# Analysis of two Nocardia brasiliensis class A β-lactamases (BRA-1 and BRS-1) and related resistance to β-lactam antibiotics

Aimee Songo, Hervé Jacquier, Maxime Danjean, Fabrice Compain, Delphine Dorchène, Zainab Edoo, Paul-Louis Woerther, Michel Arthur, David Lebeaux

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## To cite this version:

Aimee Songo, Hervé Jacquier, Maxime Danjean, Fabrice Compain, Delphine Dorchène, et al. Analysis of two Nocardia brasiliensis class A β-lactamases (BRA-1 and BRS-1) and related resistance to β-lactam antibiotics. Journal of Global Antimicrobial Resistance, 2025, 42, pp.135-141. 10.1016/j.jgar.2025.01.022. hal-05110019

HAL Id: hal-05110019

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# Analysis of two *Nocardia brasiliensis* class A *β*-lactamases (BRA-1 and BRS-1) and related resistance to *β*-lactam antibiotics

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Aimee Songo<sup>a,†</sup>, Hervé Jacquier<sup>b,c,†</sup>, Maxime Danjean<sup>b,c</sup>, Fabrice Compain<sup>a</sup>, Delphine Dorchène<sup>a</sup>, Zainab Edoo<sup>a</sup>, Paul-Louis Woerther<sup>b,c</sup>, Michel Arthur<sup>a</sup>, David Lebeaux<sup>a,d,e,*</sup>

## ARTICLE INFO

**Article history:**
Received 1 September 2024
Revised 21 January 2025
Accepted 31 January 2025
Available online 11 February 2025

Editor: Prof Wen-Chien Ko

**Keywords:**

*Nocardia brasiliensis*
*β*-lactam resistance
Class A
*β*-lactamase
BRA-1
BRS-1

## ABSTRACT

**Objective:** Molecular determinants of *β*-lactam resistance are poorly explored for most *Nocardia* species, such as *Nocardia brasiliensis*. In this study, we characterised resistance mediated by two *β*-lactamases in the reference strain *N. brasiliensis* HUJEG-1 and extended our analysis to nine *N. brasiliensis* clinical strains.

**Methods:** The susceptibility of *N. brasiliensis* HUJEG-1 was determined by measuring the MIC via microdilution for five *β*-lactam antibiotics that were or were not associated with *β*-lactamase inhibitors (clavulanate and avibactam, 4 µg/mL). Two putative class A *β*-lactamase-encoding genes (*bla*<sub>BRA-1</sub> and *bld*<sub>BRS-1</sub>) were identified in the HUJEG-1 genome. The kinetic parameters of purified BRA-1 and BRS-1 were determined by spectrophotometry. Measurement of *β*-lactam resistance was then extended to nine clinical strains. These phenotypic data were compared with the genomic diversity of whole genomes (next-generation sequencing).

**Results:** *N. brasiliensis* HUJEG-1 was resistant to amoxicillin, cefuroxime, and cefotaxime, but susceptible to their combination with clavulanate or avibactam. This strain was resistant to imipenem (with or without inhibitors) and susceptible to meropenem. BRA-1 showed high catalytic efficiencies against penams (penicillin, ampicillin) and cephems (cephaloridine, cephalothin, and cefamandole), but not against penems (imipenem, meropenem), suggesting that imipenem resistance was mediated by another mechanism. The hydrolytic activity of BRS-1 was 100–1000-fold lower than that of BRA-1 for all *β*-lactams tested, suggesting that BRS-1 has a minor contribution to *β*-lactam resistance. Analysis of the nine clinical strains showed variations in susceptibility to cefotaxime, as well as diversity in genetic backgrounds and BRA-1 sequences.

**Conclusions:** *N. brasiliensis* HUJEG-1 resistance to penams and cephems is mainly due to the class A *β*-lactamase BRA-1.

© 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

*Nocardia* species are Gram-positive bacilli found in various natural environments such as decaying vegetation, soil, and water [1]. Nocardiosis occurs after direct inoculation of *Nocardia* spp.

through the skin or inhalation, subsequently causing various diseases ranging from skin and soft tissue infections (following cutaneous trauma) to invasive diseases with frequent lung and central nervous system involvement [1]. Following repeated cutaneous trauma, a chronic infection called actinomycetoma is most frequently caused by *Nocardia brasiliensis* and has been associated with a high medico-economic burden in Mexico [2,3].

The initial and probabilistic treatment of nocardiosis mostly relies on four antibiotic classes; namely, *β*-lactam antibiotics, amino-glycosides, sulphonamides, and oxazolidinones [1,4]. However, the

<footnote><sup>a</sup> Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, U1138, LRMA, Equipe 12, Paris, France</footnote>

<footnote><sup>b</sup> EA DYNAMYC 7380, Faculté de Santé, Université Paris-Est Créteil, Créteil, France</footnote>

<footnote><sup>c</sup> Unité de Bactériologie, Département de Prévention, Diagnostic et Traitement des Infections, APHP, GHU Henri Mondor, Créteil, France</footnote>

<footnote><sup>d</sup> Université Paris Cité, Paris, France</footnote>

<footnote><sup>e</sup> Service de Microbiologie, Unité Mobile d'Infectiologie, APHP, Hôpital Européen Georges Pompidou, Paris, France</footnote>

<footnote>* Corresponding author. Mailing address: LRMA, Équipe 12, Centre de Recherche des Cordeliers, 15 rue de l'École de Médecine, 75006 Paris, France.</footnote>

<footnote>E-mail addresses: david.lebeaux@aphp.fr, david.lebeaux@yahoo.fr (D. Lebeaux).</footnote>

<footnote>† These authors contributed equally to this study.</footnote>
toxicity of either or both aminoglycosides and sulphonamides (such as cotrimoxazole, the combination of sulfamethoxazole and trimethoprim), leads to a preference for treatments based on β-lactam antibiotics, third-generation cephalosporins, or carbapenems, as these drugs have demonstrated in vivo bactericidal activity [1,4-6]. However, 9.5-51% of *Nocardia* isolates exhibit resistance to imipenem (depending upon the species tested and on the microbiological method used), notably *Nocardia farcinica*, *Nocardia abscessus* complex, *Nocardia transvalensis* complex, and, more strikingly, *N. brasiliensis* (85-100% of resistance) [7-9]. Resistance to third-generation cephalosporins is also frequent in *N. farcinica* and *N. brasiliensis* (55-100% and 23-90%, respectively) [7-10].

This far, the resistance of *N. brasiliensis* to β-lactam antibiotics has been poorly studied. A preliminary study performed on *N. brasiliensis* revealed that β-lactamase activity in crude bacterial extracts hydrolysed benzylpenicillin, ampicillin, cephalothin, and cefamandole, whereas the hydrolysis of carbapenems or third-generation cephalosporins was not studied [11]. Few years later, these authors suggested in vivo selection to the amoxicillin-clavulanate combination and studied the inhibitory effect of imipenem [12]. Here, we studied β-lactamase-mediated resistance to β-lactam antibiotics of the reference strain *N. brasiliensis* HUJEG-1 and extended our findings to a collection of nine clinical strains of *N. brasiliensis*.

## 2. Materials and methods

### 2.1. Bacterial strains

*N. brasiliensis* HUJEG-1 is a clinical strain whose genome has been fully sequenced (accession no. CP003876) [3]. Nine other *N. brasiliensis* clinical strains were randomly selected from the "Observatoire Français des Nocardioses" collection generated between 2014 and 2015 (Supplementary Table S1) [7].

### 2.2. In vitro susceptibility testing

Minimum inhibitory concentrations were determined by broth microdilution in 96-well plates in cation-adjusted Mueller-Hinton broth (CA-MHB), according to 2018 Clinical and Laboratory Standards Institute (CLSI) standard M62 [13]. Inocula were obtained from a 4-day fresh culture at 37 °C in 10 mL of CA-MHB containing 10 glass beads (diameter = 1 cm) for culture homogenisation. After 1/10 dilution in fresh CA-MHB, bacteria were inoculated in 96-well plates containing β-lactam antibiotics and inhibitors to reach a final concentration of 5 × 10⁵ CFU/ml. Microplates were incubated for 3-4 days at 37 °C, depending on bacterial growth in control wells. Breakpoints used for susceptible (S), intermediate (I), and resistant (R) strains were adapted from the CLSI and are reported in Supplementary Table S2. MICs were performed in triplicate.

### 2.3. Whole-genome sequencing, assembly, and quality control of bacterial strains

Pure bacterial suspensions in sterile water were obtained from a 4-day fresh culture on Tryptone Soya Agar plates at 37 °C. A two-step DNA extraction procedure was performed with bead beating and the DSP DNA mini kit (Qiagen, Germany). Libraries were prepared with the Nextera XT kit (Illumina San Diego, CA, USA) and pair-end sequenced on a NovaSeq System (Illumina). Reads were assembled with shovill 1.1.0 (https://github.com/tseemann/shovill) using standard parameters. Two-step quality control was performed. First, we computed assembly metrics using quast [14]. Then, we evaluated genome completeness with CheckM 1.0.11 [15], with 95% defined as the inclusion threshold.

### 2.4. In silico identification of N. brasiliensis putative β-lactamases

For HUJEG-1, we searched the sequences for β-lactamase-encoding genes using Resfinder [16] and abricate (https://github.com/tseemann/abricate), with a minimum nucleotide sequence identity and target coverage of 20%. We then aligned putative β-lactamase sequences with sequences from the NCBI Bacterial Antimicrobial Resistance Reference Gene Database (BioProject PRJNA313047) using both nucleotide (blastN) and protein (blastP) sequences to check for matches with known β-lactamases. We also searched for amino acid sequence identity against the entire UniRef90 database [17] (accessed January 2023), including protein sequences unrelated to resistance. For the nine clinical strains, we searched for putative β-lactamase-encoding genes using blastN with β-lactamase sequences from HUJEG-1 as reference.

### 2.5. Core genome, single-nucleotide polymorphisms, and protein-based phylogenies

We computed the core genome with Roary 3.13.0 [18], and the protein sequences of putative β-lactamases were aligned with MAFFT [19]. Core genomes and residue alignments were used to draw distinct maximum-likelihood phylogenies with FastTree 2.1.10 [20]. Trees were plotted with iTol [21].

### 2.6. Amino acid sequence alignment

Sequences were compared with the closely related β-lactamases AST-1 and FAR-1, respectively, from *N. asteroides* and *N. farcinica* [22,23], and to that of TEM-1 and CTX-M-15 from the Resfinder database [16]. Well-studied distant and nearby β-lactamase sequences were chosen to optimise our alignment with Ambler numbering [24].

### 2.7. Production and purification of putative N. brasiliensis β-lactamases

Primers used for amplification/cloning of putative β-lactamase-encoding genes were designed according to the *N. brasiliensis* HUJEG-1 genome [3], as previously described [25]. The following primers were used: bra1F (5' AAACATATGGCACAGACCGAATCA 3') / bra1R (5' AAACTCGAGTCAGCGCAGCTTTTC 3'), and brs1F (5' AAAACATATGGCGCCGACCACGCAG 3') / brs1R (5' AAACTCGAGTCAAGCCAACGCACT 3') (Supplementary Table S3). PCR products were cloned into the plasmid vector pET-TEV to generate recombinant plasmids (pET-TEVΩblaBRA-1 and pET-TEVΩblaBRS-1). The resulting proteins were composed of amino acid residues 32-306 (BRA-1) and 34-299 (BRS-1), and a vector-encoded N-terminal 6 × His tag followed by a TEV cleavage site (MGSSHHHHHHSSGENLYFQGHM). Putative β-lactamases were produced in *Escherichia coli* BL21 and purified by affinity (Ni-nitrilotriacetic acid resin, Sigma-Aldrich, St-Louis, USA) and size-exclusion chromatography (Superdex 200 HL26/60 column, Amersham Pharmacia Biotech, Upsalla, Sweden) in 25 mM Tris-HCl (pH 7.5) [25,26].

### 2.8. Determination of kinetic parameters for β-lactams

The hydrolytic activity of putative β-lactamases was evaluated by spectrophotometry (Carry-100 Bio; Varian), as previously described [25,27,28]. All kinetic experiments were performed at 20 °C in 100 mM 2-(N-morpholino)ethanesulphonic acid buffer at pH 6.4 to ensure comparison with previously published data [25,29,30]. The kinetic parameters $k_{cat}$ and $K_m$ were deduced from the initial hydrolysis rate at different antibiotic substrate concentrations (wavelengths and absorption coefficients are displayed in Supplementary Table S4). Clavulanate and avibactam were evaluated
as β-lactamase inhibitors using nitrocefin (100 µM) as the substrate. Various concentrations of clavulanate or avibactam were pre-incubated with 0.1 nM purified BRA-1 or BRS-1 for 5 min at 30 °C before adding nitrocefin, as previously described [25,31]. Experiments were performed at 20 °C by spectrophotometry in 100 mM 2-(N-morpholino)ethanesulphonic acid buffer at pH 6.4, monitoring hydrolysis of the β-lactam ring at 486 nm. The IC₅₀ was defined as the inhibitor concentration reducing the initial nitrocefin hydrolysis rate by 50%.

Both putative β-lactamases harboured conserved amino acid motifs present in class A β-lactamases, namely: S⁷⁰TFK, S¹³⁰DG/N, and K²³⁴T/SG (Fig. 1) [32]. These sequences were compared with a dataset of 130 β-lactamases well described phenotypically [33]. The amino acid sequence of BRA-1 shared 60.7% identity with class A β-lactamase AST-1 from *N. asteroides* [23] and that of BRS-1 shared 50.5% identity with class A β-lactamase BlaC from *Mycobacterium tuberculosis* strain H37Rv [28]. BRA-1 and BRS-1 shared 53.3% amino acid identity.

## 3. Results

### 3.1. Identification of sequences corresponding two putative β-lactamases from the N. brasiliensis HUJEG-1 genome

Two genes coding for hypothetical β-lactamases were found in the genome of *N. brasiliensis* HUJEG-1, namely *bla*<sub>BRA-1</sub> and *bla*<sub>BRS-1</sub>, encoding BRA-1 (31.2 kDa) and BRS-1 (30.8 kDa), respectively.

### 3.2. Antibiotic susceptibility testing of N. brasiliensis HUJEG-1

MICs obtained via the microdilution method showed that *N. brasiliensis* HUJEG-1 was resistant to all tested β-lactam antibiotics (MIC range = 128–256 µg/mL) except for meropenem (MIC = 4 µg/mL) (Table 1). The addition of 4 µg/mL clavulanate or avibactam restored susceptibility to amoxicillin, cefuroxime, and

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** (A) Alignment of the amino acid sequences of BRA-1 (strain HUJEG-1) with those of the most closely related β-lactamases AST-1 and FAR-1 (from *Nocardia asteroides* and *Nocardia farcinica*, respectively), and of prototypic TEM-1 and CTX-M-15. (B) Alignment of the amino acid sequences of BRS-1 (strain HUJEG-1), of the most closely related β-lactamase BLAC, from *Mycobacterium tuberculosis*, and of TEM-1 and CTX-M-15. Numbering according to the numbering scheme described by Ambler et al. [24]. Identical residues are indicated by dots. Signal peptides are shaded grey. Conserved elements of class A β-lactamases (S⁷⁰XXK, S¹³⁰DN/G, and K²³⁴S/TG) are boxed in red, and the omega loop is in blue.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 1. Continued</figcaption></figure>

**Table 1**

Nocardia brasiliensis HUJEG-1 MICs of amoxicillin, cefotaxime, cefuroxime, imipenem, and meropenem (in µg/mL) with or without a fixed concentration of clavulanate or avibactam (4 µg/mL). Data are medians of three independent experiments.

<table><thead><tr><th rowspan="2"></th><th colspan="3">MIC (µg/ml) of indicated β-lactams with or without β-lactamase inhibitors</th></tr><tr><th>No β-lactamase inhibitor</th><th>Clavulanate</th><th>Avibactam</th></tr></thead><tbody><tr><td>Amoxicillin</td><td>256</td><td>2</td><td>2</td></tr><tr><td>Cefuroxime</td><td>128</td><td>16</td><td>4</td></tr><tr><td>Cefotaxime</td><td>128</td><td>8</td><td>8</td></tr><tr><td>Imipenem</td><td>256</td><td>256</td><td>256</td></tr><tr><td>Meropenem</td><td>4</td><td>4</td><td>4</td></tr></tbody></table>

ceftazidime. In contrast, the MIC of imipenem (256 µg/mL) was not affected by the addition of 4 µg/mL clavulanate or avibactam.

1000-fold lower than that of BRA-1 for all tested β-lactam antibiotics (Table 2).

### 3.3. Kinetic parameters for β-lactam hydrolysis by BRA-1 and BRS-1

BRA-1 effectively hydrolysed cephems, such as nitrocefin, cephalothin, cefuroxime, and cefotaxime, with $k_{cat}/K_m$ values ranging from $6.0 \times 10^4$ to $8.1 \times 10^6$ M$^{-1}$s$^{-1}$ (Table 2). As an exception, ceftazidime displayed a low $k_{cat}/K_m$ value of $34 \pm 1$ M$^{-1}$s$^{-1}$ due to both high $K_m$ and low $k_{cat}$ values. Regarding penems (penicillin, ampicillin), $k_{cat}/K_m$ were higher than $2.0 \times 10^5$ and $k_{cat}$ values obtained with benzylpenicillin, amoxicillin, and piperacillin (between $7.9$ and $14.9$ s$^{-1}$) were comparable to those obtained with cefalotin, cefuroxime, and cefotaxime (Table 2). Conversely, BRA-1 poorly hydrolysed carbapenems with low $k_{cat}$ values of $4.0 \pm 0.4 \times 10^{-3}$ s$^{-1}$ and $3.2 \pm 0.5 \times 10^{-4}$ s$^{-1}$ for imipenem and meropenem, respectively. The catalytic efficacy of BRS-1 was 100-

### 3.4. Inhibition of BRA-1 and BRS-1 by clavulanate and avibactam

Considering the distinct reaction schemes for β-lactamase inhibition by clavulanate and avibactam [25], we determined the $IC_{50}$ (i.e. the concentration of inhibitor required to reduce the initial hydrolysis rate of nitrocefin by 50%) to compare the efficacy of inhibition of BRA-1 and BRS-1 by these β-lactamase inhibitors, as previously described [25]. $IC_{50}$ values (Table 3) indicated that avibactam was more effective than clavulanate for inhibition of both BRA-1 ($IC_{50} = 0.014 \pm 0.001$ µM vs. $0.080 \pm 0.010$ µM) and BRS-1 (0.47 ± 0.13 µM vs. 2.50 ± 0.08 µM). Avibactam and clavulanate were more effective against BRA-1 than against BRS-1.
<table><caption>**Table 2**
Kinetic parameters for the hydrolysis of β-lactam antibiotics by BRA-1 and BRS-1.</caption><thead><tr><th rowspan="2"></th><th colspan="3">BRA-1</th><th colspan="3">BRS-1</th></tr><tr><th>K<sub>m</sub> (µM)</th><th>k<sub>cat</sub> (s<sup>-1</sup>)</th><th>k<sub>cat</sub>/K<sub>m</sub> (M<sup>-1</sup> s<sup>-1</sup>)</th><th>K<sub>m</sub> (µM)</th><th>k<sub>cat</sub> (s<sup>-1</sup>)</th><th>k<sub>cat</sub>/K<sub>m</sub> (M<sup>-1</sup> s<sup>-1</sup>)</th></tr></thead><tbody><tr><td><strong>Penams</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Benzylpenicillin*</td><td>&lt;40</td><td>9.1 ± 2.8</td><td>&gt;2.3 × 10<sup>5</sup></td><td>&lt;40</td><td>0.2 ± 0.1</td><td>&gt; 5.2 × 10<sup>3</sup></td></tr><tr><td>Amoxicillin*</td><td>&lt;40</td><td>15 ± 3</td><td>&gt;3.7 × 10<sup>5</sup></td><td>&lt;40</td><td>0.1 ± 0.1</td><td>&gt; 3.6 × 10<sup>3</sup></td></tr><tr><td>Piperacillin*</td><td>&lt;40</td><td>S</td><td>&gt;2.0 × 10<sup>5</sup></td><td>&lt;40</td><td>0.2 ± 0.1</td><td>&gt; 5.4 × 10<sup>3</sup></td></tr><tr><td><strong>Cephems</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Nitrocefin</td><td>77 ± 11</td><td>620 ± 30</td><td>8.1 ± 1.6 × 10<sup>6</sup></td><td>&gt;1800</td><td>&gt;6.0</td><td>1.6 ± 0.8 × 10<sup>3</sup></td></tr><tr><td>Cefalotin</td><td>16 ± 1</td><td>12.7 ± 0.2</td><td>7.8 ± 0.8 × 10<sup>5</sup></td><td>&gt;1500</td><td>&gt;5.0</td><td>3.5 ± 0.2 × 10<sup>3</sup></td></tr><tr><td>Cefuroxime</td><td>105 ± 17</td><td>18.5 ± 0.9</td><td>1.8 ± 0.4 × 10<sup>5</sup></td><td>&gt;900</td><td>&gt;0.1</td><td>2.5 ± 0.3 × 10<sup>2</sup></td></tr><tr><td>Cefotaxime</td><td>106 ± 23</td><td>6.3 ± 0.5</td><td>6.0 ± 1.7 × 10<sup>4</sup></td><td>&gt;800</td><td>&gt;0.1</td><td>1.3 ± 0.1 × 10<sup>2</sup></td></tr><tr><td>Ceftazidime</td><td>&gt;500</td><td>&gt; 0.1</td><td>34.5 ± 1.2</td><td>&gt;500</td><td>&gt;8.0 × 10<sup>-3</sup></td><td>12.1 ± 1.2</td></tr><tr><td><strong>Penems</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Imipenem*</td><td>&lt;10</td><td>4.0 ± 0.4 × 10<sup>-3</sup></td><td>&gt;4.0 × 10<sup>2</sup></td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Meropenem*</td><td>&lt;10</td><td>3.2 ± 0.5 × 10<sup>-4</sup></td><td>&gt;3.2 × 10<sup>1</sup></td><td>&lt;10</td><td>1.5 ± 0.1 × 10<sup>-4</sup></td><td>&gt;1.5 × 10<sup>1</sup></td></tr><tr><td><strong>Monobactam</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Aztreonam</td><td>&gt;1100</td><td>&gt; 3</td><td>2.7 ± 0.1 × 10<sup>3</sup></td><td>&gt;1100</td><td>&gt;0.1</td><td>1.4 ± 0.1 × 10<sup>2</sup></td></tr><tr><td><strong>Oxapenam</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Clavulanate</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr></tbody></table>

ND, not detected.

* For some antibiotics, the value of $k_{cat}/K_m$ could not be directly measured when $K_m$ was lower than the lowest tested substrate concentration (in that case, the lowest tested substrate concentration already reached the $k_{cat}$ value). In this situation, $k_{cat}/K_m$ were calculated by dividing the measured $k_{cat}$ by the smallest tested antibiotic concentration.

<table><caption>**Table 3**
Comparison of concentrations of inhibitor required to reduce the initial hydrolysis rate of nitrocefin by 50% (IC₅₀s, µM) of clavulanate or avibactam for *Nocardia brasiliensis* HUJEG-1 β-lactamases BRA-1 and BRS-1 after 5 min incubation at 30 °C.</caption><thead><tr><th rowspan="2">Inhibitor</th><th colspan="2">IC₅₀ (± SD), µM</th></tr><tr><th>BRA-1</th><th>BRS-1</th></tr></thead><tbody><tr><td>Clavulanate</td><td>0.080 ± 0.010</td><td>2.5 ± 0.1</td></tr><tr><td>Avibactam</td><td>0.014 ± 0.001</td><td>0.47 ± 0.13</td></tr></tbody></table>

### 3.5. Antibiotic susceptibility of nine *N. brasiliensis* clinical isolates to β-lactam antibiotics with or without avibactam

Nine additional clinical isolates from the "Observatoire Français des Nocardioses" collection were studied (Table 4). The nine clinical isolates were resistant to amoxicillin, but with lower MICs (12–256 µg/mL) than those determined for *N. brasiliensis* HUJEG-1. The addition of avibactam reduced the MIC of amoxicillin against all isolates. All *N. brasiliensis* isolates were resistant to imipenem, with no significant effect of avibactam. One of these isolates was not susceptible to meropenem. Six out of nine isolates were susceptible to cefotaxime. Among the three cefotaxime-resistant isolates, the addition of avibactam reduced the MIC of cefotaxime from 256 to 32 µg/mL for strain 3789 and from 64 to 1 µg/mL for isolate 3807. The addition of avibactam had no impact on the remaining isolate (3790; MIC = 256 µg/mL), suggesting the presence of an additional non-β-lactamase-mediated resistance mechanism.

### 3.6. Genomes of the nine *N. brasiliensis* strains and HUJEG-1

The size of the complete genomes ranged between 7.7 and 9.9 Mb, with a GC content of 68.0 ± 0.2%. The 10 genomes included 5378 core genes and 14 686 accessor genes. Both *bla*<sub>BRA-1</sub>- and *bla*<sub>BRS-1</sub>-related genes were present in the genomes of the nine *N. brasiliensis* strains. Sequence diversity was detected in BRA-1- and BRS-1-related β-lactamases (variants BRA-1b to -1f and BRS-1b to -1j; Supplementary Figs. S1 and S2). BRA-1 and BRS-1 amino acid sequences of *N. brasiliensis* HUJEG-1 were different from those found in the nine clinical strains. For BRA-1 and BRS-1 variants identified in the nine clinical isolates, no correlation was found between amino acid sequence polymorphisms and resistance to cefotaxime. A comparison of 400-pb sequences located upstream from

the *bla*<sub>BRA-1</sub> and *bla*<sub>BRS-1</sub> genes did not identify any polymorphism in putative promoters or translation signals that could affect the level of expression of the genes (data not shown).

To go further, we explored the genetic diversity of the different strains of *N. brasiliensis*. Core genome phylogeny allowed clustering of the susceptibility to cefotaxime (Fig. 2), suggesting a potential impact of other molecular determinants associated with this genetic background on susceptibility to cefotaxime.

## 4. Discussion

Susceptibility of *Nocardia* spp. to β-lactam antibiotics is variable and the molecular determinants of resistance remain uncharacterised for most species, such as *N. brasiliensis*. In this study, we explored resistance to β-lactam antibiotics of the reference strain *N. brasiliensis* HUJEG-1 and extended our analysis to a collection of nine clinical strains belonging to the same species. An in silico approach enabled the identification of two putative class A β-lactamase-encoding genes designated *bla*<sub>BRA-1</sub> and *bla*<sub>BRS-1</sub>. Penam and cephem resistance was mediated by BRA-1, which was inhibited by clavulanate or avibactam. Concerning imipenem resistance, an absence of the effect of β-lactamase inhibitors on MICs and the low hydrolytic activity toward imipenem suggest a non-enzymatic mechanism. Analysis of imipenem hydrolysis on a crude extract, not performed in this work, could reinforce this hypothesis. Hence, other factors such as low-affinity penicillin-binding proteins may be involved in imipenem resistance.

Analysis of the nine other clinical strains displayed variations in cefotaxime susceptibility as well as diversity in BRA-1 sequences and genetic backgrounds, suggesting that cefotaxime resistance appears to partially depend on BRA-1 variants and/or on another mechanism.

Epidemiological studies indicated that *N. brasiliensis* is frequently resistant to imipenem, with 56–100% of strains being resistant depending on the method of antibiotic susceptibility testing [7–9,34]. Two studies used broth microdilution [35] or antibiotic disk diffusion [7] to compare the activity of imipenem and meropenem against *N. brasiliensis*, and showed that meropenem was more frequently active against *N. brasiliensis* (52% and 58% resistance, respectively) than imipenem (85% and 100% resistance, respectively), as we also observed [7,35]. Resistance to cefotaxime (13–23%) has also been previously noted [7–9,36].
<caption>**Table 4**</caption>

MICs of amoxicillin, cefotaxime, imipenem, and meropenem for 10 clinical isolates of *Nocardia brasiliensis* with or without a fixed concentration of avibactam (4 µg/mL).

<table><thead><tr><th rowspan="3">Strain</th><th colspan="8">MIC (µg/mL) of indicated β-lactam with or without avibactam</th></tr><tr><th colspan="2">Amoxicillin</th><th colspan="2">Cefotaxime</th><th colspan="2">Imipenem</th><th colspan="2">Meropenem</th></tr><tr><th>None</th><th>Avibactam</th><th>None</th><th>Avibactam</th><th>None</th><th>Avibactam</th><th>None</th><th>Avibactam</th></tr></thead><tbody><tr><td>HUJEG-1</td><td>256</td><td><b>2</b></td><td>128</td><td><b>8</b></td><td>256</td><td>256</td><td><b>4</b></td><td><b>4</b></td></tr><tr><td>3773</td><td>32</td><td><b>2</b></td><td><b>4</b></td><td><b>4</b></td><td>64</td><td>128</td><td><b>2</b></td><td><b>4</b></td></tr><tr><td>3774</td><td>16</td><td><b>4</b></td><td><b>4</b></td><td><b>0.5</b></td><td>128</td><td>256</td><td><b>2</b></td><td><b>8</b></td></tr><tr><td>3777</td><td>16</td><td><b>2</b></td><td><b>8</b></td><td><b>2</b></td><td>32</td><td>16</td><td><b>8</b></td><td><b>4</b></td></tr><tr><td>3779</td><td>16</td><td><b>4</b></td><td><b>2</b></td><td><b>8</b></td><td>256</td><td>256</td><td><b>4</b></td><td><b>8</b></td></tr><tr><td>3786</td><td>64</td><td><b>2</b></td><td><b>4</b></td><td><b>4</b></td><td>32</td><td>64</td><td><b>4</b></td><td><b>4</b></td></tr><tr><td>3789</td><td>64</td><td><b>4</b></td><td>256</td><td>32</td><td>64</td><td>32</td><td>16</td><td><b>4</b></td></tr><tr><td>3790</td><td>16</td><td><b>2</b></td><td>256</td><td>256</td><td>128</td><td>128</td><td>8</td><td><b>4</b></td></tr><tr><td>3791</td><td>16</td><td><b>1</b></td><td><b>4</b></td><td><b>2</b></td><td>128</td><td>128</td><td>8</td><td><b>4</b></td></tr><tr><td>3807</td><td>128</td><td><b>2</b></td><td>64</td><td><b>2</b></td><td>256</td><td>256</td><td><b>4</b></td><td><b>4</b></td></tr></tbody></table>

Data are medians of three independent experiments. Values in bold are "susceptible" to the given β-lactam antibiotic.

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Core genome maximum-likelihood tree of strain HUJEG-1 and the nine other clinical strains of *Nocardia brasiliensis*. Susceptibility to different antibiotics with or without avibactam are represented by a colour code (red for resistance, green for susceptibility). Variants of BRA-1 and BRS-1 are indicated on the right side of the figure.</figcaption></figure>

To date, the molecular determinants involved in resistance to β-lactams have been poorly studied in *N. brasiliensis*. A previously published paper studying a crude extract from four *N. brasiliensis* strains showed that penams (penicillin, ampicillin) and cephems (cephaloridine, cephalothin, and cefamandole) were hydrolysed by *N. brasiliensis* [11]. The authors also showed that clavulanate restored the activity of amoxicillin, as we also observed [11]. Strikingly, we observed that avibactam was fivefold more efficacious than clavulanate at inhibiting BRA-1 and BRS-1, as previously demonstrated with *N. farcinica*, suggesting a possible therapeutic role of the combination of amoxicillin and avibactam [25].

We further explored the mechanisms of resistance to β-lactam antibiotics in *N. brasiliensis* HUJEG-1. We showed that BRA-1 contributes to penam and cephem resistance. As mentioned above, resistance of *N. brasiliensis* to cefotaxime is variable between isolates, and we wanted to explore molecular determinants associated with cefotaxime resistance in nine other isolates of *N. brasiliensis*. Of the nine isolates, all harboured the *bla*<sub>BRA-1</sub> and *bla*<sub>BRS-1</sub> genes, but only three were resistant to cefotaxime. It is noteworthy that different alleles of these genes were found in these strains, and phylogenetic analyses revealed genetic diversity. BRA-1 was the main determinant associated with cefotaxime resistance in *N. brasiliensis* HUJEG-1, but the presence of *bla*<sub>BRA-1</sub> was not sufficient to confer resistance to cefotaxime in *N. brasiliensis*, suggesting that resistance to cefotaxime could be associated with another molecular determinant.

Comparison of genotypic and phenotypic data suggest that cefotaxime resistance may be associated with certain genetic backgrounds. Thus, study of a larger number of strains would be necessary to attempt to predict cefotaxime susceptibility from genomic data.

This work paves the way to explore molecular determinants involved in β-lactam resistance in *Nocardia* spp., and the identification of molecular signatures needs to be explored to predict resistance to β-lactam antibiotics.

**Acknowledgements:** The authors would like to thank Veronica Rodriguez-Nava for kindly providing the *N. brasiliensis* clinical strains and Mario César Salinas Carmona for providing *N. brasiliensis* HUJEG-1.

**Funding:** None.

**Declaration of competing interests:** None.

**Ethical approval:** Not required.

**Supplementary materials**

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jgar.2025.01.022.
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