Detection of Colistin-resistant Genes in Gram-negative Bacilli Isolated From Patients With COVID-19

1. Introduction
Respiratory-associated coinfections are a significant contributor to the mortality of hospitalized COVID-19 patients [1]. Factors such as prolonged intubation, extensive catheter use, and compromised immune systems in patients with respiratory complications elevate the risk of secondary bacterial and fungal infections. Consequently, antimicrobial agents were excessively utilized in critical care settings, resulting in emerging drug-resistant pathogens [2]. Infection caused by multidrug-resistant (MDR) pathogens is now a worldwide issue, considering the wide distribution of MDR isolates. SARS-CoV-2 mutations, cytokine storms following the immune response to infection, comorbidities, and the immunogenetic condition of COVID-19 patients render these patients vulnerable to secondary infections [3]. A high rate of morbidity and mortality was associated with bacterial coinfections in COVID-19 cases [4]. 
The surge in antimicrobial resistance among COVID-19 patients predominantly stems from the dissemination of high-risk clones, particularly gram-negative bacteria including Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter spp. [4]. Gram-negative bacteria have exhibited antibiotic resistance due to broad-spectrum beta-lactamases [5]. The widespread antibiotic resistance to first- and second-line antibiotics, such as cephalosporin resistance observed in hospital-associated infections caused by A. baumannii, P. aeruginosa, and K. pneumoniae, particularly carbapenemase-producing strains, presents a significant healthcare challenge [6]. 
Bacterial resistance leads to various complications, including urinary tract infections, septicemia, pneumonia, and intra-abdominal infections, affecting patients across different hospital departments. The emergence of high resistance to carbapenems and colistin mediated by mobile genetic elements poses a severe public health concern [7]. The mcr and pmr genes confer colistin resistance and are significantly expressed in colistin-resistant isolates [8]. Colistin is used as a last-resort antibiotic to treat infections caused by MDR gram-negative bacteria, but bacteria can develop resistance to it through various mechanisms including modification of lipopolysaccharide (LPS) structure, alteration of outer membrane proteins, activation of efflux pumps, mutation in regulatory genes, and horizontal transfer of resistance genes such as mcr genes. These resistance mechanisms reduce colistin's efficacy, highlighting the urgent need for prudent antibiotic use, infection control measures, and ongoing research into alternative treatment strategies to address the public health threat posed by colistin-resistant bacteria [9]. Given the critical importance of colistin as a last-resort antibiotic and the escalating threat of resistance, this study aimed to determine the prevalence of colistin resistance, both phenotypically and genotypically, and to identify the associated mcr-1, mcr-2, mcr-3, and pmrAB genes among Gram-negative bacterial isolates from hospitalized COVID-19 patients in Isfahan, Iran.

2. Materials and Methods
2.1. Study design
This study was conducted on 74 hospitalized patients with COVID-19 who were confirmed by a positive reverse transcription polymerase chain reaction (RT-PCR)test or the presence of ground glass opacity on a computed tomography (CT) scan from different units (intensive care unit (ICU), internal, and surgical) at Al-Zahra Hospital, Isfahan, Iran, in 2022. Data on the age and gender of the patients were recorded from their medical archives. All patients gave their consent to participate in the study. The Islamic Azad University Ethics Committee confirmed the study. 

2.2. Bacterial isolation and identification
Seventy-four bacterial isolates from the trachea of COVID-19 patients were obtained and cultured. Identification of isolates was confirmed using biochemical tests, including TSI, Urease, Oxidase, SIM, MRP, O/F, DNase, and Simmons citrate.

2.3. Antibiotic susceptibility tests 
According to the Clinical and Laboratory Standards Institute (CLSI-M100-2021) [10], the antibiotic susceptibility test followed the Kirby-Bauer protocol. The antibiotics tested (Padtan Teb Co., Iran) were cefepime (30 μg), amoxicillin-clavulanic acid (20/10 μg), ampicillin (10 μg), levofloxacin (5 μg), cotrimoxazole (1.25/23.75 μg), amikacin (30 μg), ceftazidime (30 μg), imipenem (10 μg), gentamicin (10 μg), tazobactam (10 μg), meropenem (10 μg), and ciprofloxacin (5 μg).
Colistin stock with a volume of 1 mL, a concentration of 5120 µg/mL, and a potency of 980 µg/mg was prepared to determine the minimum inhibitory concentration (MIC) of colistin through the microdilution method, according to CLSI M07-A10 [11]. Different concentrations of colistin were applied (0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 µg/mL). According to the CLSI protocol [11], colistin resistance thresholds for Enterobacteriaceae and non-fermenting gram-negative bacilli were as follows: MIC ≤2 µg/mL was considered susceptible, and MIC ≥4 µg/mL was considered resistant. All tests were performed in triplicate to ensure reproducibility of results.

2.4. Detection of colistin resistance genes 
Bacterial DNA was extracted using Sina Gene kit (Sina Gene Co., Iran) following the manufacturer’s instructions, and the quantity of extracted DNA was measured using a NanoDrop. To evaluate the colistin resistance genes mcr-1, mcr-2, mcr-3, and pmrAB, a PCR test was performed using previously designed primers (Sina Colon Co., Iran) [12] (Table 1).

Detection of mcr-1, mcr-2, mcr-3 genes was performed using 12.5 μL of PCR master mix (Sina Gene Co., Iran) and a PCR thermocycler (Eppendorf, Germany) according to the following procedure: one cycle of initial denaturation at 95 °C for 3 min; 30 cycles including 20 sec of denaturation at 94 °C, 15 sec of annealing at 54 °C (mcr-1), 58 °C (mcr-2), and 50 °C (mcr-3), and 15 sec of extension at 72 °C; and one cycle for the final extension at 72 °C for 1 min. Expression of the pmr genes was evaluated by PCR with one cycle at 95 °C for 3 min for initial denaturation; 30 cycles for denaturation (30 sec at 94 °C), annealing (30 sec at 54 °C), and extension (45 sec at 72 °C); and one cycle for 1 min at 72 °C for the final extension. The quality of the final PCR product was confirmed by the presence of a sharp band on the gel following electrophoresis.

2.5. Statistical analysis 
All data were analyzed using SPSS version 20 and reported as numbers, percentages, and Mean±SD. Antibiogram results were analyzed using WHONET V.5.6 software. Sequence confirmation was performed by Macrogen (Macrogen, Inc., Korea), and the results were analyzed using the National Center for Biotechnology Information (NCBI) database, Chromas, Mega4, and GeneRunner software. 

3. Results
3.1. Studied population and bacterial isolates
The mean age of COVID-19 patients was 53.08±26.12 years (1-92 years), and 41 of the 74 isolates were collected from males (55.4%). About 31% (n=23) of patients were admitted to the ICU, 31% (n=23) to the internal unit, 13.5% (n=10) to the surgical unit, and 24% (n=18) were in other units. Among gram-negative bacilli, 80% belonged to the family of non-fermenting gram-negative bacilli, including 52(70%) isolates of Acinetobacter spp., 5(7%) isolates of P. aeruginosa, 1(1.5%) isolate of Achromobacter denitrificans, and 1(1.5%) isolate of Stenotrophomonas maltophilia. About 20% of isolates belonged to the Enterobacteriaceae, including 12(16%) isolates of K. pneumonia and 3(4%) isolates of E. coli.

3.2. Antibiotic susceptibility tests
Gram-negative bacilli showed the highest resistance to ampicillin with a frequency of 94.6% and the lowest resistance to gentamicin and ceftazidime with a frequency of 74.3%. Acinetobacter spp. isolates demonstrated the highest resistance to amoxicillin-clavulanic acid (98.1%), meropenem (94.5%), ampicillin, and cefepime (92.6%) and were sensitive to ceftazidime. P. aeruginosa isolates showed high resistance to ampicillin (100%) and were sensitive to gentamicin, amikacin, and ceftazidime. Among E. coli isolates, the highest resistance was observed to ampicillin, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanic acid (100%), and the lowest resistance to levofloxacin, piperacillin-tazobactam, amikacin, and gentamicin was observed. K. pneumonia isolates were resistant to ampicillin (100%) and sensitive to amikacin, gentamicin, and meropenem (Table 2).

The mean MIC for colistin was 13.7 µg/mL. According to our findings, 20(64.5%) isolates of Acinetobacter spp., 6(19.3%) isolates of K. pneumoniae, 4(12.9%) isolates of P. aeruginosa, and 1(2.3%) isolate of A. denitrificans were resistant to colistin (MIC ≥4 µg/mL), while no isolates of E. coli or S. maltophilia showed resistance to colistin (Table 3).

3.3. Molecular detection
Genotyping results showed that 20% (n=6) of the isolates had the pmrAB gene, and none of the gram-negative bacillus isolates had mcr-1, mcr-2, or mcr-3 genes. Among the isolates resistant to colistin, 8(26.6%) isolates expressed pmrA (five isolates were Acinetobacter spp., and three isolates were P. aeruginosa), and pmrB was detected in six (20%) isolates (four isolates were Acinetobacter spp., and two isolates were P. aeruginosa). None of the K. pneumoniae and E. coli isolates demonstrated any of the colistin resistance genes. A total of 10 isolates with high resistance to colistin and a sharp band on gel electrophoresis of PCR products of the pmrAB gene were sequenced and confirmed by Gene Fanavaran company (FNM Co., Iran).

4. Discussion
The emergence of the COVID-19 pandemic has brought unprecedented challenges to global healthcare systems, with a profound impact on patient management and treatment strategies. Among the numerous complications associated with COVID-19, secondary antibiotic-resistant infections have emerged as significant clinical concerns, particularly among hospitalized patients [13, 14]. Colistin, a last-resort antibiotic, has been increasingly relied upon for managing MDR bacterial infections. However, reports of colistin-resistant bacteria isolated from the trachea of COVID-19 patients underscore the urgency of addressing antimicrobial resistance in this global health crisis [14, 15].
The most detected isolates from the trachea were from ICU patients, of which Acinetobacter spp. and P. aeruginosa from non-fermenting gram-negative bacilli and K. pneumoniae from Enterobacteriaceae had the highest prevalence among detected isolates. Viral respiratory infections, including COVID-19 and influenza, disrupt the host’s innate and adaptive immune defenses, leading to secondary infections. These secondary infections are often linked to more severe outcomes, particularly in debilitated patients with underlying comorbidities [16]. Costa et al. reported a high prevalence of bacterial infection associated with ventilator and tracheitis among hospitalized COVID-19 patients in the ICU. Among those with secondary infections after hospitalization (29.8%), A. baumannii, P. aeruginosa, and K. pneumoniae were more prevalent than others. Over half of the A. baumannii, P. aeruginosa, and K. pneumoniae isolates were MDR [17]. Shah et al. demonstrated a high prevalence of bacterial infection in the respiratory system, bloodstream, and other sterile body sites. The most isolated bacteria from the respiratory system were P. aeruginosa, K. pneumoniae, and E. coli. They reported an increased mortality rate in COVID-19 patients with bacterial infections [18]. 
We observed that most Acinetobacter spp., P. aeruginosa, K. pneumoniae, and E. coli isolates were resistant to ampicillin. Musuuza et al. found that 24% of COVID-19 patients had coinfections with gram-negative bacteria, a factor significantly associated with adverse outcomes such as prolonged hospitalization or increased mortality [19]. Studies reported a high prevalence of individuals with COVID-19 receiving antibiotic treatment, including broad-spectrum regimens, without conclusive evidence of secondary bacterial infection [20], which resulted in emerging MDR isolates. In the study by Ahmed et al., E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, and gram-positive bacteria were more prevalent among isolates from COVID-19 patients, respectively. They reported that most E. coli and K. pneumoniae isolates were resistant to ampicillin, while P. aeruginosa isolates were resistant to ciprofloxacin, and A. baumannii isolates represented a wide spectrum of resistance to amikacin, ciprofloxacin, ceftazidime, levofloxacin, cotrimoxazole, piperacillin-tazobactam, and tetracycline [21]. Another study by Pourajam et al. reported that about 10% of respiratory samples from COVID-19 patients demonstrated bacterial infections. All E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii isolates were resistant to ciprofloxacin, and E. coli, K. pneumoniae, and P. aeruginosa isolates showed a high resistance to ampicillin [22]. 
The emergence of highly resistant strains significantly challenges the management of gram-negative bacterial infections, and colistin has become one of the main treatment options, particularly for nosocomial infections [23]. In the current study, Acinetobacter spp. illustrated a high resistance (64.5%) to colistin, followed by K. pneumoniae (19.3%), P. aeruginosa (12.9%), and Achromobacter denitrificans, respectively, while E. coli and Stenotrophomonas maltophilia were sensitive to colistin. Studies reported various rates of gram-negative bacteria resistance to colistin. Colistin-resistant isolates were highly prevalent in Asia and Europe, from 0.2% to 17.5% [24]. The variation in reported findings could stem from variances in geographic regions, methodologies for studying resistance, diversity in sample types, sample size, patient health statuses, antibiotic prescription practices, and adherence to infection control protocols. 
Moosavian et al. reported that 13.6% of Enterobacteriaceae isolates were resistant to colistin with MIC values >2 μg/mL. Among these E. coli and K. pneumoniae isolates, about 1.7% of them expressed the mcr-1 gene [25]. Among the mcr-1, mcr-2, mcr-3, and pmrAB resistance genes, we observed that 20% of the isolates carried the pmrAB gene, with the majority being A. baumannii isolates, followed by P. aeruginosa. Rout et al. reported that among A. baumannii strains isolated from hospital infections, 5.9% of isolates were resistant to colistin, and these isolates expressed pmrA and pmrB genes [26]. Osama et al. demonstrated that among 30 carbapenem-resistant isolates, five isolates were resistant to colistin. The results of genotyping for mcr-1, pmrB, and pmrA genes showed that one isolate carried the pmrA gene, one isolate had mcr-1, pmrA, and pmrB genes, while three isolates carried pmrA and pmrB genes [27]. 
Since we did not find any mcr genes among the isolates, the resistance to colistin in these isolates may be caused by other mutations, other bacterial resistance mechanisms, and resistance mechanisms associated with the pmrAB efflux pump. The lower resistance to aminoglycoside antibiotics in these samples suggested that they could serve as viable alternatives to beta-lactam antibiotics. The study's strengths lie in providing a comprehensive perspective on colistin-resistant gram-negative bacterial isolates, antibiotic resistance patterns, and associated genes. However, limitations include the potential impact of the study's sample size on generalizability, its single-center design limiting broader applicability, and possible biases in sample collection and patient selection. While genotyping provides molecular insights, its coverage may not encompass all resistance mechanisms, and the absence of comparison groups hinders contextualization within broader epidemiological trends.

5. Conclusion
This study highlighted the prevalence of antibiotic resistance among gram-negative bacilli, emphasizing the need for careful antibiotic prescription. While certain antibiotics showed lower resistance rates, significant proportions of isolates, notably Acinetobacter spp., exhibited resistance to colistin. Continuous research is essential to address these challenges and develop effective treatment strategies.

Acknowledgements
The authors are grateful for the support of this study by Falavarjan Branch, Islamic Azad University, Isfahan, Iran.
Compliance with ethical guidelines
This study was approved by the Research Ethics Committee of Falavarjan Branch, Islamic Azad University, Isfahan, Iran (Code: IR.IAU.FALA.REC.1401.006).

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Conceptualization, study design, and writing the original draft:    Laleh Hoveida; Review and editing: Soodabeh Rostami; Statistical analysis, data acquisition, experiments, data interpretation, supervision, administrative, technical, and material support: All authors.

Conflict of interest
The authors declared no conflict of interest.

Data availability
Data are available upon request from the author.

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