# Colistin resistance in ESBL- and Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Cambodia

Mallorie Hide, Soda Meng, Sokleaph Cheng, Anne-Laure Bañuls, Santy Ky, Chantana Yay, Denis Laurent, Gauthier Delvallez

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Mallorie Hide, Soda Meng, Sokleaph Cheng, Anne-Laure Bañuls, Santy Ky, et al.. Colistin resistance in ESBL- and Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Cambodia. Journal of Global Antimicrobial Resistance, 2024, 38, pp.236-244. 10.1016/j.jgar.2024.06.017. hal-04675726

HAL Id: hal-04675726

https://hal.science/hal-04675726v1

Submitted on 5 Feb 2025

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<figure><img src="image_1.png" /></figure>

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permeability. It has been shown that colistin increases permeability, not only by altering the bilayer structure but also by acting on membrane fluidity, having an effect on the reduction of efflux pump activities [2].

From a mechanistic point of view, the resistance to colistin could be due to both *mcr* genes and the two-component system (TCS) modifications. The dissemination of colistin resistance has received increased attention since a mobilized gene conferring resistance to colistin, *mcr-1*, was described in *Enterobacteriaceae* from humans and food-producing animals in China in 2015 [4]. Additional gene homologs have since been identified (*mcr-1* to *mcr-10*) [5-7]. The *mcr* genes encode phosphoethanolamine (PEtN) transferase enzyme that adds PEtN to lipid A, and consequently results in a more cationic lipopolysaccharide (LPS). Through horizontal and vertical transfers, *mcr* genes have spread widely into diverse environmental niches. These ecosystems constitute underestimated vast reservoirs ("phantom resistome") of these *mcr* genes [5]. In addition, various chromosomal mutations associated with colistin resistance leading to modifications in PmrA/PmrB and PhoQ/PhoP proteins (TCS), and the inactivation of the MgrB protein decreases the affinity of colistin for the LPS [8]. The 144-bp *mgrB* gene encodes a small transmembrane protein that exerts negative feedback on the above-mentioned pathway by interaction with the sensor kinase PhoQ at the periplasmic level [9].

Worldwide, colistin is still widely used in livestock as treatment, metaphylaxis, prophylaxis, and growth promotion [10] and the indiscriminate use of colistin has led to the development of resistance. In Cambodia, the circulation of colistin resistance in the human population is still poorly studied and colistin is (i) one of the most commonly used antibiotics in small-scale urban pig farms and (ii) in poultry farms in a packaged amoxicillin-colistin mixture for respiratory and gastrointestinal infections [11-14]. From a mechanistic point of view, up to now, few mechanisms

of colistin resistance were reported in Cambodia, only *mcr-1* and *mcr-3* have been identified from MDR *Escherichia coli* (*E. coli*) and *Klebsiella pneumoniae* (*K. pneumoniae*) isolated from humans, poultry, and pigs, and no data are available concerning TCS [14,15].

In this context, we aim to fill a knowledge gap by exploring colistin resistance and the molecular determinants contributing to colistin resistance among a collection of Extended Spectrum beta-lactamase (ESBL) and Carbapenemase producing (CP) *E. coli* and *K. pneumoniae* isolates from the Medical Biology Laboratory of the Institut Pasteur du Cambodge (MBL-IPC), Cambodia between 2016 and 2020.

## 2. Materials and methods

### 2.1. Bacterial isolates

A panel of 262 ESBL and/or CP isolates of *E. coli* and *K. pneumoniae* was isolated at the MBL-IPC from patients who presented at various public and private health care facilities in Cambodia. ESBL-*E. coli* (*n* = 119), ESBL-*K. pneumoniae* (*n* = 23), CP-*E. coli* (*n* = 104), and CP-*K. pneumoniae* (*n* = 16) isolates represent all the ESBL-*E. coli*/*K. pneumoniae* strains isolated at MBL-IPC in 2020, and all the CP-*E. coli*/*K. pneumoniae* strains isolated in 2016 to 2020 (Fig. 1). The isolates were grown from various sample types including urine, blood culture, sputum, and pus and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Bruker Daltonics). Phenotypic Antibiotic Susceptibility Testing was performed by the disc diffusion method in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for the following antibiotics (Bio-Rad): amoxicillin (20 µg), amoxicillin-clavulanate (20-10 µg), ticarcillin (75 µg), mecillinam (10 µg), cefotaxime (5 µg), ceftazidime (10 µg),

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Samples collection and experimental design. All the ESBL-E and CPE isolates pertaining to *E. coli* (*n* = 223) or *K. pneumoniae* (*n* = 39) species were submitted to colistin susceptibility testing using MIC and DNA of the 18 colistin resistant isolates were tested for both *mcr* variants and TCS genes. Abbreviations: MBL-IPC, Medical Biology Laboratory of the Institut Pasteur du Cambodge; ESBL-E, Extended Spectrum beta-lactamase producing *Enterobacteriaceae*; CPE, carbapenemase producing *Enterobacteriaceae*; EC, *Escherichia coli*; KP, *Klebsiella pneumoniae*; MIC, minimum inhibitory concentration; *mcr*, mobile colistin resistance; TCS, two-component system.</figcaption></figure>
imipenem (10 µg), ertapenem (10 µg), amikacin (30 µg), gentamycin (10 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), co-trimoxazole 1,25-23,75 µg), nitrofurantoin (100 µg), and fosfomycin (200 µg) [16]. In case of resistance to cefotaxime and/or ceftazidime, both of them pertaining to third-generation cephalosporin (3GC), a second-line antibiotic panel was performed including ticarcillin-clavulanate (75-10 µg), piperacillin-tazobactam (30-6 µg), aztreonam (30 µg), cefepime (30 µg), and tigecycline (15 µg) (Bio-Rad). The phenotypic detection of ESBL was performed by double-disc synergy test: discs containing 3GC (including cefotaxime 5 µg and ceftazidime 10 µg,) and cefepime 30 µg were applied to Mueller-Hinton plates 30 mm next to an amoxicillin-clavulanic acid disc (20-10 µg). A positive result is indicated when the inhibition zones around any of the cephalosporin discs are augmented in the direction of the disc containing clavulanic acid. The same protocol is applied on Mueller-Hinton agar supplemented with 250 mg/L cloxacillin (cephalosporinase inhibitor) to facilitate the detection of ESBL in case of hyper-production of cephalosporinase. All the strains resistant to ertapenem and/or imipenem were previously tested by polymerase chain reaction (PCR) for the presence of genes encoding for carbapenemases (OXA-48-like, NDM, and KPC; Table 1) [17]. All isolates were maintained for prolonged storage at -80 °C in Difco skim milk (Becton Dickinson) medium and glycerol and subcultured for colistin susceptibility testing and DNA extraction.

## 2.2. Colistin susceptibility testing

The minimum inhibitory concentration (MIC) of colistin was determined using broth microdilution according to Clinical Laboratories Standards Institute guidelines [18]. Colistin sulphate powder from Sigma-Aldrich was resuspended at 100 mg/mL and bacterial inoculums were grown at $5 \times 10^5$ CFU/mL in Mueller-Hinton broth 2 (Sigma, reference item 90922) for 24 h with different colistin concentrations (ranging from 0 to 0.25, 0.5, 1, 2, 4, 8, 16 and 32 µg/mL) [19,20]. Bacterial growth was determined by measuring the optical density of the cultures at 600 nm in Multiskan FC Microplate Photometer (ThermoFisher). *Pseudomonas aeruginosa* ATCC 27853 and *E. coli* ATCC 25922 were used as controls.

## 2.3. Molecular determinants

To identify the mechanisms of resistance, chromosomal mutations on *mgrB*, *crrB* (only for *K. pneumoniae*), *pmrA*, *pmrB*, *pmrD* (only for *E. coli*), *phoQ*, and *phoP* genes (TCS), and the presence of *mcr* genes, *mcr-1* to *mcr-9* were investigated by PCR and sequencing for isolates exhibiting MICs superior or equal to the epidemiological cutoff values of 4 µg/mL for colistin. DNA extraction was performed on bacterial colonies by the direct boiling method. The colony was diluted in 100 microliters of distilled water in a microtube, incubated at 95 °C for 10 min in a water bath and centrifuged at 10 000 g for 10 min. The DNA was collected by pipetting the supernatant. DNA concentration was estimated by spectrophotometry using a NanoDrop™ 2000 (Thermo Scientific) and stored at -20 °C. *mcr-1* to *mcr-5* were amplified according to Rebelo at al. (2018) and *mcr-6* to *mcr-9* according to Borowiak et al. (2020) [7,21]. Concerning chromosome-mediated colistin resistance (*pmrA*, *pmrB*, *phoP*, *phoQ*, *mgrB*, *pmrD*, and *crrB*), genes were amplified in simplex PCR using the primers described in the Supplementary File S1 [22-25]. Positive PCR obtained in this study were used as positive controls PCR for all the amplifications. PCR products of *mcr* genes and TCS genes were sent to Macrogen, Korea, for capillary electrophoresis sequencing in order to identify *mcr* variants and modifications on the TCS. Sequence data were analysed using MegaX software and submitted to the GenBank database [26].

## 2.4. Statistical analysis

Comparison of categorical variables was performed using a Chi-squared test. A *P* value < 0.05 was considered significant.

# 3. Results

Among the 262 isolates, 223 were identified as *E. coli*, whereas 39 were identified as *K. pneumoniae*. There were 44.4% of the patients who were men, and the average age was 36 years old (ranging from 0 to 104 y). Patients came from 21 different provinces out of a total of 25 (Phnom Penh being the most represented [46.8%]), and presented at 20 different health care facilities. There were 71.4% of the strains that were isolated from urine samples, and 48.7% of the patients have received antibiotic therapy in the last 3 months, including 30.8% of them receiving 3GC and 15.4% receiving carbapenems, whereas 13.5% of them reported travel outside of Cambodia in the last 6 months.

## 3.1. Colistin susceptibility testing

Among the ESBL isolates, 7 isolates of *E. coli* (5.9%) and 8 isolates of *K. pneumoniae* (34.8%) had an MIC superior or equal to 4 µg/mL for colistin, whereas in the CP isolates, colistin-resistance represents 1.0% (n = 1, *bla*<sub>OXA48</sub>) for *E. coli* and 12.5% (n = 2, *bla*<sub>NDM</sub>) for *K. pneumoniae* (Table 2). In total, 18 isolates showed a colistin-resistant phenotype (8 *E. coli* and 10 *K. pneumoniae*), mainly associated with the ESBL phenotype (*P* = 0.02) and the *K. pneumoniae* species (*P* < 0.001). MICs of colistin within our samples ranged from 4 to > 32 µg/mL with higher MICs for *K. pneumoniae* compared to *E. coli*.

## 3.2. Characteristics of colistin-resistant isolates

The 18 colistin-resistant isolates were collected within 4 years (from 2017 to 2020) from patients aged 1 y to 73 y and living in 10 provinces (Table 1). Regarding antibiotic treatment, information was available for 15 of the 18 resistant isolates, and 9 patients (60%) had received antibiotic treatment prior to bacterial isolation. Unfortunately, the specific antibiotic classes and names were not available. Concerning travel history, only 1 of the 15 patients (6.7%) reported traveling outside of Cambodia in the last 6 months. Travel information was missing for three patients. Among the colistin-resistant isolates, amikacin was the only antibiotic that remained constantly active on both *E. coli* and *K. pneumoniae*, whereas tigecycline, fosfomycin, and nitrofurantoin remained active on *E. coli* (ECAST breakpoints for *K. pneumoniae* are currently not available for these 3 drugs; Table 1). Concerning beta-lactam antibiotics among ESBL isolates, only amoxicillin - clavulanate and mecillinam remained inconsistently active (46.7% of resistance for amoxicillin - clavulanate, 36.4% for mecillinam). All the colistin-resistant isolates were also resistant to cefepime, aztreonam (except one CP-*E. coli*) and trimethoprim/sulfamethoxazole.

## 3.3. Molecular determinants

We identified molecular determinants associated with colistin-resistance among the 18 isolates (Table 3). All sequences have been submitted to the GenBank database, and the accession numbers are listed in Supplementary File S2. Our results allowed identifying *mcr*-like genes for 7 among 8 *E. coli* (87.5%) resistant isolates and 6 among 10 *K. pneumoniae* (60%) with the presence of *mcr-1* (*E. coli* + *K. pneumoniae*), *mcr-3* (*E. coli*), and *mcr-8.2* (*E. coli* + *K. pneumoniae*) variants. Among the 6 *mcr*-like genes detected for *K. pneumoniae*, we have been unable to identify 2 of them (ESBL-KP4 and ESBL-KP5) because the sequencing of their multiplex *mcr-6/9*
<table><caption>**Table 1**

Epidemiological data and antibiotic susceptibility testing of the 18 colistin resistant isolates.</caption><thead><tr><th>Isolate</th><th>MIC µg/mL</th><th>Year</th><th>Sample</th><th>Sex</th><th>Age</th><th>Residence</th><th>Clinic</th><th>Health centre</th><th>Bacterial species</th><th>RM</th><th>AMX</th><th>AMC</th><th>TIC</th><th>TCC</th><th>PTZ</th><th>MEC</th><th>CTZ</th><th>CTX</th><th>FEP</th><th>ATM</th><th>IPM</th><th>ETP</th><th>AN</th><th>GM</th><th>CIP</th><th>LVX</th><th>SXT</th><th>NIF</th><th>FOS</th><th>TGC</th></tr></thead><tbody><tr><td>ESBL-KP8</td><td>16</td><td>2020</td><td>Urine</td><td>M</td><td>8 y</td><td>Kandal</td><td>UTI</td><td>2</td><td>KP</td><td>ESBL</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>ESBL-KP7</td><td>16</td><td>2020</td><td>Urine</td><td>M</td><td>13 y</td><td>Pursat</td><td>Leukaemia</td><td>2</td><td>KP</td><td>ESBL</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>ESBL-KP6</td><td>8</td><td>2020</td><td>Urine</td><td>M</td><td>10 y</td><td>Kampot</td><td>Leukaemia</td><td>2</td><td>KP</td><td>ESBL</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>ESBL-KP5</td><td>4</td><td>2020</td><td>Urine</td><td>M</td><td>10 y</td><td>Kampot</td><td>Leukaemia</td><td>2</td><td>KP</td><td>ESBL</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>ESBL-KP4</td><td>8</td><td>2020</td><td>Urine</td><td>M</td><td>1 y</td><td>Kampong Speu</td><td>UTI</td><td>2</td><td>KP</td><td>ESBL</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>ESBL-KP3</td><td>&gt;32</td><td>2020</td><td>Urine</td><td>F</td><td>1 y</td><td>Siem Reap</td><td>Hydronephrosis</td><td>3</td><td>KP</td><td>ESBL</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>S</td><td>S</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>ESBL-KP2</td><td>&gt;32</td><td>2020</td><td>Sputum</td><td>M</td><td>48 y</td><td>Kampong Cham</td><td>Pneumonia</td><td>1</td><td>KP</td><td>ESBL</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>ND<sup>1</sup></td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>ESBL-KP1</td><td>8</td><td>2020</td><td>Sputum</td><td>F</td><td>48 y</td><td>Phnom Penh</td><td>Pneumonia</td><td>1</td><td>KP</td><td>ESBL</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>ND<sup>1</sup></td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>ESBL-EC7</td><td>16</td><td>2020</td><td>Urine</td><td>M</td><td>4</td><td>Phnom Penh</td><td>Encephalopathy</td><td>2</td><td>EC</td><td>ESBL</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td></tr><tr><td>ESBL-EC6</td><td>8</td><td>2020</td><td>Urine</td><td>F</td><td>1</td><td>Prey Veng</td><td>Icterus</td><td>2</td><td>EC</td><td>ESBL</td><td>R</td><td>S</td><td>R</td><td>S</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td></tr><tr><td>ESBL-EC5</td><td>8</td><td>2020</td><td>Urine</td><td>F</td><td>1 y</td><td>Prey Veng</td><td>Teratoma</td><td>2</td><td>EC</td><td>ESBL</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td></tr><tr><td>ESBL-EC4</td><td>4</td><td>2020</td><td>Urine</td><td>F</td><td>2 y</td><td>Kampong Thom</td><td>UTI</td><td>3</td><td>EC</td><td>ESBL</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td></tr><tr><td>ESBL-EC3</td><td>8</td><td>2020</td><td>Urine</td><td>F</td><td>18 y</td><td>Siem Reap</td><td>UTI</td><td>3</td><td>EC</td><td>ESBL</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td></tr><tr><td>ESBL-EC2</td><td>4</td><td>2020</td><td>Blood</td><td>F</td><td>42 y</td><td>Phnom Penh</td><td>Sepsis</td><td>1</td><td>EC</td><td>ESBL</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>ND<sup>1</sup></td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>ND<sup>1</sup></td><td>S</td><td>S</td></tr><tr><td>ESBL-EC1</td><td>8</td><td>2020</td><td>Pus</td><td>F</td><td>68 y</td><td>Phnom Penh</td><td>Abscess</td><td>1</td><td>EC</td><td>ESBL</td><td>R</td><td>S</td><td>R</td><td>R</td><td>S</td><td>ND<sup>1</sup></td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>R</td><td>ND<sup>1</sup></td><td>S</td><td>S</td></tr><tr><td>CP-KP2</td><td>8</td><td>2020</td><td>Urine</td><td>F</td><td>4 y</td><td>Svay Rieng</td><td>Acute renal failure</td><td>2</td><td>KP</td><td>CPE (NDM)</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>CP-KP1</td><td>&gt;32</td><td>2017</td><td>Urine</td><td>M</td><td>73 y</td><td>Phnom Penh</td><td>UTI</td><td>1</td><td>KP</td><td>CPE (NDM)</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>R</td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td><td>NA<sup>2</sup></td></tr><tr><td>CP-EC1</td><td>8</td><td>2017</td><td>Urine</td><td>F</td><td>36 y</td><td>Phnom Penh</td><td>UTI</td><td>1</td><td>EC</td><td>CPE (OXA-48)</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>R</td><td>S</td><td>R</td><td>R</td><td>R</td><td>S</td><td>S</td><td>ND</td></tr></tbody></table>

Abbreviations: AMX, amoxicillin; AMC, amoxicillin-clavulanic acid; AN, amikacin; ATM, aztreonam; CIP, ciprofloxacin; CP, carbapenemase producing *Enterobacteriaceae*; CTX, cefotaxime; CTZ, ceftazidime; EC, *Escherichia coli*; ESBL, extended-spectrum beta-lactamase; ETP, ertapenem; F, female; FEP, cefepime; FOS, fosfomycin; GM, gentamicin; IPM, imipenem; KP, *Klebsiella pneumoniae*; LVX, levofloxacin; M, male; MEC, mecillinam; MIC, minimum inhibitory concentration; NA, not applicable; ND, no data; NIF, nitrofurantoin; PTZ, piperacillin-tazobactam; R, resistant; RM, resistance mechanism; S, susceptible; SXT, trimethoprim-sulfamethoxazole; TCC, ticarcillin-clavulanic acid; TIC, ticarcillin; TGC, tigecycline; UTI, urinary tract infection.

ND¹, mecillinam, and nitrofurantoin have only been tested on strains isolated from urine samples.

NA², TGC, NIF, and FOS EUCAST breakpoints for *Klebsiella pneumoniae* are currently not available.
<table><caption>**Table 2**
Rates of colistin resistance within 262 ESBL and CPE isolated at Institut Pasteur du Cambodge (Medical Biology Laboratory).</caption><thead><tr><th rowspan="2"></th><th colspan="2">Escherichia coli (n = 223)</th><th colspan="2">Klebsiella pneumoniae (n = 39)</th><th rowspan="2">Total resistance (n = 262)</th></tr><tr><th>Susceptible</th><th>Resistant</th><th>Susceptible</th><th>Resistant</th></tr></thead><tbody><tr><td>ESBL (n = 142)</td><td>112</td><td>7</td><td>15</td><td>8</td><td>15</td></tr><tr><td>%</td><td>94.1</td><td>5.9</td><td>65.2</td><td>34.8</td><td>10.6</td></tr><tr><td>CPE (n = 120)</td><td>103</td><td>1</td><td>14</td><td>2</td><td>3</td></tr><tr><td>%</td><td>99.0</td><td>1.0</td><td>87.5</td><td>12.5</td><td>2.5</td></tr><tr><td>Total (n = 262)</td><td>215</td><td>8</td><td>29</td><td>10</td><td>18</td></tr><tr><td>%</td><td>96.4</td><td>3.6</td><td>74.4</td><td>25.6</td><td>6.9</td></tr></tbody></table>

Abbreviations: CPE, carbapenemase producing Enterobacteriaceae; ESBL, Extended Spectrum beta-lactamase.

products gave a mixed sequence making it impossible to analyse [7].

Investigations on TCS modifications revealed several chromosome-mediated colistin resistances (Table 3). For *E. coli*, six among eight colistin-resistant isolates carried non-synonymous mutations within five among the six studied loci already described in previous studies, leading to amino acid alterations in MgrB, PmrA, PmrB, PmrD, and PhoQ [24,27-31]. The two remaining isolates were only *mcr-1* (ESBL-EC6) and *mcr-8.2* (ESBL-EC7) positive. In addition, we found four novel non-synonymous mutations leading to amino acid alterations in PhoP (I47V), PhoQ (N352K), PmrB (G19R), and PmrD (G85R) that might potentially be related to colistin resistance of *E. coli*. For *K. pneumoniae*, all the colistin-resistant isolates (n = 10) revealed insertion or non-synonymous mutations leading to amino acid alterations already described in previous studies within MgrB, PmrA, PmrB, and CrrB but none within *phoP* and *phoQ* [25,32-39]. It is important to note the co-occurrence of both *mcr* genes and colistin resistance conferring TCS mutations in 5 of 8 *E. coli* and 6 of 10 *K. pneumoniae*.

## 4. Discussion

The aim of this study was to explore the colistin resistance within ESBL- and carbapenemase-producing *E. coli* and *K. pneumoniae* isolated at the Medical Biology Laboratory of the Institut Pasteur du Cambodge (MBL-IPC), Cambodia, between 2016 and 2020 and to characterize the molecular basis of colistin resistance by exploring both *mcr* variants and TCS genes. Among 262 *E. coli* and *K. pneumoniae* isolates, 18 (6.87%) were colistin-resistant, 13 of which carried *mcr*-like genes. Even though some isolates may not be clinically relevant, as they were obtained from the urine of patients hospitalized for non-infectious reasons, it is noteworthy that these enterobacteria are being carried, sometimes by very young patients.

### 4.1. Frequency of colistin-resistant isolates

Based on the available patient data, resistant isolates are distributed across various Cambodian provinces and cannot be linked to specific antibiotic treatments or travel activities. Additionally, information regarding patient occupation and specific locations are not available. In the ESBL isolates, 5.9% of *E. coli* and 34.8% of *K. pneumoniae* had a MIC superior or equal to 4 µg/mL for colistin, whereas in the carbapenemase producing Enterobacteriaceae (CPE), colistin-resistance represents 1% for *E. coli* and 12.5% for *K. pneumoniae*. We detected one CPE-*E. coli* (*bla*<sub>OXA48</sub>) and 2 CPE-*K. pneumoniae* (*bla*<sub>NDM</sub>) (Table 2) resistant to colistin representing an emerging threat for clinical management and infection control. Colistin resistance in carbapenem-resistant Enterobacterales has been showed in Southeast Asia [39]. Other authors showed that, for *E. coli*, the resistance rate is lower than for *K. pneumoniae* and the most common carbapenemase was NDM metallo-β-lactamases and OXA β-lactamases (OXA-48) [14]. The predom-

inance of *K. pneumoniae* colistin resistance in our Cambodian samples compared to *E. coli* colistin resistance is in agreement with studies in other countries, such as China, Iran, and India [40-42]. According to Atterby et al., *E. coli* and *K. pneumoniae* harbouring ESBL genes are common in rural Cambodian communities, especially in women, small children, poultry, and pigs [43]. In their review focused on Southeast Asia, Malchione et al., showed that the rate of carbapenem and colistin resistance among *E. coli* is low in Cambodia (< 1%) whereas no estimation could have been made for *K. pneumoniae* due to lack of data in this country [14]. Our study has helped to fill the gap and revealed that, in our sample, colistin resistance was ca. 3.6% among *E. coli* and 25.6% among *K. pneumoniae*. In India, authors estimated, respectively, 3.5% and 18.5% were the percentages of colistin-resistant *E. coli* and *K. pneumoniae* within MDR samples [41].

### 4.2. Susceptibility profiles

Amikacin is the only antibiotic for which both *K. pneumoniae* and *E. coli* colistin-resistant isolates remained fully susceptible. *E. coli* colistin-resistant isolates remained fully susceptible to tigecycline, fosfomycin, and nitrofurantoin. However, the use of these antibiotics is limited as current guidelines recommend that amikacin should be used in combination with other antibiotics for severe infections [44], whereas nitrofurantoin can be used in monotherapy for the treatment of resistant bacteria but limited to uncomplicated lower urinary tract infections [45]. Fosfomycin is an effective antibiotic and is recommended by intravenous use to treat severe infections when other antibiotic treatments are not suitable, and by oral use for uncomplicated urinary tract infections. However, its use to treat severe infections should be limited in order to delay the development of resistance [44,45].

### 4.3. Molecular determinants

All the 18 of 262 Enterobacteriaceae resistant to colistin harboured chromosomal mutations in the TCS and/or *mcr* genes (*mcr-1* and its variants). The co-occurrence of both *mcr* genes and mutations in the TCS observed in 11 out of 18 colistin-resistant isolates aligns with the findings of Premnath et al. [46]. They reported the presence of *mcr* genes alongside alterations in *mgrB* in colistin-resistant enterobacteria isolated from chicken meat in India.

#### 4.3.1. *mcr* variants

We identified 13 *mcr* among the 18 colistin-resistant isolates, corresponding to variants *mcr-1*, *mcr-3*, and *mcr-8.2*. The localization of *mcr* variants on plasmid and/or chromosome will be determined in a further study. Despite sparse data, polymyxin resistance was documented in 8 of 11 Southeast Asian nations, with *mcr-1* being the predominant genotype (including Cambodia) [14]. From a mechanistic point of view, according to these authors, the frequency of colistin resistance in Cambodia is low and only *mcr-1* and *mcr-3* have been identified before our study. In Cambodia, the
<table><caption>**Table 3**
Mcr variants and mutations in TCS genes leading to amino acid changes in the 18 colistin resistant isolates.</caption><thead><tr><th rowspan="2">Isolate</th><th rowspan="2">Year</th><th rowspan="2">Resistance</th><th rowspan="2">Species</th><th rowspan="2">MIC µg/mL</th><th colspan="2">mcr</th><th colspan="2">MgrB</th><th colspan="2">PhoP</th><th colspan="2">PhoQ</th><th colspan="2">PmrA</th><th colspan="2">PmrB</th><th colspan="2">PmrD</th><th colspan="2">CrrB</th></tr><tr><th>Variant</th><th>Ref</th><th>Mutation</th><th>Ref</th><th>Mutation</th><th>Ref</th><th>Mutation</th><th>Ref</th><th>Mutation</th><th>Ref</th><th>Mutation</th><th>Ref</th><th>Mutation</th><th>Ref</th><th>Mutation</th><th>Ref</th><th>Mutation</th><th>Ref</th></tr></thead><tbody><tr><td>ESBL-KP8</td><td>2020</td><td>ESBL</td><td>KP</td><td>16</td><td>mcr-8.2</td><td>[49]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>D86E</td><td>[38]</td><td>G345E</td><td>[36]</td><td>ND</td><td>-</td><td>-</td><td>-</td></tr><tr><td>ESBL-KP7</td><td>2020</td><td>ESBL</td><td>KP</td><td>16</td><td>mcr-8.2</td><td>[49]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>G345E</td><td>[36]</td><td>ND</td><td>-</td><td>-</td><td>-</td></tr><tr><td>ESBL-KP6</td><td>2020</td><td>ESBL</td><td>KP</td><td>8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>R256G</td><td>[34]</td><td>ND</td><td>-</td><td>-</td><td>-</td></tr><tr><td>ESBL-KP5</td><td>2020</td><td>ESBL</td><td>KP</td><td>4</td><td>mcr-6-9</td><td>[7]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>G53R</td><td>[33]</td><td>R256G</td><td>[34]</td><td>ND</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>ESBL-KP4</td><td>2020</td><td>ESBL</td><td>KP</td><td>8</td><td>mcr-6-9</td><td>[7]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>G53R</td><td>[33]</td><td>A246T</td><td>[25]</td><td>ND</td><td>DELETION</td><td>[25]</td><td>I66V</td><td>[25,39]</td></tr><tr><td>ESBL-KP3</td><td>2020</td><td>ESBL</td><td>KP</td><td>&gt; 32</td><td>-</td><td>-</td><td>IS5/IS1182</td><td>[32]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>R256G</td><td>[34]</td><td>ND</td><td>Q239H</td><td>[25,39]</td><td>T276A</td></tr><tr><td>ESBL-KP2</td><td>2020</td><td>ESBL</td><td>KP</td><td>&gt; 32</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>A41T</td><td>[25]</td><td>L213M</td><td>[25]</td><td>ND</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>ESBL-KP1</td><td>2020</td><td>ESBL</td><td>KP</td><td>8</td><td>mcr-8.2</td><td>[49]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>T157P</td><td>[25,35]</td><td>ND</td><td>DELETION</td><td>[25]</td><td></td><td></td></tr><tr><td>ESBL-EC7</td><td>2020</td><td>ESBL</td><td>EC</td><td>16</td><td>mcr-8.2</td><td>[49]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>ESBL-EC6</td><td>2020</td><td>ESBL</td><td>EC</td><td>8</td><td>mcr-1</td><td>[4]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>ESBL-EC5</td><td>2020</td><td>ESBL</td><td>EC</td><td>8</td><td>mcr-8.2</td><td>[49]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>N11D</td><td>-</td><td>-</td><td>-</td></tr><tr><td>ESBL-EC4</td><td>2020</td><td>ESBL</td><td>EC</td><td>4</td><td>mcr-1</td><td>[4]</td><td>-</td><td>I47V</td><td>This study</td><td>E464D</td><td>A482T</td><td>[24,27]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>M20K</td><td>[24]</td><td>ND</td><td>-</td></tr><tr><td>ESBL-EC3</td><td>2020</td><td>ESBL</td><td>EC</td><td>8</td><td>mcr-3</td><td>[59]</td><td>-</td><td>-</td><td>N352K*</td><td>This study</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>S71C</td><td>[31]</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>ESBL-EC2</td><td>2020</td><td>ESBL</td><td>EC</td><td>4</td><td>mcr-1</td><td>[4]</td><td>V8A</td><td>[28]</td><td>-</td><td>-</td><td>-</td><td>T31S</td><td>[30]</td><td>E123D</td><td>[28-30]</td><td>D14N</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>ESBL-EC1</td><td>2020</td><td>ESBL</td><td>EC</td><td>8</td><td>mcr-1</td><td>[4]</td><td>V8A</td><td>[28]</td><td>-</td><td>-</td><td>-</td><td>T31S</td><td>[30]</td><td>V351I</td><td>[28-30]</td><td>S71C</td><td>[31]</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>CP-KP2</td><td>2020</td><td>CPE</td><td>KP</td><td>8</td><td>mcr-1</td><td>[4]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>A217V</td><td>[37]</td><td>-</td><td>-</td><td>-</td><td>ND</td><td>-</td><td>-</td><td>-</td></tr><tr><td>CP-KP1</td><td>2017</td><td>CPE</td><td>KP</td><td>&gt; 32</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>R256G</td><td>[34]</td><td>ND</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>CP-EC1</td><td>2017</td><td>CPE</td><td>EC</td><td>8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>N346K</td><td>[29]</td><td>-</td><td>-</td><td>-</td><td>G19R*</td><td>This study</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

Abbreviations: CPE, carbapenemase producing Enterobacteriaceae; EC, Escherichia coli; ESBL, extended spectrum beta-lactamase; KP, Klebsiella pneumoniae; mcr, mobile colistin resistance; MIC, minimum inhibitory concentration; ND, not done; TCS, two-component system; -, no detected mutations.

<footnote>* : novel mutations revealed in this study.</footnote>
presence of *mcr-1* has been first identified in faeces from a hospitalized child [47]. A few colistin resistance genes (*mcr-1* and *mcr-3*) have also been detected from both MDR *E. coli* and *K. pneumoniae* isolated from humans, poultry, and pigs. In 2019, both *E. coli* and *K. pneumoniae* resistant isolates to colistin (*mrc* like genes) have been identified in the Kampong Cham Province [42]. Our study also confirmed the presence of *mcr-1* and *mcr-3* and interestingly allowed the first detection of *mcr-8.2* in both *E. coli* / *K. pneumoniae* isolated in the human population in Cambodia. Concerning *mcr-8*, this variant has been first detected in China in 2018 in both poultry and faecal carriage [48]. One year later, *mcr-8.2* was identified in pig stool samples in China, soon after being detected in various clinical and environmental samples and recently in paediatric sepsis [25,49,50]. According to the NCBI database, *mcr-8.2* has been recently detected in Southeast Asia, especially in Vietnam (*K. pneumoniae* from patients in intensive care unit, Ho Chi Minh City - BioSample: SAMN15541446). To our knowledge, *mcr-8.2* has not been detected in *E. coli* or in Cambodia outside the present study.

In our study, *mcr-1* and 8.2 were detected in both *E. coli* and *K. pneumoniae* whereas *mcr-3* was only present in *E. coli*. In total, 87.5% colistin-resistant *E. coli* and 60% of colistin-resistant *K. pneumoniae* contained *mcr* genes. This is in agreement with other studies showing that the presence of *mcr* mainly concerns *E. coli* [42]. Among the six *mcr*-like genes detected for *K. pneumoniae*, two of them could not be sequenced, even after several trials [7]. The next step to identify these two variants could be to perform molecular cloning for these mixed sequences. This could be due to the presence of several variants of *mcr* genes in a single isolate, as already shown by other authors [51]. Last, it should be noted that the presence of *mcr-10*, identified in 2020, was not explored in this study [6].

### 4.3.2. TCS modifications

In *E. coli*, we identified mutations already described in previous studies within *mgrB*, *pmrA*, *pmrB*, *pmrD*, and *phoQ* [24,27-31] as well as four novel non-synonymous mutations in *phoP* (PhoP147V), *phoQ* (PhoQ N352K), *pmrB* (PmrB G19R), and *pmrD* (PmrD G85R) that might be related to colistin resistance. *PhoP* does not seem to be involved in colistin resistance for our sample because no known mutations have been identified. However, we can notice that we detected a mutation leading to an amino acid substitution (*PhoP* I47V - ESBL-EC4) at a position previously identified within *Enterobacter cloacae* (I47F) but leading to another amino acid change [52]. Concerning the substitution G19R on *PmrB*, the amino acid 19 has been associated with colistin resistance in *E. coli* by other authors but the mutation led to a different amino acid (G19E) [53].

For *K. pneumoniae*, we detected mutations within *mgrB*, *pmrA*, *pmrB*, and *crrB*. These mutations are single-nucleotide polymorphisms or insertion and deletion and have been previously associated with colistin resistance of *K. pneumoniae* [25,32-39]. Nine among 10 *K. pneumoniae* resistant isolates revealed mutations at different locations on *pmrB*. Contrarily to *E. coli* sample, all *K. pneumoniae* resistant isolates carried at least one chromosomal mutation associated with colistin resistance. To our knowledge, this is the first description of TCS modifications in *E. coli* / *K. pneumoniae* isolates from patients in Cambodia because only *mcr-1* and *mcr-3* have been identified in this country [42].

### 4.4. Colistin use in Cambodia

In Cambodia, interviews with farmers, veterinarians, and animal feed retailers and field observations identified a broad range of antibiotics used in food animal farms including beta-lactam, fluoroquinolones, tetracycline, colistin, and lincosamides [12]. The antimicrobials most commonly mentioned or kept in small-scale urban pig farming were amoxicillin, tylosin, gentamicin, and colistin.

A previous study showed that within 261 *E. coli* isolated from pigs, 20% of phenotypic resistance to colistin were associated with *mcr-1* and *mcr-3*-like genes [11]. Another study examined colistin resistance in *E. coli* from pigs, pig carcasses, and pork in Thailand, Lao PDR, and Cambodia border provinces and the highest resistance rate was found in *E. coli* (17.8%) from Cambodia with the presence of *mcr-1*, 3, and 5 [54]. Our data revealed a lower percentage of colistin resistant isolates in humans (3.6%) but considering only ESBL/CP- *E. coli*. Dissemination of *mcr* in *Enterobacterales* among humans, animals, and the environment is a public health issue and this study clearly demonstrates the increase of colistin resistance in the Cambodian human population infected by MDR *Enterobacteriaceae*. Since in Cambodia, there is an uncontrolled use of colistin in veterinary medicine contrarily to human health, the transfer of *mcr* genes, or bacteria carrying *mcr* genes from animals (or contaminated environment) to humans are the most likely. Two recent studies on chicken meat in Vietnam revealed that 49.7% to 52% have colistin-resistant *E. coli* (mostly associated with *mcr-1*) [55,56]. The authors suggested direct transmission of colistin resistance genes between farm animals and humans through environmental factors, such as flies or birds [56]. In China, the spread of *mcr-10*-carrying plasmids was observed among chickens, slaughterhouse workers, and healthy residents [57].

Putting together with evidence that colistin resistance circulates within humans, food, wild animals, environment, flies, and wastewater and that already 10 different *mcr* isoforms have been detected since 2015 in addition to mutations in TCS, there is a huge need to reconsider the use of this antimicrobial in medical veterinary use and to mitigate the international trade of this molecule listed on the World Health Organization Critically Important Antimicrobials for Human Medicine [10]. Efforts to monitor and control the spread of colistin resistance in Cambodia and neighbouring countries are crucial to preserving the effectiveness of antibiotics.

### 4.5. Limitations

Our study has certain limitations, as our sample is not exhaustive, and because it is based on a retrospective study of bacteria isolated at the MBL-IPC, with a small number of CPE. To better describe colistin-resistant bacteria circulating in Cambodia, it would be interesting to complete this study by including enterobacteria from various compartments (humans, animals, and environment), identify plasmid type carrying *mcr*, explore *mcr-10*, and additional chromosomal loci involved in colistin resistance such as genes of the AcrAB-TolC efflux systems, *crrA*, *qseB/qseC*, *yciM*, and *lpxM*. A global genomic approach could help as well to explore virulence factors including aerobactin, salmochelin, and yersiniabactin, especially within colistin resistant CPE [58].

## 5. Conclusion

This study explored the presence of colistin resistance among ESBL-CP- enterobacteria in humans in Cambodia. The presence of multiple *mcr* variants, emergence of *mcr-8.2*, chromosomal mutations in TCS and co-occurrence of both *mcr* genes and TCS modifications on resistant isolates underscore the need for continuous surveillance, antimicrobial stewardship, and infection control measures to mitigate the spread of colistin resistance.

## Ethics statement

Permission for the study was obtained from the National Ethics Committee for Health Research (NECHR) in Cambodia (Approval letter number 074NECHR).
# Declaration of competing interest

None.

# Acknowledgments

This study was supported by the International Joint Laboratory "Drug resistance in Southeast Asia" (French National Research Institute for Sustainable Development [IRD]).

# Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jgar.2024.06.017.

# References

[1] Paterson DL, Harris PNA. Colistin resistance: a major breach in our last line of defence. Lancet Infect Dis 2016;16:132-3. doi:10.1016/S1473-3099(15) 00463-6.

[2] Armengol E, Domènech O, Fusté E, Pérez-Guillén I, Borrell JH, Sierra JM, et al. Efficacy of combinations of colistin with other antimicrobials involves membrane fluidity and efflux machinery. Infect Drug Resist 2019;12:2031-8. doi:10.2147/IDR.S207844.

[3] Armengol E, Asunción T, Viñas M, Sierra JM. When combined with colistin, an otherwise ineffective rifampicin-linezolid combination becomes active in *Escherichia coli*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii*. Microorganisms 2020;8:86. doi:10.3390/microorganisms8010086.

[4] Liu Y-Y, Wang Y, Walsh TR, Yi L-X, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016;16:161-8. doi:10.1016/S1473-3099(15)00424-7.

[5] Anyanwu MU, Jaja IF, Nwobi OC. Occurrence and characteristics of mobile colistin resistance (*mcr*) gene-containing isolates from the environment: a review. Int J Environ Res Public Health 2020;17:1028. doi:10.3390/ijerph17031028.

[6] Wang C, Feng Y, Liu L, Wei L, Kang M, Zong Z. Identification of novel mobile colistin resistance gene *mcr-10*. Emerg Microbes Infect 2020;9:508-16. doi:10.1080/22221751.2020.1732231.

[7] Borowiak M, Baumann B, Fischer J, Thomas K, Deneke C, Hammerl JA, et al. Development of a novel *mcr-6* to *mcr-9* multiplex PCR and assessment of *mcr-1* to *mcr-9* occurrence in colistin-resistant *Salmonella enterica* isolates from environment, feed, animals and food (2011-2018) in Germany. Front Microbiol 2020;11:80. doi:10.3389/fmicb.2020.00080.

[8] Giordano C, Barnini S. Rapid detection of colistin-resistant Klebsiella pneumoniae using MALDI-TOF MS peak-based assay. J Microbiol Methods 2018;155:27-33. doi:10.1016/j.mimet.2018.11.008.

[9] Lippa AM, Goulian M. Feedback inhibition in the PhoQ/PhoP signaling system by a membrane peptide. PLoS Genet 2009;5:e1000788. doi:10.1371/journal.pgen.1000788.

[10] Umair M, Hassan B, Farzana R, Ali Q, Sands K, Mathias J, et al. International manufacturing and trade in colistin, its implications in colistin resistance and One Health global policies: a microbiological, economic, and anthropological study. Lancet Microbe 2023;4 E264-76. doi:10.1016/S2666-5247(22)00387-1.

[11] Ström G, Boqvist S, Albihn A, Fernström L-L, Andersson Djurfeldt A, Sokerya S, et al. Antimicrobials in small-scale urban pig farming in a lower middle-income country – arbitrary use and high resistance levels. Antimicrob Resist Infect Control 2018;7:35. doi:10.1186/s13756-018-0328-y.

[12] Om C, McLaws M-L. Antibiotics: practice and opinions of Cambodian commercial farmers, animal feed retailers and veterinarians. Antimicrob Resist Infect Control 2016;5:42. doi:10.1186/s13756-016-0147-y.

[13] Ström Hallenberg G, Börjesson S, Sokerya S, Sothyra T, Magnusson U. Detection of mcr-mediated colistin resistance in *Escherichia coli* isolates from pigs in small-scale farms in Cambodia. Antimicrob Agents Chemother 2019;63 e02241-18/aac/63/3/AAC.02241-18.atom. doi:10.1128/AAC.02241-18.

[14] Reed TAN, Krang S, Miliya T, Townell N, Letchford J, Bun S, et al. Antimicrobial resistance in Cambodia: a review. Int J Infect Dis 2019;85:98-107. doi:10.1016/j.ijid.2019.05.036.

[15] Malchione MD, Torres LM, Hartley DM, Koch M, Goodman JL. Carbapenem and colistin resistance in Enterobacteriaceae in Southeast Asia: review and mapping of emerging and overlapping challenges. Int J Antimicrob Agents 2019;54:381-99. doi:10.1016/j.ijantimicag.2019.07.019.

[16] European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint points for interpretation of MICs and zone diameters 2020.

[17] Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011;70:119-23. doi:10.1016/j.diagmicrobio.2010.12.002.

[18] Clinical Laboratories Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing. 30th ed; 2020.

[19] Li Y, Lin X, Yao X, Huang Y, Liu W, Ma T, et al. Synergistic antimicrobial activity of colistin in combination with rifampin and azithromycin against *Escherichia coli* producing MCR-1. Antimicrob Agents Chemother 2018;62 e01631-18/aac/62/12/e01631-18.atom. doi:10.1128/AAC.01631-18.

[20] Song Y, Yu L, Zhang Y, Dai Y, Wang P, Feng C, et al. Prevalence and characteristics of multidrug-resistant *mcr-1-positive Escherichia coli* isolates from broiler chickens in Tai'an, China. Poult Sci 2020;99:1117-23. doi:10.1016/j.psj.2019.10.044.

[21] Rebelo AR, Bortolaia V, Kjeldgaard JS, Pedersen SK, Leekitcharoenphon P, Hansen IM, et al. Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, *mcr-1*, *mcr-2*, *mcr-3*, *mcr-4* and *mcr-5* for surveillance purposes. Eurosurveillance 2018;23. doi:10.2807/1560-7917.ES.2018.23.6.17-00672.

[22] Cannatelli A, D'Andrea MM, Giani T, Di Pilato V, Arena F, Ambretti S, et al. *In vivo* emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP *mgrB* regulator. Antimicrob Agents Chemother 2013;57:5521-6. doi:10.1128/AAC.01480-13.

[23] Jayol A, Poirel L, Brink A, Villegas M-V, Yilmaz M, Nordmann P. Resistance to colistin associated with a single amino acid change in protein PmrB among Klebsiella pneumoniae isolates of worldwide origin. Antimicrob Agents Chemother 2014;58:4762-6. doi:10.1128/AAC.00084-14.

[24] Kim S, Woo JH, Kim N, Kim MH, Kim SY, Son JH, et al. Characterization of chromosome-mediated colistin resistance in *Escherichia coli* isolates from livestock in Korea. Infect Drug Resist 2019;12:3291-9. doi:10.2147/IDR.S225383.

[25] Yang T-Y, Wang S-F, Lin J-E, Griffith BTS, Lian S-H, Hong Z-D, et al. Contributions of insertion sequences conferring colistin resistance in Klebsiella pneumoniae. Int J Antimicrob Agents 2020;105894. doi:10.1016/j.ijantimicag.2020.105894.

[26] Kumar S, Stecher G, Li M, Knyaz C, Tamura KMEGA X. molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 2018;35:1547-9. doi:10.1093/molbev/msy096.

[27] Al-Mir H, Osman M, Drapeau A, Hamze M, Madec J-Y, Haenni M. WGS analysis of clonal and plasmidic epidemiology of colistin-resistance mediated by *mcr* genes in the poultry sector in Lebanon. Front Microbiol 2021;12:624194. doi:10.3389/fmicb.2021.624194.

[28] Delannoy S, Le Devencel L, Jouy E, Fach P, Drider D, Kempf I. Characterization of colistin-resistant *Escherichia coli* isolated from diseased pigs in France. Front Microbiol 2017;8:2278. doi:10.3389/fmicb.2017.02278.

[29] Luo Q, Yu W, Zhou K, Guo L, Shen P, Lu H, et al. Molecular epidemiology and colistin resistant mechanism of *mcr-positive* and *mcr-negative* clinical isolates *Escherichia coli*. Front Microbiol 2017;8:2262. doi:10.3389/fmicb.2017.02262.

[30] Mbelle NM, Feldman C, Osei Sekyere J, Maningi NE, Modipane L, Essack SY. The resistome, mobilome, virulome and phylogenomics of multidrug-resistant *Escherichia coli* clinical isolates from Pretoria, South Africa. Sci Rep 2019;9:16457. doi:10.1038/s41598-019-52859-2.

[31] Zakaria AS, Edward EA, Mohamed NM. Genomic insights into a colistin-resistant uropathogenic *Escherichia coli* strain of O23:H4-ST641 lineage harboring *mcr-1* on a conjugative IncH12 plasmid from Egypt. Microorganisms 2021;9:799. doi:10.3390/microorganisms9040799.

[32] Azam M, Gaind R, Yadav G, Sharma A, Upmanyu K, Jain M, et al. Colistin resistance among multiple sequence types of Klebsiella pneumoniae is associated with diverse resistance mechanisms: a report from India. Front Microbiol 2021;12:609840. doi:10.3389/fmicb.2021.609840.

[33] Bourrel AS, Poirel L, Royer G, Darty M, Vuillemin X, Kieffer N, et al. Colistin resistance in Parisian inpatient faecal *Escherichia coli* as the result of two distinct evolutionary pathways. J Antimicrob Chemother 2019;74:1521-30. doi:10.1093/jac/dkz090.

[34] Cheng Y-H, Lin T-L, Pan Y-J, Wang Y-P, Lin Y-T, Wang J-T. Colistin resistance mechanisms in Klebsiella pneumoniae strains from Taiwan. Antimicrob Agents Chemother 2015;59:2909-13. doi:10.1128/AAC.04763-14.

[35] Choi J-M, Ko KS. Mutant prevention concentrations of colistin for *Acinetobacter baumannii*, *Pseudomonas aeruginosa* and Klebsiella pneumoniae clinical isolates. J Antimicrob Chemother 2014;69:275-7. doi:10.1093/jac/dkt315.

[36] Ekelund O, Klokkhammer Hetland MA, Hoyland Löhr I, Schön T, Somajo S. Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS. J Antimicrob Chemother 2021;76:3183-91. doi:10.1093/jac/dkab328.

[37] Esposito EP, Cervoni M, Bernardo M, Crivaro V, Cuccurullo S, Imperi F, et al. Molecular epidemiology and virulence profiles of colistin-resistant Klebsiella pneumoniae blood isolates from the hospital agency "Ospedale dei Colli," Naples, Italy. Front Microbiol 2018;9:1463. doi:10.3389/fmicb.2018.01463.

[38] Samuelsen Ø, Overballe-Petersen S, Bjørnholt JV, Brisse S, Doumith M, Woodford N, et al. Molecular and epidemiological characterization of carbapenemase-producing Enterobacteriaceae in Norway, 2007 to 2014. PLoS One 2017;12:e0187832. doi:10.1371/journal.pone.0187832.

[39] Liu X, Wu Y, Zhu Y, Jia P, Li X, Jia X, et al. Emergence of colistin-resistant hypervirulent Klebsiella pneumoniae (Cor-HVkP) in China. Emerg Microbes Infect 2022;11:648-61. doi:10.1080/22221751.2022.2036078.

[40] Gysin M, Hon PY, Tan P, Sengduangphachan A, Simmalavong M, Hinthongth P, et al. Apramycin susceptibility of multidrug-resistant Gram-negative blood culture isolates in five countries in Southeast Asia. Int J Antimicrob Agents 2022;60:106659. doi:10.1016/j.ijantimicag.2022.106659.

[41] Kaza P, Mahindroo J, Veeraraghavan B, Mavuduru RS, Mohan B, Taneja N. Evaluation of risk factors for colistin resistance among urtopathogenic isolates of *Escherichia coli* and Klebsiella pneumoniae: a case-control study. J Med Microbiol 2019;68:837-47. doi:10.1099/jmm.0.000986.
[42] Wang X, Liu Y, Qi X, Wang R, Jin L, Zhao M, et al. Molecular epidemiology of colistin-resistant Enterobacteriaceae in inpatient and avian isolates from China: high prevalence of mcr-negative Klebsiella pneumoniae. Int J Antimicrob Agents 2017;50:536-41. doi:10.1016/j.ijantimicag.2017.05.009.

[43] Atterby C, Osbjer K, Tepper V, Rajala E, Hernandez J, Seng S, et al. Carriage of carbapenemase- and extended-spectrum cephalosporinase-producing Escherichia coli and Klebsiella pneumoniae in humans and livestock in rural Cambodia; gender and age differences and detection of blaOXA-48 in humans. Zoonoses Public Health 2019;66:603-17. doi:10.1111/zph.12612.

[44] Hanberger H, Edlund C, Furebring M, C GGiske, Å Melhus, Nilsson LE, et al. Rational use of aminoglycosides—review and recommendations by the Swedish Reference Group for Antibiotics (SRGA). Scand J Infect Dis 2013;45:161-75. doi:10.3109/00365548.2012.747694.

[45] Gardiner BJ, Stewardson AJ, Abbott LJ, Peleg AY. Nitrofurantoin and fosfomycin for resistant urinary tract infections: old drugs for emerging problems. Aust Prescr 2019;42:14. doi:10.18773/austprescr.2019.002.

[46] Premnath MAC, Prabakaran K, Sivasankar S, Boppe A, Sriramajayam L, Jeyaraj S. Occurrence of mcr genes and alterations in mgrB gene in intrinsic colistin-resistant Enterobacterales isolated from chicken meat samples. Int J Food Microbiol 2023;404:110323. doi:10.1016/j.ijfoodmicro.2023.110323.

[47] Stoesser N, Mathers AJ, Moore CE, Day NP, Crook DW. Colistin resistance gene mcr-1 and pHNSHP45 plasmid in human isolates of Escherichia coli and Klebsiella pneumoniae. Lancet Infect Dis 2016;16:285-6. doi:10.1016/S1473-3099(16)00010-4.

[48] Wang X, Wang Y, Zhou Y, Li J, Yin W, Wang S, et al. Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae. Emerg Microbes Infect 2018;7:1-9. doi:10.1038/s41426-018-0124-z.

[49] Yang X, Liu L, Wang Z, Bai L, Li R. Emergence of mcr-8.2-bearing Klebsiella quasipneumoniae of animal origin. J Antimicrob Chemother 2019;74:2814-17. doi:10.1093/jac/dkz213.

[50] Liu K-D, Jin W-J, Li R-B, Zhang R-M, Sun J, Liu Y-H, et al. Prevalence and molecular characteristics of mcr-1-positive Escherichia coli isolated from duck farms and the surrounding environments in coastal China. Microbiol Res 2023;270:127348. doi:10.1016/j.micres.2023.127348.

[51] Timmermans M, Wattiau P, Denis O, Boland C. Colistin resistance genes mcr-1 to mcr-5, including a case of triple occurrence (mcr-1, -3 and -5), in Escherichia coli isolates from faeces of healthy pigs, cattle and poultry in Belgium, 2012-2016. Int J Antimicrob Agents 2021;57:106350. doi:10.1016/j.ijantimicag.2021.106350.

[52] Nawfal Dagher T, Al-Bayssari C, Chabou S, Baron S, Hadjadj L, Diene SM, et al. Intestinal carriage of colistin resistant Enterobacteriaceae at Saint Georges Hospital in Lebanon. J Glob Antimicrob Resist 2020;21:386-90. doi:10.1016/j.jgar.2019.12.001.

[53] Janssen AB, Van Hout D, Bonten MJM, Willems RJL, Van Schaik W. Microevolution of acquired colistin resistance in Enterobacteriaceae from ICU patients receiving selective decontamination of the digestive tract. J Antimicrob Chemother 2020;75:3135-43. doi:10.1093/jac/dkaa305.

[54] Pungpian C, Lee S, Trongjit S, Sinwat N, Angkititrakul S, Prathan R, et al. Colistin resistance and plasmid-mediated mcr genes in Escherichia coli and Salmonella isolated from pigs, pig carcass and pork in Thailand, Lao PDR and Cambodia border provinces. J Vet Sci 2021;22:e68. doi:10.4142/jvs.2021.22.e68.

[55] Nakayama T, Le Thi H, Thanh PN, Minh DTN, Hoang ON, Hoai PH, et al. Abundance of colistin-resistant Escherichia coli harbouring mcr-1 and extended-spectrum β-lactamase-producing E. coli co-harbouring blaCTX-M-55 or -65 with blaTEM isolates from chicken meat in Vietnam. Arch Microbiol 2022;204:137. doi:10.1007/s00203-021-02746-0.

[56] Nguyen PTL, Tran HTM, Tran HA, Pham TD, Luong TM, Nguyen TH, et al. Carriage of plasmid-mediated colistin resistance-1-positive Escherichia coli in humans, animals, and environment on farms in Vietnam. Am J Trop Med Hyg 2022;107:65-71. doi:10.4269/ajtmh.21-1203.

[57] Xu L, Wan F, Fu H, Tang B, Ruan Z, Xiao Y, et al. Emergence of colistin resistance gene mcr-10 in Enterobacterales isolates recovered from fecal samples of chickens, slaughterhouse workers, and a nearby resident. Microbiol Spectr 2022;10:e00418-22. doi:10.1128/spectrum.00418-22.

[58] Liu Y, Lin Y, Wang Z, Hu N, Liu Q, Zhou W, et al. Molecular mechanisms of colistin resistance in Klebsiella pneumoniae in a tertiary care teaching hospital. Front Cell Infect Microbiol 2021;11:673503. doi:10.3389/fcimb.2021.673503.

[59] Yin W, Li H, Shen Y, Liu Z, Wang S, Shen Z, et al. Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli. mBio 2014;8. e00543-17 www.mbio.org/8/3/e00543-17.atom.