# Antibiotic resistance profiles of gram-negative bacteria in southern Tunisia: Focus on ESBL, carbapenem and colistin resistance

Mariem Yengui, Rahma Trabelsi, Radhouane Gdoura, Aïcha Hamieh, Hanane Zerrouki, Jean-Marc Rolain, Linda Hadjadj

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## To cite this version:

Mariem Yengui, Rahma Trabelsi, Radhouane Gdoura, Aïcha Hamieh, Hanane Zerrouki, et al. Antibiotic resistance profiles of gram-negative bacteria in southern Tunisia: Focus on ESBL, carbapenem and colistin resistance. Infection, Genetics and Evolution, 2025, 133, pp.105787. 10.1016/j.meegid.2025.105787. hal-05375445

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# Antibiotic resistance profiles of gram-negative bacteria in southern Tunisia: Focus on ESBL, carbapenem and colistin resistance

Mariem Yengui<sup>a,b,c</sup>, Rahma Trabelsi<sup>a</sup>, Radhouane Gdoura<sup>a</sup>, Aïcha Hamieh<sup>b,c</sup>, Hanane Zerrouki<sup>b,c</sup>, Jean-Marc Rolain<sup>c,d</sup>, Linda Hadjadj<sup>b,c,*</sup>

## ARTICLE INFO

**Keywords:**

Urine

ESBL

Carbapenemase

Colistin

## ABSTRACT

The main objective of this cross-sectional study was to investigate the prevalence of beta-lactam (cephalosporins or carbapenems) or colistin resistant bacteria. Those were isolated from urine samples in two private polyclinics located in the Sfax region, in southern Tunisia. From September 2021 to August 2022, 116 strains resistant to β-lactams or colistin were isolated, identified by MALDI-TOF, and their antibiotic susceptibility was assessed by disk diffusion method. Resistance genes were detected by real-time PCR, standard PCR, and sequencing. The results revealed that the 116 strains consisted predominantly of Enterobacteriaceae (92.2 %) and non-fermenting bacteria (7.8 %). Among these strains, 21 (18.1 %) were resistant to carbapenems, three (2.7 %) to colistin, including two strains of Klebsiella pneumoniae (1.7 %) exhibiting resistance to both carbapenems and colistin. In Enterobacteriaceae, bla<sub>CTX-A</sub>, bla<sub>SHV</sub>, and bla<sub>TEM</sub> were found in 79.5 %, 46.7 %, and 40.2 % of strains, respectively. For these strains, the minimum inhibitory concentrations (MICs) of imipenem and ertapenem ranged from >32 to 6 µg/mL and > 32 to 2 µg/mL, respectively, with bla<sub>OXA-48</sub> and bla<sub>NDM</sub> detected in 21.7 % and 19.6 % of isolates, respectively. Seven A. baumannii isolates resistant to imipenem and meropenem (MICs >32 µg/mL and 8 µg/mL, respectively) carried bla<sub>OXA-23</sub> (n = 5) and bla<sub>OXA-24</sub> (n = 2). In addition, mutations in the *mgrB* gene conferring colistin resistance were identified in two isolates. Two K. pneumoniae were colistin-resistant and carried the bla<sub>OXA-48</sub> gene. These results highlight the urgency of developing new strategies for the identification and surveillance of pathogenic strains in humans to effectively combat this growing public health threat in Tunisia.

## 1. Introduction

Urinary tract infections (UTIs) are a significant public health concern. In 2019, approximately 404.61 million cases of UTIs were reported globally, with a particularly high incidence among women (Yang et al., 2022). These infections are mainly induced by gram-negative bacteria with the prevalence of Enterobacteriaceae such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and K. aerogenes (Flores-Mireles et al., 2015).

The production of β-lactamases is a primary mechanism of antibiotic resistance in Gram-negative bacteria. Multidrug-resistant bacteria were

initially treated with third-generation cephalosporins (C3Gs), but the emergence of resistance led to the use of carbapenems, which are generally more effective against both Gram-positive and Gram-negative bacteria (Meletis, 2016). Currently, the emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) in the population is a major and widespread health problem (Dziri et al., 2020).

The spread of CRE, frequently calls into question the effectiveness of carbapenem therapies as a last resort treatment to combat multidrug-resistant (MDR) organisms. In gram-negative bacteria, carbapenemases have been frequently described worldwide (Dziri et al., 2020). Currently, more than 350 types of carbapenemase enzymes have been

<footnote><sup>a</sup> Department of Life Sciences, Research Laboratory of Environmental Toxicology, Microbiology and Health (LR17ES06), Faculty of Sciences of Sfax, University of Sfax, BP 1171, 3000 Sfax, Tunisia</footnote>

<footnote><sup>b</sup> Aix Marseille Univ, MEPHI, Faculté de Médecine et de Pharmacie, Marseille, France</footnote>

<footnote><sup>c</sup> IHU-Méditerranée Infection, Marseille, France</footnote>

<footnote><sup>d</sup> Aix Marseille Univ, APHM, MEPHI, Faculté de Médecine et de Pharmacie, Marseille, France</footnote>

<footnote>* Corresponding author at: Linda Hadjadj, Aix Marseille Univ, MEPHI, IHU Méditerranée Infection, Faculté de Médecine et de Pharmacie, 19-21 boulevard Jean Moulin, 13385, Marseille CEDEX 05, France.</footnote>

<footnote>E-mail address: linda.hadjadj@univ-amu.fr (L. Hadjadj).</footnote>
characterised, with a prevalence of KPC, NDM, VIM, IMP, and OXA type (Dziri et al., 2020). The metallo-β-lactamase enzyme NDM-1, which was first identified in New Delhi in 2009, helps bacteria hydrolyse all antibiotics belonging to the β-lactam family, with the exception of monobactam (Yong et al., 2009). The presence and distribution of isolates producing NDM-1 has been documented in several countries, including the United States, the United Kingdom, France, Africa, and Australia (Chen et al., 2011). In Tunisia, this gene was found to be disseminated in all regions of the country, within the hospital environment and in the community (Mansour et al., 2019). Since OXA-48 carbapenemases were first isolated in Turkey in 2001, they have been frequently reported throughout the world (Mhaya et al., 2020a). The blaOXA-48 gene is predominant in Europe, particularly in France, Spain, and Switzerland. Additionally, North African countries, particularly Tunisia, Egypt and Libya, as well as in various countries in the Middle East consist of that predominance (Boyd et al., 2022).

The spread of carbapenemase-producing *A. baumannii* in clinical settings has become a significant threat to public health. This resistance is most commonly mediated by oxacillinases, such as OXA-23, OXA-24 and OXA-58 (Ramirez et al., 2020). The class B β-lactamase gene blaNDM has also been reported in *A. baumannii*. A concerning characteristic of blaNDM is its location on mobile genetic elements, facilitating its transfer between different bacterial species (Berrazeg et al., 2014).

The emergence and spread of carbapenemase-producing bacteria requires clinicians to introduce colistin for the treatment of infections caused by pathogens (Berglund, 2019). Resistance to colistin is induced following a modification of lipopolysaccharides (LPS), causing a reduction in the negative charge of LPS, which consequently reduces its affinity for this antibiotic. Mutations in two-part regulatory systems, such as mutations occuring in the *mgrB*, *phoP/Q*, *pmrA/B*, *pmrC* and *crrABC* genes, or through plasmid-mediated resistance genes (*mcr-1* to *mcr-10*) are at the base of these changes (Moffatt et al., 2019).

Our study aimed to evaluate the prevalence of ESBLs, carbapenemases, and colistin resistance in Gram-negative bacteria isolated from urine samples. These samples were collected from two polyclinics in the Sfax region of southern Tunisia between September 2021 and August 2022. In addition, antibiotic resistance profile were analyzed and mechanisms responsible for this resistance were identified.

# 2. Materials and methods

## 2.1. Sampling and bacterial isolates

From September 2021 to August 2022, a cross-sectional study was conducted using urine samples from patients hospitalized in two private Polyclinics (Polyclinic 1 and Polyclinic 2) in the Sfax region, southern Tunisia. Clinically significant strains were isolated from these samples based on the following phenotypic criteria: either a synergistic effect observed using the disk diffusion synergy method, or resistance to third-generation cephalosporins (Cefotaxime and Ceftazidime), carbapenems, or colistin. These strains, initially identified as part of routine bacteriological diagnostics in the two medical analysis laboratories, were stored in 50 % glycerol in Tunisia and subsequently analyzed retrospectively at the IHU-Méditerranée Infection research laboratory in Marseille, France. Bacterial identification was performed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonik, Bremen, Germany) (Seng et al., 2009).

## 2.2. Antibiotic susceptibility profile

The antibiotic susceptibility pattern of all isolates was visualised using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (bioMérieux, Marcy l'Etoile, France). The antibiotics (i2A®, Montpellier, France) tested for *Enterobacteriaceae* were amoxicillin (20 µg), amoxicillin/clavulanic acid (20/10 µg), cefepime (30 µg), piperacillin/tazobactam (36 µg), mecillinam (10 µg), ceftriaxone (30 µg), ertapenem

(10 µg), imipenem (10 µg), nitrofurantoin (100 µg), trimethoprim/sulfamethoxazole (25 µg), amikacin (30 µg), ciprofloxacin (5 µg), tetracycline (30 µg), and gentamicin (10 µg). Different antibiotics were tested on all non-fermenting gram-negative bacteria, including ticarcillin (75 µg), ticarcillin-clavulanic acid (85 µg), piperacillin/tazobactam (36 µg), aztreonam (30 µg), ceftazidime (10 µg), cefepime (30 µg), meropenem (10 µg), imipenem (10 µg), amikacin (30 µg), gentamicin (10 µg), ciprofloxacin (5 µg), rifampicin (100 µg), minocycline (30 µg), and trimethoprim-sulfamethoxazole (1.25/23.75 µg). For isolates showing a resistance phenotype by disc diffusion method, the minimum inhibitory concentration (MIC) was determined by the E-test method (bioMérieux, Marcy l'Etoile, France) for ertapenem, imipenem, and meropenem and by the UMIC microdilution method (Biocentric, Bandol, France) for colistin. The data was interpreted according to the European Committee on Antimicrobial Susceptibility Testing EUCAST/CASFM 2022 (Breakpoint Tables for Interpretation of MICs and Zone Diameters Version 12.0, 2022). Moreover, β-CARBA test (Bio-Rad, Hercules, CA, USA) was performed to detect carbapenemase production.

## 2.3. Molecular detection of ESBL genes and carbapenem, and colistin resistance genes

Bacterial DNA extraction was carried out by heat shock (Dashti et al., 2009). ESBL resistance genes blaTEM, blaSHV, blaCTX-A (blaCTX-M-1 and blaCTX-M-9 group), and blaCTX-B (blaCTX-M-2 and blaCTX-M-8/25 group), and carbapenemase (blaOXA-23, blaOXA-24, blaOXA-48, blaOXA-58, blaKPC, blaNDM, blaVIM) were detected by real-time PCR (RT-PCR) (Bio-Rad, CFX96 Touch TM, Hercules, CA, USA) (El-Sawalhi et al., 2023). The blaIMP gene was screened by standard PCR (2720 Thermal Cycler, Applied Biosystems, Waltham, MA, USA). RT-PCR was also used to screen for colistin resistance genes such as mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, and mcr-8 in colistin-resistant isolates (El-Sawalhi et al., 2023) (Touati et al., 2020). The primers used for qPCR and conventional PCR were selected from validated references, ensuring high specificity without cross-amplification (El-Sawalhi et al., 2023) (Touati et al., 2020). Sequencing of all carbapenemase resistance genes was carried out by an ABI 3130 automated sequencer (PE Applied Biosystems, Foster City, CA, USA) using a BigDye terminator cycle sequencing kit. The data were analyzed using ChromasPro software and the gene sequences were subsequently compared to the NCBI reference sequence database. Concerning the colistin-resistant and mcr-negative strains, the detection of mutations in genes involved in colistin resistance such as pmrA, pmrB, phoP, phoQ and mgrB was carried out by amplification and sequencing. The sequences of these genes were compared with the sequences of the colistin-sensitive reference strain K. pneumoniae MGH78578.

## 2.4. Statistical analysis

Statistical analysis was carried out using SPSS version 23 software. The chi-squared test was used to study the difference in resistance compared to each polyclinic. P values < .05 were considered to be statistically significant.

# 3. Results

## 3.1. Bacteria isolated

Between September 2021 and August 2022, 116 g-negative isolates were collected according to the criteria described above, including 44 from Polyclinic 1 and 72 from Polyclinic 2 (Table 1). These strains represent 2.2 % of the total number isolated (5236) from 5236 patients testing for UTIs, including 2389 at Polyclinic 1 and 2847 at Polyclinic 2, both located in the Sfax region, southern Tunisia. The distribution of isolates by age, sex, and year are detailed in Table 1.
<table><caption>**Table 1**
Distribution of isolates per polyclinic.</caption><thead><tr><th rowspan="2">Bacterial Species</th><th colspan="2">Age (years)</th><th colspan="2">Sex</th><th colspan="2">Months</th><th colspan="2">Number of isolates</th></tr><tr><th>(20-40)</th><th>(41-85)</th><th>Men</th><th>Women</th><th>Sep 2021-Feb 2022</th><th>Mar 2022-Aug 2022</th><th>Polyclinic 1</th><th>Polyclinic 2</th></tr></thead><tbody><tr><td><strong>Enterobacteriaceae</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>E. coli</em><br>(n = 52)</td><td>13</td><td>39</td><td>19</td><td>33</td><td>24</td><td>28</td><td>16</td><td>36</td></tr><tr><td><em>K. pneumoniae</em> (n = 46)</td><td>20</td><td>26</td><td>14</td><td>32</td><td>21</td><td>25</td><td>18</td><td>28</td></tr><tr><td><em>E. cloacae</em><br>(n = 5)</td><td>1</td><td>4</td><td>3</td><td>2</td><td>3</td><td>2</td><td>3</td><td>2</td></tr><tr><td><em>K. aerogenes</em> (n = 2)</td><td>0</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td><em>M. morganii</em><br>(n = 1)</td><td>0</td><td>1</td><td>1</td><td>0</td><td>1</td><td>0</td><td>0</td><td>1</td></tr><tr><td><em>P. mirabilis</em><br>(n = 1)</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td></tr><tr><td><strong>Non-fermenting bacilli</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>A. baumannii</em> (n = 8)</td><td>2</td><td>6</td><td>5</td><td>3</td><td>5</td><td>3</td><td>4</td><td>8</td></tr><tr><td><em>P. aeruginosa</em> (n = 1)</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>1</td></tr><tr><td>Polyclinic</td><td>Polyclinic 1</td><td>14</td><td>30</td><td>14</td><td>30</td><td>23</td><td>21</td><td>-</td></tr><tr><td></td><td>Polyclinic 2</td><td>22</td><td>50</td><td>31</td><td>41</td><td>32</td><td>40</td><td>72</td></tr></tbody></table>

Sep: September, Feb: February, Mar: March, Aug; August.

## 3.2. Antibiotic resistance phenotype

For all *Enterobacteriaceae* included in this study, the antibiotic sensitivity profile revealed that all strains are resistant to amoxicillin. The antibiotic sensitivity profile revealed 90.7 % resistance to amoxicillin/clavulanic acid, 74.8 % resistance to cefepime, 74.6 % resistance to ceftriaxone, 13.1 % resistance to ertapenem, and 12.1 % resistance to imipenem. Specifically, in Polyclinic 1, isolates showed 79.5 % resistance to amoxicillin/clavulanic acid, 64.1 % resistance to cefepime, 69.2 % resistance to ceftriaxone, and 12.8 % resistance to ertapenem and imipenem (Table 2a). In Polyclinic 2, *Enterobacteriaceae* showed 97.1 % resistance to amoxicillin/clavulanic acid, 80.9 % resistance to cefepime, 77.9 % resistance to ceftriaxone, 13.2 % resistance to ertapenem, and 11.7 % resistance to imipenem. Resistance phenotypes for *Enterobacteriaceae* isolates are detailed in Supplementary Table S1. The chi-squared statistical test shows that there was no significant difference in resistance for each polyclinic.

For non-fermenting bacilli, resistance phenotypes showed 100 % resistance to aztreonam, ceftazidime, and ciprofloxacin as well as 88.9 % resistance to ticarcillin, ticarcillin-clavulanic acid, piperacillin/tazobactam, and gentamicin. We found seven *A. baumannii* isolates resistant to both imipenem and meropenem (Table 2b).

<table><caption>**Table 2a**
Antibiotic resistance profiles of *Enterobacteriaceae*.</caption><thead><tr><th>Antibiotics</th><th>Polyclinic 1<br>(n = 39)</th><th>Polyclinic 2<br>(n = 68)</th><th>Total<br>(n = 107)</th></tr></thead><tbody><tr><td>AMX</td><td>39</td><td>68</td><td>107</td></tr><tr><td>AMC</td><td>31</td><td>66</td><td>97</td></tr><tr><td>FEP</td><td>25</td><td>55</td><td>80</td></tr><tr><td>TPZ</td><td>16</td><td>36</td><td>52</td></tr><tr><td>MEC</td><td>1</td><td>1</td><td>2</td></tr><tr><td>CRO</td><td>27</td><td>53</td><td>80</td></tr><tr><td>ETP</td><td>5</td><td>9</td><td>14</td></tr><tr><td>IMP</td><td>5</td><td>8</td><td>13</td></tr><tr><td>FT</td><td>6</td><td>15</td><td>21</td></tr><tr><td>SXT</td><td>18</td><td>60</td><td>78</td></tr><tr><td>AK</td><td>8</td><td>15</td><td>23</td></tr><tr><td>CIP</td><td>27</td><td>42</td><td>69</td></tr><tr><td>TET</td><td>27</td><td>37</td><td>64</td></tr><tr><td>CS</td><td>0</td><td>3</td><td>3</td></tr><tr><td>GN</td><td>15</td><td>26</td><td>41</td></tr></tbody></table>

AMX = amoxicillin, AMC = amoxicillin/clavulanic acid, FEP = cefepime, TPZ = piperacillin/tazobactam, MEC = mecillinam, CRO = ceftriaxone, ETP = ertapenem, IPM = imipenem, FT = nitrofurantoin, SXT = trimethoprim/sulfamethoxazole, AK = amikacin, CIP = ciprofloxacin, TET = tetracycline, CS = colistin, and GN = gentamicin.

<table><caption>**Table 2b**
Antibiotic resistance profiles of non-fermenting bacilli.</caption><thead><tr><th rowspan="2">Antibiotics</th><th colspan="2">A. baumannii<br>(n = 8)</th><th rowspan="2">P. aeruginosa<br>(n = 1)</th><th rowspan="2">Total<br>(n = 9)</th></tr><tr><th>Polyclinic 1<br>(n = 4)</th><th>Polyclinic 2<br>(n = 4)</th></tr></thead><tbody><tr><td>TIC</td><td>3</td><td>4</td><td>1</td><td>8</td></tr><tr><td>TCC</td><td>3</td><td>4</td><td>1</td><td>8</td></tr><tr><td>TPZ</td><td>3</td><td>4</td><td>1</td><td>8</td></tr><tr><td>ATM</td><td>4</td><td>4</td><td>1</td><td>9</td></tr><tr><td>CAZ</td><td>4</td><td>4</td><td>1</td><td>9</td></tr><tr><td>FEP</td><td>2</td><td>3</td><td>1</td><td>6</td></tr><tr><td>MER</td><td>3</td><td>4</td><td>0</td><td>7</td></tr><tr><td>IMP</td><td>3</td><td>4</td><td>0</td><td>7</td></tr><tr><td>RA</td><td>1</td><td>2</td><td>1</td><td>4</td></tr><tr><td>SXT</td><td>2</td><td>4</td><td>1</td><td>7</td></tr><tr><td>AK</td><td>3</td><td>3</td><td>1</td><td>7</td></tr><tr><td>CIP</td><td>4</td><td>4</td><td>1</td><td>9</td></tr><tr><td>MIN</td><td>2</td><td>3</td><td>1</td><td>6</td></tr><tr><td>CS</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>GN</td><td>4</td><td>3</td><td>1</td><td>8</td></tr></tbody></table>

TIC = ticarcillin, TCC = ticarcillin-clavulanic acid, TPZ = piperacillin/tazobactam, ATM = aztreonam, CAZ = ceftazidime, FEP = cefepime, MER = meropenem, IMP = imipenem, AK = amikacin, GN = gentamicin, CIP = ciprofloxacin, RA = rifampicin, MIN = minocycline, SXT = trimethoprim-sulfamethoxazole and CS = colistin.

In this study, phenotypic characterisation of the isolates revealed that 21 strains were resistant to carbapenems (13 *K. pneumoniae*, 7 *A. baumannii*, and 1 *E. cloacae*). Among *Enterobacteriaceae*, the MICs values for imipenem ranged from 6 µg/mL to >32 µg/mL, and for ertapenem, from 2 µg/mL to >32 µg/mL. For *A. baumannii*, the MICs were greater than 32 µg/mL for imipenem and at 8 µg/mL for meropenem. The β-CARBA test was positive for all carbapenem-resistant strains except for one *K. pneumoniae* isolate (UR22). We identified three *K. pneumoniae* strains resistant to colistin, two of which were also resistant to carbapenems (Table 3).

## 3.3. Molecular characterisation of isolates

### 3.3.1. Distribution of the ESBL gene

Real-time PCR results showed that the *bla*<sub>CTX-A</sub> gene was the most predominant type in *Enterobacteriaceae* (n = 85/116) followed by *bla*<sub>SHV</sub> (n = 50/116) and *bla*<sub>TEM</sub> (n = 44/116). These genes were detected in isolates from both polyclinics (Table 4). Among the eight strains of *A. baumannii*, only one strain presented the *bla*<sub>TEM</sub> gene. The distribution
<table><caption>**Table 3**

Characterisation of strains resistant to carbapenems and/or colistin.</caption><thead><tr><th>Strains</th><th>β-CARBA Test</th><th>ERT MIC (µg/mL)</th><th>IMP MIC (µg/mL)</th><th>MER MIC (µg/mL)</th><th>CS MIC (µg/mL)</th><th>Carbapenem resistance genes</th><th>Mutations of the associated colistin-resistance proteins</th></tr></thead><tbody><tr><td>E. cloacae UR77</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>ND</td><td>bla<sub>NDM-1</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR50</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>ND</td><td>bla<sub>NDM-1</sub>/bla<sub>OXA-48</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR54</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>ND</td><td>bla<sub>NDM-5</sub>/bla<sub>OXA-48</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR85</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>ND</td><td>bla<sub>NDM-1</sub>/bla<sub>OXA-48</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR58</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>ND</td><td>bla<sub>NDM-5</sub>/bla<sub>OXA-48</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR94</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>ND</td><td>bla<sub>OXA-48</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR95</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>ND</td><td>bla<sub>NDM-5</sub>/bla<sub>OXA-48</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR55</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>ND</td><td>bla<sub>NDM-5</sub>/bla<sub>OXA-48</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR35</td><td>+</td><td>&gt; 32</td><td>24</td><td>ND</td><td>ND</td><td>bla<sub>OXA-48</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR79</td><td>+</td><td>12</td><td>2</td><td>ND</td><td>ND</td><td>bla<sub>NDM-1</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR113</td><td>+</td><td>8</td><td>6</td><td>ND</td><td>ND</td><td>bla<sub>NDM-1</sub></td><td>ND</td></tr><tr><td>K. pneumoniae UR22</td><td>—</td><td>4</td><td>0.19</td><td>ND</td><td>ND</td><td>AB</td><td>ND</td></tr><tr><td>K. pneumoniae UR88</td><td>+</td><td>&gt; 32</td><td>&gt; 32</td><td>ND</td><td>32</td><td>bla<sub>OXA-48</sub></td><td>mgrB (L9R)</td></tr><tr><td>K. pneumoniae UR74</td><td>+</td><td>&gt; 32</td><td>2</td><td>ND</td><td>32</td><td>bla<sub>OXA-48</sub></td><td>pmrB (HATPAse_c:R256G)</td></tr><tr><td>K. pneumoniae UR19</td><td>—</td><td>ND</td><td>ND</td><td>ND</td><td>64</td><td>AB</td><td>mgrB (C39Y)</td></tr><tr><td>A. baumannii UR86</td><td>+</td><td>ND</td><td>&gt; 32</td><td>&gt; 8</td><td>ND</td><td>bla<sub>OXA-23</sub></td><td>ND</td></tr><tr><td>A. baumannii UR57</td><td>+</td><td>ND</td><td>&gt; 32</td><td>&gt; 8</td><td>ND</td><td>bla<sub>OXA-24</sub></td><td>ND</td></tr><tr><td>A. baumannii UR59</td><td>+</td><td>ND</td><td>&gt; 32</td><td>&gt; 8</td><td>ND</td><td>bla<sub>OXA-23</sub></td><td>ND</td></tr><tr><td>A. baumannii UR56</td><td>+</td><td>ND</td><td>&gt; 32</td><td>&gt; 8</td><td>ND</td><td>bla<sub>OXA-24</sub></td><td>ND</td></tr><tr><td>A. baumannii UR105</td><td>+</td><td>ND</td><td>&gt; 32</td><td>&gt; 8</td><td>ND</td><td>bla<sub>OXA-23</sub></td><td>ND</td></tr><tr><td>A. baumannii UR69</td><td>+</td><td>ND</td><td>&gt; 32</td><td>&gt; 8</td><td>ND</td><td>bla<sub>OXA-23</sub></td><td>ND</td></tr><tr><td>A. baumannii UR90</td><td>+</td><td>ND</td><td>&gt; 32</td><td>&gt; 8</td><td>ND</td><td>bla<sub>OXA-23</sub></td><td>ND</td></tr></tbody></table>

MER = meropenem, IMP = imipenem, ETP = ertapenem, CS = colistin, MIC = minimum inhibitory concentration, ND: Not done, AB: Absent.

<table><caption>**Table 4**

Frequencies and distribution of antibiotic genes in the two polyclinics.</caption><thead><tr><th rowspan="2">Bacterial Species</th><th rowspan="2"></th><th colspan="7">Antibiotic Resistance Genes</th></tr><tr><th>bla<sub>SHV</sub></th><th>bla<sub>TEM</sub></th><th>bla<sub>CTX-A</sub></th><th>bla<sub>NDM</sub></th><th>bla<sub>OXA-48</sub></th><th>bla<sub>OXA-23</sub></th><th>bla<sub>OXA-24</sub></th></tr></thead><tbody><tr><td rowspan="10">Enterobacteriaceae</td><td>E. coli<br>(n = 52)</td><td>1</td><td>21</td><td>45</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>K. pneumoniae (n = 46)</td><td>46</td><td>19</td><td>38</td><td>9</td><td>10</td><td>0</td><td>0</td></tr><tr><td>E. cloacae<br>(n = 5)</td><td>1</td><td>1</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>K. aerogenes<br>(n = 2)</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M. morganii<br>(n = 1)</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>P. mirabilis<br>(n = 1)</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Non-fermenting bacilli</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>A. baumannii<br>(n = 8)</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>5</td><td>2</td></tr><tr><td>P. aeruginosa<br>(n = 1)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="2">Polyclinic</td><td>Polyclinic 1</td><td>19</td><td>24</td><td>38</td><td>3</td><td>5</td><td>3</td><td>0</td></tr><tr><td>Polyclinic 2</td><td>31</td><td>20</td><td>47</td><td>6</td><td>5</td><td>2</td><td>2</td></tr></tbody></table>

of all genes among different species is summarised in Table 4. None of our strains harboured *bla*<sub>CTX-B</sub> genes.

### 3.3.2. Carbapenem resistance genes

The sequences obtained showed that the *bla*<sub>OXA-48</sub> and *bla*<sub>NDM</sub> genes were detected in the 13 CREs (ten strains with *bla*<sub>OXA-48</sub> and nine strains with *bla*<sub>NDM</sub>) (Table 3). In this study, four K. pneumoniae isolates harboured *bla*<sub>OXA-48</sub> and *bla*<sub>NDM-5</sub> genes, and two K. pneumoniae isolates carrying both the *bla*<sub>NDM-1</sub> and *bla*<sub>OXA-48</sub> gene. The *bla*<sub>OXA-48</sub> gene was detected in five isolates at Polyclinic 1 and in five isolates at Polyclinic 2 (Table 4). The *bla*<sub>NDM</sub> gene was detected in three isolates at Polyclinic 1 and in six isolates at Polyclinic 2. Among the seven isolates of A. baumannii resistant to carbapenem, the *bla*<sub>OXA-23</sub> gene was found in five isolates, while the *bla*<sub>OXA-24</sub> gene was detected in two A. baumannii (Table 3). None of these strains contained the *bla*<sub>OXA-58</sub>, *bla*<sub>KPC</sub>, *bla*<sub>VIM</sub>, and *bla*<sub>IMP</sub> genes.

### 3.3.3. Colistin resistance mechanisms

In this study, none of our isolates contained the *mcr-1*, *mcr-2*, *mcr-3*, *mcr-4*, *mcr-5*, or *mcr-8* genes. Three strains of K. pneumoniae were colistin-resistant, with mutations in the *mgrB* and *pmrB* genes, and none in the *pmrA*, *phoP* and *phoQ* genes (Table 3). Comparison of the sequences obtained with reference sequences revealed false-sense mutations in the *mgrB* gene with the replacement of cysteine by tyrosine at position 39 (C39Y) and of leucine by arginine at position 9 (L9R) for strains UR19 and UR88, respectively. Strain UR74 presented a mutation (R256G) in the *pmrB* gene specifically in the HATPase C domain (250–358). These mutations were found in isolates from Polyclinic 2 (Table 4). Additionally, two of these strains (UR88 and UR74) were both resistant to colistin and producers of the *bla*<sub>OXA-48</sub> gene (Table 3).
## 4. Discussion

UTIs are considered to be a serious public health problem worldwide and have a significant impact on the quality of life of those affected (Flores-Mireles et al., 2015). Currently, antibiotics such as trimethoprim-sulfamethoxazole, ampicillin, and ciprofloxacin are the most commonly recommended medications for the treatment of UTIs (Flores-Mireles et al., 2015). However, the burden that these common infections place on society could be significantly increased due to increasing rates of antibiotic resistance and high rates of recurrence (Mancuso et al., 2023). Monitoring antimicrobial resistance prevalence is therefore crucial for adapting and improving treatment strategies based on epidemiological data.

Our study focused on various strains, isolated from urine samples of hospitalized patients, that exhibited resistance to C3G, imipenem, and/or colistin. Antibiotic sensitivity testing revealed a high rate of antimicrobial resistance, with 2.2 % of the isolated bacteria classified as multidrug-resistant strains. In southwest Tunisia, a 2016 study at a Gafsa hospital reported that 3.6 % of *Enterobacteriaceae* causing UTIs were ESBL producers (Guermazi-Toumi et al., 2018). However, in the governorate of Sfax this prevalence had risen from 1.6 % to 10.2 % between 2004 and 2015 (Smaoui et al., 2015). In France, 6.7 % of UTIs are attributed to ESBL-producing *Enterobacteriaceae*, whereas these strains can account for up to 40 % of *Enterobacteriaceae* isolates in some countries, such as the United States (Farfour et al., 2022). Among *Enterobacteriaceae*, resistance to fourth-generation cephalosporins accounted for 64.1 % in polyclinic 1 and 80.9 % in polyclinic 2. The rate of antibiotic resistance is relatively similar between the two polyclinics. ESBL genes, such as *bla*<sub>TEM</sub> and/or *bla*<sub>SHV</sub>, were found in 44 and 50 isolates, respectively, while *bla*<sub>CTX-A</sub> genes were detected in 85 isolates. Furthermore, a previous study conducted within a hospital in southwestern Tunisia on *Enterobacteriaceae* isolated from urine samples showed that the CTX-M-1 group and the CTX-M-9 group were detected in 53.6 % and 3.6 %, respectively, of C3G-resistant *Enterobacteriaceae* isolates (Smaoui et al., 2015). In congruence with other reports, our study confirms that the CTX-M-1 group is the most dominant CTX-M group among bacteria causing UTIs in Tunisia, particularly in *E. coli* and *K. pneumoniae* isolates (Farfour et al., 2022).

ESBL-producing bacteria often harbor additional resistance mechanisms to other antibiotics, thereby limiting treatment options for infections caused by these organisms. Carbapenems are typically the antibiotics of choice for treating infections caused by these multidrug-resistant strains. However, the increased use of carbapenems has led to the emergence and global spread of carbapenemase-producing bacteria (Dziri et al., 2020). In this study, we detected the presence of the *bla*<sub>OXA-48</sub> gene in ten carbapenem-resistant *K. pneumoniae* strains. In Tunisia, carbapenem resistance in *K. pneumoniae* was mainly mediated by the *bla*<sub>OXA-48</sub> gene (Ben Hassena et al., 2022). Between 2014 and 2016, a study carried out at a military hospital in Tunis showed that out of 102 *Enterobacteriaceae* resistant to carbapenems, 84 isolates produced the *bla*<sub>OXA-48</sub> gene (Navon-Venezia et al., 2017). Sallemi et al. also reported that the carbapenemase OXA-48 is the most frequently reported enzyme in strains isolated from the intestines of food handlers in Sfax (Grami et al., 2016). These findings raise concerns about the asymptomatic carriage of this pathogen not only in hospital environments but also in the community, which facilitates the spread of antibiotic resistance genes.

We also showed that nine CREs harboured the *bla*<sub>NDM</sub> gene, including eight *K. pneumoniae* isolate and one *E. cloacae* isolate. The *bla*<sub>NDM-1</sub> gene was detected in carbapenem-resistant *K. pneumoniae* from a sternal pus sample of a 73-year-old Libyan patient who was admitted to the intensive care unit of a hospital in Tunis (Ben Helal et al., 2018). In 2018, the *bla*<sub>NDM-1</sub> gene was detected for the first time in Tunisia, in a strain isolated from a urine sample originating from a community patient (Sallem et al., 2022). This study points to a carbapenem resistance within *Enterobacteriaceae* that is attributed to the presence of the *bla*<sub>OXA-48</sub> and

*bla*<sub>NDM</sub> genes, consistent with existing literature. In Africa between 2000 and 2022, the most frequently detected carbapenemases were NDM (43.1 %) and OXA-48-like (42.9 %) (Ben Nasr et al., 2013). It should be noted that two strains of *K. pneumoniae* were positive for the *bla*<sub>NDM-1</sub> gene and the *bla*<sub>OXA-48</sub> gene. This coexistence has already been reported in a carbapenem-resistant *K. pneumoniae* detected in central Tunisia (Mhaya et al., 2020b), although to our knowledge, the co-occurrence of the *bla*<sub>OXA-48</sub> and *bla*<sub>NDM-5</sub> genes was first observed in Tunisia in our study. In our study, carbapenemase *bla*<sub>OXA-23</sub> and *bla*<sub>OXA-24</sub> were identified in five and two carbapenem-resistant *A. baumannii*, respectively. In Tunisia, the *bla*<sub>OXA-23</sub> gene was detected for the first time in 2008 in a hospital environment in carbapenem-resistant *A. baumannii* isolates (Kedisaletse et al., 2023). The OXA-23 enzyme has been considered to be the main mechanism of carbapenem resistance in *A. baumannii* in Tunisia as well as in other North African countries (Kindu et al., 2020). According to this review, in Africa, the most frequently detected carbapenemases in *A. baumannii* were the *bla*<sub>OXA-23</sub> gene, and the enzymes IMP, NDM and OXA-24 were also identified (Kindu et al., 2020). Although several studies in Tunisia have screened for *bla*<sub>OXA-24</sub>, none of them have reported the detection of this gene. (Cheikh et al., 2018).

In Tunisia, beyond carbapenem resistance, epidemiological studies have documented the spread of colistin-resistant Gram-negative bacteria from diverse sources, including humans, animals, natural environments, and food products (Dziri et al., 2021). Sequencing analysis showed that the *K. pneumoniae* strains UR19 and UR88 showed a mutation in the *mgrB* gene, leading to amino acid substitutions (C39Y and L9R), which are linked to reduced susceptibility to colistin (Nordmann et al., 2016). C39 plays an essential role in the function and structural stability of the MgrB protein, particularly in its inhibition of the PhoQ/PhoP signaling pathway. Mutations at C39 are associated with colistin-resistant *Klebsiella* isolates, further highlighting its crucial functional role (Yadavalli et al., 2020). Haeili et al. also demonstrated, using complementation assays, that a substitution at C39stop in *mgrB* contributes to colistin resistance in *K. pneumoniae* isolates. (Haeili et al., 2017). Our results also showed that the *K. pneumoniae* strain (UR19) has a mutation in the *mgrB* gene, resulting in amino acid substitutions (L9R). Olaitan et al. reported that this mutation leads to inactivation of *mgrB* gene by creating a premature stop codon at position 9 (L9), based on whole-genome sequencing comparison of a colistin-sensitive *K. pneumoniae* strain and an in vitro-selected colistin-resistant clone of the same strain (Olaitan et al., 2014). MgrB alterations play a major role in colistin resistance in *K. pneumoniae* isolates (Haeili et al., 2017). This resistance has been reported worldwide, including in African countries such as Algeria, Libya and Burkina Faso (Hassen et al., 2022). The *pmrB* (R256G) mutation gene was detected in a strain of *K. pneumoniae* (UR74), although this mechanism of colistin resistance has not been clarified. This mutation was reported by Cheng et al. and Haeili et al. who suggest that this alteration alone may not contribute to colistin resistance (Haeili et al., 2017) (Cheng et al., 2015). In our study, two *K. pneumoniae* strains resistant to colistin (alteration of the *mgrB* gene) were also found to produce ESBL and OXA-48. In 2016, the same pattern was observed in Algeria, when strains of *K. pneumoniae* producing the *bla*<sub>CTX-M-15</sub>, *bla*<sub>TEM-183</sub>, *bla*<sub>SHV-106</sub> and *bla*<sub>OXA-48</sub> genes and resistant to colistin were isolated from urine samples (Yousfi et al., 2019). In northern Tunisia, a study carried out in 2022 in Tunis reported the detection of a *K. pneumoniae* isolate from a colistin-resistant urine sample following alteration of *mgrB* (G37S) and co-producer of *bla*<sub>OXA-48</sub>, *bla*<sub>NDM-1</sub> and *bla*<sub>CTX-M-15</sub> (Ben Sallem et al., 2022). To date, the frequency of colistin resistance in clinical isolates has been under-estimated. In the past year, Tunisia and other North African countries including Egypt and Algeria (Yousfi et al., 2019; Sadek et al., 2022), as well as certain European countries, have faced the emergence of pathogenic bacteria resistant to colistin (Wang et al., 2020; D'Onofrio et al., 2020). Our study has limitations due to the absence of a complete genomic analysis of the isolated strains, to characterize the complete resistome and determine the sequence types, which would have provided a better understanding of the clonal
structure and propagation of bacterial populations in Tunisia. Further research is urgently needed to better understand the prevalence and spread of antibiotic-resistant bacteria, and effective measures must be implemented to control their spread.

## 5. Conclusion

Characterisation of the resistance mechanisms of bacteria isolated from urinary samples in the Sfax region confirmed the presence of ESBL-producing *Enterobacteriaceae* and the persistence of carbapenemase genes such as OXA-48 and NDM in the Tunisian population. The presence of *K. pneumoniae* strains resistant to β-lactams, carbapenems, and colistin is a worrying phenomenon that must be monitored. Regular surveillance studies are needed to stem the increasing spread of pathogens and prevent these strains from becoming endemic in Tunisia. At the same time, strict infection prevention and control measures must be implemented.

## CRediT authorship contribution statement

**Mariem Yengui:** Writing – review & editing; Writing – original draft, Visualization, Methodology, Formal analysis, Data curation, Conceptualization. **Rahma Trabelsi:** Writing – original draft, Validation, Supervision, Project administration, Investigation, Conceptualization. **Radhouane Gdoura:** Writing – original draft, Supervision, Funding acquisition, Conceptualization. **Aïcha Hamieh:** Writing – original draft, Methodology, Formal analysis. **Hanane Zerrouki:** Writing – original draft, Supervision, Methodology, Formal analysis, Data curation. **Jean-Marc Rolain:** Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization. **Linda Hadjadj:** Writing – review & editing; Writing – original draft, Validation, Supervision, Methodology, Data curation, Conceptualization.

## Institutional review board statement

This study was approved by the CPP SUD of the Tunisian Ministry of Health (CPP SUD No. 0456/2022).

## Funding

This work was supported by the Sfax University, in collaboration with the French Government under the "Investissements d'avenir" programme (Investments for the Future) managed by the Agence Nationale de la Recherche (ANR, fr: National Agency for Research), (Méditerranée Infection 10-IAHU-03).

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We thank the pharmacist-biologists at the two polyclinics for their help in collecting the samples.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.meegid.2025.105787.

## Data availability

Data will be made available on request.

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