.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-03 11:15:43","doi":"10.21203/rs.3.rs-4317037/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5f6184ca-2667-4f1c-98ac-9562e8296daa","owner":[],"postedDate":"May 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-28T09:44:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-03 11:15:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4317037","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4317037","identity":"rs-4317037","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}