# Activity of LpxC inhibitors on carbapenemase producing Citrobacter spp. clinical isolates

Víctor Vinuesa, Raquel Cruces, Javier E Cañada-García, Jesús Oteo-Iglesias, Andrés Corral-Lugo, Michael J Mcconnell

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Víctor Vinuesa, Raquel Cruces, Javier E Cañada-García, Jesús Oteo-Iglesias, Andrés Corral-Lugo, et al. Activity of LpxC inhibitors on carbapenemase producing Citrobacter spp. clinical isolates. Journal of Global Antimicrobial Resistance, 2025, 44, pp.366-370. 10.1016/j.jgar.2025.07.020. hal-05235706

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Short Communication

# Activity of LpxC inhibitors on carbapenemase-producing Citrobacter
spp. clinical isolates

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updates

Víctor Vinuesaª, Raquel Crucesª, Javier E. Cañada-Garcíaᵇ,ᶜ, Jesús Oteo-Iglesiasᵇ,ᶜ,
Andrés Corral-Lugoᵈ,*, Michael J. McConnellᵉ,f

## ARTICLE INFO

**Article history:**

Received 28 September 2024

Revised 30 June 2025

Accepted 18 July 2025

Available online 23 July 2025

Editor: Professor A Tsakris

**Keywords:**

Citrobacter spp.

Antibiotic resistance

Carbapenemase

LpxC inhibitors

Clinical isolates

Strain collection

## ABSTRACT

**Objective:** Citrobacter spp. are commonly found in the human intestine and can cause antibiotic-resistant infections. Inhibitors of LpxC, an enzyme involved in the synthesis of lipid A, have demonstrated potent activity against multiple Gram-negative species, but their activity in Citrobacter spp. has not been well-characterised. The objective of the present study was to evaluate the antimicrobial activity of LpxC inhibitors against carbapenemase-producing Citrobacter spp. clinical isolates.

**Methods:** The MICs of LpxC-2, LpxC-4, and CHIR-090 LpxC inhibitors were determined for 61 clinical isolates with high genetic diversity. Time-kill assays with the three LpxC inhibitors were performed with two Citrobacter spp. strains, with low (Cit 52) and high (Cit 110) LpxC inhibitor MIC values.

**Results:** LpxC-4 was shown to be the most potent inhibitor (MIC₉₀ = 0.5 mg/L). In general, the three LpxC inhibitors demonstrated similar activity between Citrobacter spp. isolates harbouring type A (n = 11 isolates), B (n = 39 isolates), and D (n = 11 isolates) carbapenemases. All LpxC inhibitors showed bactericidal activity at concentrations 4× the MIC and no activity at ¼× the MIC at 24 h for the two Citrobacter spp. strains analysed.

**Conclusions:** Although LpxC-4 had a lower MIC₉₀ value compared with CHIR-090, CHIR-090 showed a bactericidal effect at short times and prevented the regrowth of both isolates in the time-kill assays. In terms of cross-resistance, all three structurally similar LpxC inhibitors showed a moderate positive correlation between their activity.

© 2025 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

## 1. Introduction

Citrobacter spp. have been identified as the causative agents for multiple infection types, most notably urinary, respiratory tract infections, and sepsis, primarily in the health care setting [1]. Similar to trends observed in other Enterobacterales species over preceding decades, there has been an increase in resistance to multiple antibiotic classes in Citrobacter spp. clinical iso-

lates [1,2]. Carbapenems have been a mainstay of treatment for infections caused by Enterobacterales species, including Citrobacter spp. Unfortunately, the emergence and dissemination of carbapenem-resistant Enterobacteriaceae have complicated the use of carbapenems for the clinical management of infections caused by these species. Recently, multiple reports have described carbapenem resistance in Citrobacter spp. clinical isolates due to the presence of carbapenemases such as VIM-1, OXA-48, KPC-2, NDM-1, and VIM-2 [2–6]. In this context, the evaluation of antimicrobials with novel mechanisms of action against carbapenemase-producing Citrobacter spp., such as LpxC inhibitors, is of interest.

Inhibitors of LpxC, a deacetylase catalysing the first irreversible step in the biosynthesis of lipid A, have been developed for infections caused by Gram-negative bacteria [7,8]. As lipid A is

<footnote>ªIntrahospital Infections Laboratory, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain</footnote>

<footnote>ᵇReference & Research Laboratory in Resistance to Antibiotics & Infections Related to Healthcare, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain</footnote>

<footnote>ᶜCIBER de Enfermedades Infecciosas (CIBERINFEC), Madrid, Spain</footnote>

<footnote>ᵈUniversity of Rennes, CNRS, Institut de Génétique et Développement de Rennes (IGDR) UMR 6290, Rennes, France</footnote>

<footnote>ᵉDepartment of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA</footnote>

<footnote>fEck Institute for Global Health, University of Notre Dame, Notre Dame, IN, USA</footnote>

<footnote>* Corresponding author. Mailing address: Institut de Génétique & Développement de Rennes, Equipe Contrôle Qualité de la Synthèse Protéique, UMR 6290 CNRS-UR, ERL Inserm U1305 Campus de Beaulieu, Bât. 13, 263 Avenue du Général Leclerc, CS 74205/35042, Rennes, Cedex, France.</footnote>

<footnote>E-mail address: andres.corallugo@gmail.com (A. Corral-Lugo).</footnote>
essential for the viability of most Gram-negative species, inhibi-
tion of LpxC results in rapid bacterial cell death. In this context,
the goal of the present study was to assess the activity of three
different LpxC inhibitors in a collection of multidrug-resistant
carbapenemase-producing Citrobacter spp. clinical isolates.

## 2. Materials and methods

### 2.1. Strains and inhibitors

A total of 61 carbapenem-producing Citrobacter spp. clinical isolates collected from Spanish hospitals between 2012 and 2015 in a previous study were used (Supplementary Table 1) [3]. The strains included 57 Citrobacter freundii and 4 Citrobacter koseri isolates. Pulsed-field gel electrophoresis of the 57 C. freundii isolates revealed 44 distinct pulsotypes. The results of antibiotic susceptibility testing of the strains using 12 clinically relevant antibiotics have been reported previously. The inhibitors LpxC-2 (PF-4,753,299, Sigma-Aldrich), LpxC-4 (PF-5,081,090, Axon MedChem), and CHIR-090 (Sigma-Aldrich) were used in this study.

### 2.2. MIC determinations

MIC values were determined for each LpxC inhibitor and each
Citrobacter spp. strain according to guidelines by the Clinical
and Laboratory-Standards Institute for antimicrobials [9]. The Es-
cherichia coli strain ATCC 25,922 was used as a control in all as-
says. No susceptibility breakpoints for LpxC inhibitors have been
determined.

### 2.3. Time-kill curves

LpxC inhibitors were evaluated at a concentration of 4×, 1×,
and ¼× the MIC value in an adjusted bacterial culture (5 × 10⁵
CFU/mL). Cultures were incubated at 37°C with agitation, and sam-
ples were taken at 0, 3, 6, and 24 h for bacterial quantification
on LB agar. Each strain/inhibitor concentration was tested at least
twice. Bactericidal activity was defined as at least a 3 log10 reduc-
tion in bacterial counts at 24 h compared with cultures with no
inhibitor.

### 2.4. Cross-resistance study

To investigate the relationship between the activity of the three LpxC inhibitors in the 61 *Citrobacter* spp. described above, scatterplots were constructed. The MIC of each strain/inhibitor combination was used, and their relationship was compared in LpxC-2 versus LpxC-4, LpxC-2 versus CHIR-090, and LpxC-4 versus

CHIR-090 using two quantitative variables for each strain. Negative and positive correlation was determined with the direction of the slope, while the slope of the curve I was used to determine the strength of the relationship, taking into account that r < 0.3 means none or very weak, 0.3 < r < 0.5 means weak, 0.5 < r < 0.7 means moderate, and r > 0.7 means a strong relationship. Validation of all correlation tests was assessed using the T-distribution test (two-tailed). P ≤ 0.05 was considered significant.

## 3. Results

### 3.1. MICs of LpxC inhibitors in carbapenemase-producing Citrobacter spp

MIC values were determined for all carbapenemase-producing
Citrobacter spp. isolates (Supplementary Table 1) by broth mi-
crodilution, and MIC₅₀ and MIC₉₀ values were determined for
the strain collection (n = 61 isolates). The three LpxC inhibitor
MIC₅₀ values ranged from 0.25 to 2 mg/L, and MIC₉₀ values
ranged from 0.5 to 8 mg/L, with LpxC-4 demonstrating the
most potent activity against isolates (Table 1). A total of 16
strains demonstrated very high susceptibility to LpxC-4, with
MIC values ≤0.125 mg/L. The highest MIC value observed was
16 mg/L in two isolates with inhibitor LpxC-2 (Supplementary
Table 1).

Associations between different carbapenemase types present in
*Citrobacter* spp. isolates and susceptibility to the three LpxC in-
hibitors was assessed (Table 1). In general, the three LpxC in-
hibitors demonstrated similar activity against *Citrobacter* spp. iso-
lates harbouring type A (n = 11 isolates), B (n = 39 isolates), and
D (n = 11 isolates) carbapenemases. However, it is noteworthy that
the MIC₅₀ and MIC₉₀ values for LpxC-2 were fourfold higher in iso-
lates harbouring type A and B carbapenemases compared with iso-
lates harbouring type D carbapenemases.

### 3.2. Time-kill analysis of LpxC inhibitors in carbapenemase-producing Citrobacter spp. isolates

In order to characterise the cell-killing dynamics of the three LpxC inhibitors within the carbapenemase-producing *Citrobacter* spp. isolates, time-kill curve analyses were performed on two selected isolates. Isolates Cit 52 (Fig. 1A, C, and E) and Cit 110 (Fig. 1B, D, and F) were selected based on their different susceptibilities to the three LpxC inhibitors. All LpxC inhibitors showed bactericidal activity (3 log decrease compared with the initial inoculum) and a 4× MIC at 24 h for the two *Citrobacter* spp. CHIR-090 also had a bactericidal effect on both strains at the MIC concentration for 24 h. In the case of LpxC-2 and LpxC-4 at

<table><caption>**Table 1**
Activity of LpxC inhibitors against carbapenemase-producing Citrobacter spp. isolates.</caption><thead><tr><th rowspan="2">Strains (n)</th><th rowspan="2">Compound</th><th colspan="3">MIC (mg/L)</th></tr><tr><th>50%</th><th>90%</th><th>Range</th></tr></thead><tbody><tr><td rowspan="3">All Citrobacter spp. isolates (61)</td><td>LpxC-2</td><td>2</td><td>8</td><td>0.25-16</td></tr><tr><td>LpxC-4</td><td>0.25</td><td>0.5</td><td>0.0625-1</td></tr><tr><td>CHIR-090</td><td>1</td><td>2</td><td>0.25-4</td></tr><tr><td rowspan="3">Carbapenemase type A Citrobacter spp. isolates (11)</td><td>LpxC-2</td><td>2</td><td>8</td><td>0.5-16</td></tr><tr><td>LpxC-4</td><td>0.25</td><td>1</td><td>0.125-1</td></tr><tr><td>CHIR-090</td><td>0.5</td><td>2</td><td>0.25-2</td></tr><tr><td rowspan="3">Carbapenemase type B Citrobacter spp. isolates (39)</td><td>LpxC-2</td><td>2</td><td>8</td><td>0.5-8</td></tr><tr><td>LpxC-4</td><td>0.25</td><td>0.5</td><td>0.125-1</td></tr><tr><td>CHIR-090</td><td>1</td><td>2</td><td>0.25-2</td></tr><tr><td rowspan="3">Carbapenemase type D Citrobacter spp. isolates (11)</td><td>LpxC-2</td><td>0.5</td><td>2</td><td>0.25-16</td></tr><tr><td>LpxC-4</td><td>0.125</td><td>0.5</td><td>0.0625-1</td></tr><tr><td>CHIR-090</td><td>0.5</td><td>2</td><td>0.25-4</td></tr></tbody></table>
A

Cit 52 LpxC-2

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

B

Cit 110 LpxC-2

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

C

Cit 52 LpxC-4

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

D

Cit 110 LpxC-4

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

E

Cit 52 CHIR-090

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

F

Cit 110 CHIR-090

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

**Fig. 1.** Time-kill curves for LpxC-2, LpxC-4, and CHIR-090 against two different carbapenemase-producing *Citrobacter* spp. clinical isolates. Bacterial load (log10 CFU/ml) of *Citrobacter* spp. strain 52 (carbapenemase type B) over 24 h in the absence and presence of ¼× MIC, 1× MIC, and 4× MIC of LpxC-2 (A), LpxC-4 (C), and CHIR-090 (E). Bacterial load (log10 CFU/ml) of *Citrobacter* spp. strain 110 (carbapenemase type D) in the absence and presence of LpxC-2, LpxC-4, and CHIR-090 are shown in (B, D, and F), respectively. The limit of detection for the time-kill assays was 10 CFU/ml.

MIC concentration, the two compounds caused decreases in the bacterial count after 6 h of incubation, although an overgrowth occurred after 24 h of incubation. LpxC-2 at ¼× MIC produced lower growth in both strains compared with the culture without inhibitor before and after 6 h of incubation. The rest of the inhibitors did not show activity against the two of *Citrobacter* spp. strains at the ¼× MIC concentration.

### 3.3. *Cross-resistance to LpxC inhibitors in carbapenemase-producing Citrobacter* spp. isolates

The MIC values in Supplementary Table 1 were plotted (Fig. 2) to study the antimicrobial activity relationship that the three struc-

turally related LpxC inhibitors (Fig. 2A) had on *Citrobacter* spp. The MICs for LpxC-2 versus LpxC-4 (Fig. 2B), LpxC-2 versus CHIR-090 (Fig. 2C), and LpxC-4 versus CHIR-090 (Fig. 2D), as well as the coefficients for the models after linear regression, were measured. A moderate positive correlation (0.5 < r < 0.7) was observed in the relationship between LpxC-4 versus LpxC-2 and CHIR-090, whereas a weak positive correlation (r < 0.5) between LpxC-2 versus CHIR-090 was observed.

### 4. Discussion

Infections caused by carbapenem-resistant Enterobacteriaceae present formidable challenges for the medical community as treat-
A

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

B

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

C

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

D

<figure><img src="image_12.png" /><figcaption>**Fig. 2.** Carbapenemase-producing *Citrobacter* spp. presents cross-resistance to structurally related LpxC inhibitors. Molecular structure of the LpxC inhibitors (A) and scatter-plots of LpxC-2 versus LpxC-4 (B), LpxC-2 versus CHIR-090 (C), and LpxC-4 versus CHIR-090 (D) inhibitors show LpxC inhibitor cross-resistance among 61 *Citrobacter* spp. clinical isolate strains in the study. The MICs (mg/L) of LpxC-2 (B and C) and LpxC-4 (D) are shown in the abscissa axis, while the MICs (mg/L) of LpxC-4 (B) and CHIR-090 (C and D) are shown in the ordinate axis.</figcaption></figure>

ment options are becoming increasingly limited. The lipopolysaccharide pathway has emerged as an attractive target for the development of new antibiotics because it is an important structure required for the viability of most Gram-negative species. Also, lipopolysaccharide biosynthesis of LpxC is a highly conserved enzyme that does not show sequence homology to any mammalian protein. LpxC inhibitors were first identified by Merck researchers in the 1990s and are structurally similar, containing moieties that mimic the natural LpxC substrate [8,9], and subsequent studies have led to more potent and broad-spectrum inhibitors, notably CHIR-090. Crystallographic and nuclear magnetic resonance analyses have elucidated the conformation and inhibitor-binding mechanisms of LpxC. These inhibitors share key features, including a zinc-binding group (e.g., hydroxamic acid) and a hydrophobic tail that mimics the enzyme's natural substrate [7]. Since the introduction of the LpxC inhibitor L-573,655, various small-molecule LpxC inhibitors targeting Gram-negative bacteria have been developed, such as TU-514, CHIR-090, ACHN-975, and TP0586532. However, only ACHN-975 progressed to phase I clinical trials before being discontinued due to safety concerns. None of these inhibitors are currently available on the market [10].

Initial studies on CHIR-90 reported that this compound has excellent antibiotic activity against *P. aeruginosa* and *E. coli*, as judged by disc diffusion assay [8]. Molecular studies indicate that CHIR-090 is a two-step slow, tight-binding inhibitor of *E. coli* LpxC with $K_i = 4.0$ nM, $K_i^* = 0.5$ nM, $k_5 = 1.9 \text{ min}^{-1}$, and $k_6 = 0.18 \text{ min}^{-1}$ [11]. Studies in clinical multidrug-resistant bacteria indicate that the $MIC_{9n}$ of CHIR-090 varies among several multidrug-resistant pathogens, including *P. aeruginosa* (4 mg/L), *K. pneumoniae* (2 mg/L), *E. coli* (0.25 mg/L), *Enterobacter aerogenes* (0.5 mg/L), *C. freundii* (0.5 mg/L), and *A. baumannii* (>64 mg/L). Notably, resistance has been observed in *A. baumannii* [12].

In this article, we report that three different LpxC inhibitors demonstrate robust activity against a collection of clinical *Citrobacter* spp.-harbouring carbapenemases [12–14]. Specifically, LpxC-2 (MIC = 0.25–16 mg/L), LpxC-4 (MIC = 0.0625–1 mg/L), and CHIR-090 (MIC = 0.25–4 mg/L) are potent inhibitors of carbapenemase-producing *Citrobacter* spp. clinical isolates that derived from a nationally representative collection taken from 27 hospitals in 14 Spanish provinces. Likewise, it should be noted that this collection has high genetic diversity, including up to 44 different pulsed-field gel electrophoresis patterns [7,13,14]. We found that these LpxC inhibitors are bactericidal in vitro at 1× and 4× MIC. LpxC-2, LpxC-4, and CHIR-090 inhibitors have been extensively studied in *P. aeruginosa* [14,15], with LpxC-4 demonstrating a clear $MIC_{90}$ (1 mg/L) advantage over the biphenyl analogue LpxC-2 when tested against the wild-type strain and a panel of 106 recent clinical isolates. This advantage was also observed when the inhibitors were tested in 98 *Klebsiella pneumoniae* clinical isolates [15]. CHIR-90 and LpxC-4 exhibited activity against *E. coli* and *Enterobacter* spp., *Burkholderia cepacia*, and *Stenotrophomonas maltophilia* but not against *Acinetobacter baumannii*. In the same study, several clinical isolates and ATCC strains of *E. coli* were analysed against CHIR-90 and its derivatives. The results showed that LpxC inhibitors ($MIC_{9n} = 0.25–4$ g/L) significantly outperformed the comparator agents ciprofloxacin and aztreonam ($MIC_{9n} > 64$ g/L). In our study, LpxC-4 ($MIC_{90} = 0.5$ mg/L) also showed better activity, both when compared with LpxC-2 ($MIC_{90} = 8$ mg/L) and with CHIR-090 ($MIC_{90} = 2$ mg/L). Likewise, when LpxC-4 was used in the time-kill experiments it was shown to be bactericidal against *P. aeruginosa* and *K. pneumoniae* at low concentrations; however, to avoid regrowth, high concentrations of the inhibitor (4× MIC) were necessary [16]. We observed a similar effect in time-kill experiments using LpxC-4 against two carbapenemase-producing Cit-
robacter spp. isolates, where ¼× MIC did not show a bactericidal effect, 1× MIC showed a bactericidal effect at early time points, and 4× MIC was needed to have an effect at 24 h. A comparable effect was also observed when LpxC-2 was used, regardless of the resistant clinical isolate analysed. Our analysis (Fig. 2) may also suggest a correlation between higher MIC values for different LpxC inhibitors within a strain. While this analysis may point to potential cross-resistance to different inhibitors, additional studies with resistant mutants that assess resistance to multiple inhibitors are necessary in order to definitively assess cross-resistance.

To the best of our knowledge, only one study has reported the use of LpxC inhibitors against C. freundii, and none against carbapenemase-producing Citrobacter spp. For C. freundii (n = 12) the antimicrobial activity of the LpxC inhibitor LPC-069 was comparable to the activity of the inhibitors used in this study against carbapenemase-producing Citrobacter spp. (MIC90 = 0.4 mg/L) [17,18]. However, it should be noted that one limitation of this study was the lack of sequence-level typing data—for example, multilocus sequence typing—on the strain used. Future studies aimed at determining activity against additional Citrobacter spp. strains with different MIC values for LpxC inhibitors and exploring combination therapies with LpxC inhibitors and currently used antimicrobials would be of interest.

Declaration of competing interest

M.J.M. is a founder and shareholder, and has received research support from the biotechnology company Vaxdyn, S.L. Vaxdyn played no role in the present study. No other competing interests are declared.

Acknowledgements

The authors thank CIBER de Enfermedades Infecciosas (CIBER-INFEC) for providing us the Citrobacter collection strains.

Funding

V.V. is supported by the Río Hortega Program from the Instituto de Salud Carlos III. A.C.L. is supported by the Atracción de Talento Program of the Community of Madrid. This project was funded by MPY380/18, awarded to M.J.M. from Instituto de Salud Carlos III.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jgar.2025.07.020.

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