Distribution of Colistin Resistance Genes Among Clinical Isolate of Gram-Negative Bacteria

This study focuses on the identification and the distribution of Colistin resistance genes among Gram-negative bacteria isolated from clinical samples at the National Ribat Teaching hospital, in Khartoum, Sudan. A total of 165 Gram-negative isolated pathogens were as follow Klebsiella pneumoniae (73/44.2%), E. coli (53/32.1%), Pseudomonas aeruginosa (33/20%), Proteus vulgaris (6/3.6%), and Citrobacter freundii (2/1.2%), isolated between December 2019 and February 2020 from clinical samples at the National Ribat Teaching hospital in Khartoum, Sudan. Stock of all isolates were stored in 20% Glycerol-broth at -80 °C for further subculture for antimicrobial susceptibility profile and resistance genes associated with Colistin resistance were identified. Colistin resistance associated genes (mcr


Introduction
Antibiotics resistance has become a major concern among Gram-negative bacterial infections because of the unavailability of alternative treatment options. The antibiotic resistance phenomenon is emerged due to antibiotics overuse and bacterial evolution [1]. Colistin is a cationic polypeptide-based antibiotic, also known as polymxin E, it's considered the reserve antibiotics against the multidrug resistance (MDR) infections caused by Gramnegative bacterial pathogens such as Enterobacteriaceae [2,3].
The antimicrobial action of Colistin is directed towards the Gramnegative bacterial cell membrane by interacting and disrupting the lipopolysaccharide (LPS) molecules in the outer membrane which results in bacterial death [4]. In the last decade, there is an increased in the resistance of bacteria against several commonly used antibiotics and the limitation in discovery of a new ones had led the Colistin as a valuable drug of choice for many MDR strains [5][6][7]. MDR bacterial strains was defined MDR as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories. Unfortunately, MDR, extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of E. coli and other Enterobacteriaceae strains were detected worldwide, and they were harboring multiple resistance mechanisms [8][9][10].
Some bacterial strains have a natural Colistin resistance ability, such as S. marcescens, Proteus spp., B. cepacia, M. morganii, Providencia spp., and Vibrio cholera [11]. However, recently, several studies have reported Colistin-resistant isolates that have acquired Colistin resistance via chromosomal genes or plasmids worldwide [12,13]. Resistance to Colistin was associated with mutation in two components regulatory system that mediated by chromosomal genes [14,15]. The gene mcr-1 is gene of the phosphoethanolamine transferase enzyme family, which reported for the first time from China in 2016 as the first plasmid-mediated Colistin resistance gene among the Escherichia coli (E. coli) strains obtained from patients, animals, and food [16][17][18][19]. Ever since, mcr-1 positive strains have been detected in Enterobacteriaceae worldwide [20][21][22][23][24]. This first plasmid-mediated gene was followed by reports of variants mcr-1 genes (mcr-1.2, mcr-1.3…) and also the description of new other 7 mcr genes (mcr-2 -mcr-8) [25][26][27][28][29]. The emergence of colistin resistance genes have the ability to cause a major therapeutic challenge in the treatment of Enterobacteriaceae infections, which led to new recommendations for clinicians and laboratory diagnosis [30][31][32]. The Colistin-resistant Gram-negative bacteria impact on the clinical outcomes is clear. Thus, an appropriate and concrete actions against this strain is urgently required. This study focuses on the identification and the distribution of Colistin resistance genes among Gram-negative bacteria isolated from clinical samples at the National Ribat Teaching hospital, in Khartoum, Sudan.

Samples Collection and Processing, Growth Conditions and Chemicals
To investigate the occurrence of Colistin resistant Gram- All the samples were collected and processed after approval of the ethical committee at the National Ribat University.

Determination of Minimal Inhibitory Concentration (In Vitro Susceptibility Testing)
Antibiotic susceptibility testing was performed by disc diffusion assay according to guidelines from the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2012). Our first approach to screen Colistin resistance among bacterial isolates, a 10 μg disc of Colistin was used and the test was performed on Mueller Hinton agar (HiMedia, India) and included a panel of 14 antibiotics belonging to different classes (Table 3). Isolates displaying an inhibition zone ≤ 10 mm (n = 61) were selected for further testing of Colistin resistance (mcr-1, mcr-2, mcr-3, mcr-4, mcr-5) genes.

Molecular Characterization
Bacterial Genomic DNA Isolation: Bacterial genomic DNA was extracted manually by phenol chloroform extraction method from Colistin resistance isolates. Extracted genomic DNA were stored at -20ºC for further analysis. Nano-Drop from Analytik Jena Thermal Cycler (Jena, Germany) was used to determine the DNA concentration and purity of the extracts.

Detection Of Colistin Resistance mcr Genes: Colistin
resistance associated genes (mcr-1, mcr-2, mcr-3, mcr-4, mcr-5) of Gram-negative isolates were determined by polymerase chain reaction (PCR) using appropriate primer sets. The primer sequences and corresponding annealing temperatures used in all PCR reactions in this study are listed in (Table 1)  were resistance to Colistin antibiotic with MIC ≥ 9-10 μg/Ml. The majority 67% (4/6) of K. pneumoniae Colistin-resistant strains were considered MDR. Although, the two P. vulgaris strains and one C. freundii strain were also considered as an MDR strains.  (Table 2). High resistance was observed against both amoxicillin (62%) and ampicillin (63%).

Discussion
In this study, the phenotypic and genomic characterized In the present study, the prevalence of mcr-1 gene was higher among K. pneumoniae 60% (6/10), and this is in contrast with those reported by Nahed Adam and Hisham N Altayb, 2017, they found that the majority of mcr-1 gene was detected among E. coli strains. This difference can be explained by the fact that they majority of their isolates were E. coli compared to this study; it was K. pneumoniae.
Surprisingly, in this study the prevalence of Colistin resistance among P. aeruginosa isolates was considered low which was similar to the reported rates from previous study [34]. Additionally, the MDR P. vulgaris and C. freundii isolates were both have maintained a very low Colistin resistance rate (Table 4). So far, a high mortality rates have been immensely associated with the colsitin-resistant Gram-negative infection [35][36][37][38][39]. Thus, an intense investigation is required to increase the therapeutic choices for such pathogens.
It seems in Gram-negative pathogens isolated from different clinical samples have acquired Colistin-resistant gene, either through mutation of the genes or acquisition of mcr gene from other bacteria. However, the relatively large number of isolates and the low prevalence of mcr gene carrying strains obtained from the patients suggests that the evolution of Colistin resistance is currently of major concern in hospital, this was the first report of mcr-1&2 genes detected among Gram-negative strain in Sudan at the National Ribat hospital in Khartoum.

Conclusion
In the past few years, the prevalence of Colistin-resistant pathogens has been emerged worldwide. In addition, Colistin

Disclosure
Authors declare that they have no conflicts of interest in this work.