First Report of the mcr-1 colistin Resistance Gene Identied in Escherichia coli Isolated from a Clinical Sample in Naples in 2020

Background: The emergence of a novel plasmid-mediated colistin resistance mechanism, encoded by the mcr-1 gene, represents a major public health concern. The mechanism of resistance to colistin, mediated by plasmids, is a serious problem, both for its ability to be transferred to other species, and for infections caused by carbapenem-resistant Gram-negative, in which colistin is used as an antimicrobial drug of last line for the treatment of these infections. The present study highlights the rst isolation and genetic evaluation of detecting plasmid-mediated resistance to colistin in a multidrug-resistant (MDR) Escherichia coli (E. coli) isolated from a clinical sample in the metropolitan city of Naples, Italy. Results: Colistin-resistant E. coli isolate was identied in August 2020 from the blood culture of a male patient with multiple comorbidities. The minimum inhibitory concentration (MIC) of colistin was 8 mg/L. In addition to colistin, the isolate was resistant to third-generation cephalosporins (cefotaxime and ceftazidime), penicillin (amoxicillin and piperacillin), aminoglycosides (gentamicin and tobramycin), and uoroquinolones (ciprooxacin and levooxacin). However, it showed susceptibility to carbapenems (ertapenem, imipenem, and meropenem), tetracyclines (tigecycline), and piperacillin-tazobactam. The results of the PCR conrmed the presence of the mcr-1 resistance gene. Conclusion: This study conrms the presence of resistance to colistin mediated by the mcr-1 gene in a clinical isolate of E. coli. Although resistance to colistin caused by the mcr-1 gene is not common in our region, it should not be ignored. Therefore, further surveillance studies are recommended to monitor the spread of plasmid-mediated colistin resistance genes in Gram-negative MDR bacteria. 5′-CGAATGGAGTGTGCGGTG-3′) 5′-AGATGGTATTGTTGGTTGCTG-3′) and

Conclusion: This study con rms the presence of resistance to colistin mediated by the mcr-1 gene in a clinical isolate of E. coli. Although resistance to colistin caused by the mcr-1 gene is not common in our region, it should not be ignored. Therefore, further surveillance studies are recommended to monitor the spread of plasmid-mediated colistin resistance genes in Gram-negative MDR bacteria.

Background
In recent years, the phenomenon of antibiotic resistance (AMR, Antimicrobial resistance) has increased signi cantly due to the inappropriate and extended use of antibiotics, both in human and veterinary medicine, causing the emergence, multiplication, and spread of strains resistant to more antibiotics [1]. It should be noted that this phenomenon often concerns healthcare-related infections, which develop and spread within hospitals and other health facilities [2,3]. Unfortunately, the emergence of multidrugresistance microorganisms progressively reduces the possibility of applying an antibiotic approach and re ects on effective and timely treatments. This leads to important implications in terms of clinical (increased morbidity, lethality, duration of the disease) as well as economic terms [4]. Carbapenems are among the rst-line antibiotics used in the treatment of infections caused by multidrug-resistant bacteria (MDR). The direct consequence of a carbapenems wide use brought to an increase in resistance rates among Enterobacteriaceae (CRE), causing much public health concern [5]. In this scenario, the last chance for a patient with a carbapenem-resistant MDRs is represented by Colistin (polymyxin E) [6].
Colistin belongs to the class of Polymyxins, polycationic peptides that selectively act on the permeability of the cytoplasmic membrane of gram-negative bacteria, causing cell lysis [7]. Resistance to colistin is mainly caused by changes in the lipopolysaccharide molecules (LPS). Indeed, the addition of cationic groups to lipid A reduces the access of colistin to the plasma membrane [8]. Speci cally, the resistance is mediated by the enzymatic activity of the phosphoethanolamine transferase, encoded by the mcr gene, which transfers phosphoethanolamine to the lipid A, modifying the lipopolysaccharides of the membrane [9]. Resistance to colistin, mediated by the mcr gene, is readily transmissible through conjugation to other pathogenic species, such as Klebsiella pneumoniae and Pseudomonas aeruginosa [10,11]. Clinical isolates of colistin-resistant E. coli have been rarely reported. The rst isolate of E. coli resistant to colistin mediated by plasmid (mcr-1) have been found in China in 2012 [12]. To date, in many countries of Europe and in many other areas of the world the mcr-1 gene has been identi ed in E. coli isolated from clinical samples [13][14][15][16]. In Italy, the mcr-1 gene was rst described in 2016, found in eight clinical isolates of E. coli, in the clinical microbiology laboratories of two Italian hospitals (Florence, central Italy; Lecco, northern Italy). Two isolates positive for mcr-1 exhibited a multidrug-resistant phenotype, including expanded spectrum cephalosporins, uoroquinolones, and trimethoprim-sulfamethoxazole, and produced extended-spectrum β-lactamase activity [17]. More recently, three cases of sepsis caused by E. coli colistin resistant, which harbored the mcr-1 gene, have been described in patients admitted to the Policlinico San Matteo of Pavia in Italy, between August 2016 and January 2017 [18]. Despite the prevalence of mcr genes among CRE or the incidence of colistin resistance is still very rare, it raises concern among experts, being a last-line antibiotic used in the treatment of infection by carbapenemresistant Gram-negative [19]. In this study, we reported a case of sepsis caused by colistin-resistant E. coli strain (mcr-1), isolated at the section of Microbiology and Virology, University Hospital "Luigi Vanvitelli", Naples, Italy.

Results
Based on the microbial identi cation of characteristic protein ngerprints of bacteria, we evaluated the E. coli isolate by MALDI-TOF MS. Among all the output results, the only microorganism with a valid score over 2.0 were taken into consideration. Figure 1 shows one of the spectra obtained from the MALDI-TOF analysis, which refers to the obtained E.coli.
After the bacterial isolation, an antibiogram evaluation was performed. The antibiotic panel resistances are summarized in Table 1. Indeed, the isolate was not susceptible to third-generation cephalosporins (cefotaxime and ceftazidime) due to ESBL (extended-spectrum beta-lactamase) production. Also, it showed resistance to penicillin (amoxicillin and piperacillin), aminoglycosides (gentamicin and tobramycin), and uoroquinolones (cipro oxacin and levo oxacin). However, it was still able to be inhibited by carbapenems (ertapenem, imipenem, and meropenem), tetracyclines (tigecycline), and piperacillin-tazobactam.

Conclusion
This report should serve as a public health warning signal, however, to intensify surveillance efforts and identify mcr-1 tanks in sensitive populations. Environmental, food and clinical monitoring are essential in order to reduce the transfer of multi-strain resistant bacteria between animals and humans Finally, increased vigilance and the implementation of antimicrobial management programs are necessary and important in the health sector to slow the spread of antimicrobial resistance [22][23][24][25].

Sample collection and preparation:
A volume of 5-10 mL of blood, obtained from peripheral vein sampling, was inoculated in blood culture bottles (aerobic bottle and anaerobic bottle). Blood culture bottles were incubated in the automated blood culture monitoring BACTEC 9240 blood culture system (Becton Dickinson Diagnostic Instrument Systems

Polymerase Chain Reaction (PCR) Ampli cation:
PCRs were performed employing the Kodaq 2X PCR MasterMix Kit (Abm, Canada). Two microliters of bacterial DNA were ampli ed in a 50 µL reaction volume with 10 µM of each primer, using an annealing temperature of 62 °C and 38 cycles. Analyses of the PCR products were conducted on 1,8% agarose gel and detected using Gel Doc™ EZ Imager (Bio-Rad, USA).

Declarations
Ethics approval and consent to participate: The study was designed and conducted in accordance with the Helsinki declaration. This study was performed in accordance with the National Guidelines System (SNLG), for the use of biological samples.
We declare that the following work was carried out following standard hospital protocols, approved by the Ethics Committee of the University of Campania "Luigi Vanvitelli" -A.O.U. "Luigi Vanvitelli". The need for written informed consent was waived because de-identi ed retrospective data were used. The permission of the corresponding author was required to access the raw data/samples.

Consent for publication:
Not applicable.
Availability of data and materials: All data generated or analysed during this study are included in this published article