Prevalence and Molecular Characterization of Carbapenem resistance Gram negative bacilli among hospitalized patients in Khartoum state

Carbapenems are broad-spectrum β-lactam antibiotics widely prescribed for the treatment of multidrug-resistant gram negative bacilli in systemic infections. In the past ten years the Carbapenem resistance among gram-negative bacilli is rapidly expanding across nosocomial infection isolates. This study was conducted to determine the prevalence and to characterize Carbapenem resistance genes among Gram negative bacilli (GNB) isolated from patients treated in hospitals in Khartoum state, Sudan. Methods A cross-sectional laboratory based study was conducted over six months at the microbiology department and Institute Endemic Diseases, University A total of 206 GNB isolates from different clinical specimens were analyzed for carbapenem resistance genes using phenotypic tests and armed by genes detection. Multiplex PCR was performed for each strain to detect the carbapenemase genes, including those encoding the NDM-, VIM-, and IMP -type Metallo-beta-lactamases, the class A KPC-type carbapenemases, and the class D OXA-48 enzyme. In addition to CTXM, TEM and SHV. DNA sequencing and bioinformatics analysis were used to detect genes subtypes.


Introduction
Carbapenems are important broad-spectrum β-lactam antibiotics widely prescribed for the curing of multidrug-resistant gram negative bacilli in systemic infections. Carbapenems have been considered as a robust antibiotic to treat the extended spectrum β-lactamases (ESBLs) in the past ten years [1]. ESBLs are one of the most common resistant genes distributed among gram negative bacilli through plasmids and transposons [2] and the novel β-lactamases with direct carbapenem-hydrolyzing activity has contributed to an increased prevalence of carbapenem resistant Enterobacteriaceae (CRE), which is causing therapeutic failure worldwide [3]. Carbapenemase enzymes including New Delhi Metallo-beta-lactamase ( NDM), veron integron metallo-beta-lactamases ( VIM), imipenemase ( IMP), Klebsiella pneumoniae carbapenemases ( KPC), and oxacillinase-48 ( OXA-48) [4]. These enzymes are encoded by what is termed carbapenem resistance determining genes (CRDG), which hydrolyze β-lactam drugs including Carbapenems and other β-lactam agents [5]. Moreover resistant to carbapenem can occur by other mechanisms including overproduction of ESBL or AmpC enzyme in combination with porin mutations by reduced outer membrane permeability and activation of multidrug e ux pumps in response to antibiotic exposure [6]. Carbapenem resistance genes are enhancing mechanism of antibiotic resistance among the family Enterobacteriaceae and non-lactose fermenting gram-negative bacilliin consequence of the selective pressure assessed by inadequate use of carbapenem and third generation cephalosporins [7]. Moreover plasmids coding for carbapenemase enzymes may carry co-resistance genes for other β-lactam and non β-lactam antibiotics [5]. Detection of carbapenemase production by clinical microbiology laboratory is essential to guide the clinicians to provide appropriate therapy and update treatment guild lines furthermore provide evidence on clinically observed treatment failure using molecular analysis.
The major problem in Sudan is the lack of control over antimicrobial use and despite the lack of speci c data about the incidence of AMR in Sudan. This study was designed to describe the current situation of carbapenemase producing Gram-negative bacteria and determine the resistance genes involved in Khartoum state, Sudan.

Study design and clinical strains
A cross-sectional laboratory based study was conducted at the microbiology department in Soba University Hospital and institute of Endemic Diseases, University of Khartoum; involving gram negative clinical bacterial isolates, that suspected as carbapenemase producing strains based on carbapenem sensitivity testing zone inhibition (zone size less than 20mm). These were isolated from a variety of clinical specimens: blood, urine, swabs, sputum, a tip of catheters, and body uids, between October 2016 and February 2017 from hospitalized patients in Soba University Hospital. Quality control strains used in antimicrobial susceptibility testing and the biochemical test had been used for primary identi cation [8].
Molecular identi cation (PCR) using species speci c primers for Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Universal primers (16SrRNA) were used for more con rmation Table.1. All these strains stored in 20% glycerol at -20 °C until use.
Phenotypic screening and con rmatory test for carbapenemase The bacterial isolates screened for carbapenemase production according to CLSI guidelines (CLSI, 2017). In this method, carbapenem antibiotics, meropenem (MEM) and imipenem (IPM) discs (10 g, each) (Mast Diagnostic, UK) were used and Isolates that showed a zone of inhibition ≤ 21 mm in diameter for meropenem were considered as suspected carbapenemase producers. Phenotypic con rmatory test for carbapenemases production by Boronic acid synergy test for class A β-lactamases, the EDTA synergy for Metallo-β-lactamase and the Modi ed Hodges Test (MHT) for detecting KPC and OXA-48 producers [10].

Detection of Carbapenemase encoding genes
The PCR was carried out using thermal cycler (analytikjena® Biometra TADVANCED, Germany), by using the following primers (Macrogen, Korea), bla VIM, bla IMP, bla NDM, bla NDM-1, bla KPC, bla OXA-48, blaTEM, bla SHV and bla CTX-M genes Table 2. The reaction was carried out in a total reaction volume of 25 μl (5μl Master mix of Maxime RT premix kit (iNtRON BIOTECHNOLOGY, Seongnam, Korea),, 0.6 μl of forward primer, 0.6 μl of reverse primer, 2μl of DNA and 16.8 μl deionized sterile water). The reaction was conducted for 30 cycles [6]. The purity and integrity of each PCR product were evaluated electrophoresis in a 2% agarose gel in TBE 1X, that contain 2.5 μl of (20mg/ml) ethidium bromide at 100V for 40 min.

DNA Sequencing
The PCR product of bla NDM genes and 16srRNA were puri ed and Sanger sequencing was performed by Macrogen Company (Seoul, Korea)..

Bioinformatics Analysis
First of all we ensure the ambiguous sites are correctly called and determined the overall quality of the sequences proofed the nucleotides chromatogram by using Finch TV software version 1.4.0 (http://www.geospiza.com/Products/ nchtv.shtml). Then nucleotides sequences of the NDM genes achieved were searched for sequence similarity using nucleotide BLAST [11] (http: //blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignment for highly similar sequences, which retrieved from NCBI using the MEGA version 7 software [12]. Phylogenetic tree of bla NDM genes and their evolutionary relationship with those obtained from database were conducted using MEGA version 7 [12].

Statistical analysis
Data were analyzed using SPSS software version 20.0. Cross tabulation was used to present the relations between data, qualitative data were performed through χ2test, and signi cance was set at p≤ 0.05.
Prevalence of carbapenemase producing gram-negative bacilli based on phenotypic tests Carbapenemase activity was detected in 171 (83%) of the 206 clinical isolates were positive for the production of one or more carbapenemase enzymes by phenotypic methods as the following 24 (14%) by MHT method and Boronic acid screen, 105 (61.5%) by the EDTA test and 42 (24.5%) of the isolates were positive for both EDTA and Boronic acid methods. Details of the carbapenemase activity among different strains by phenotypic tests are shown in Table 3. That mean the MBL type is the most prevalent type of carbapenemase hydrolysis enzyme among Gram-negative bacilli in Khartoum state, OXA and KPC types are present at a low level.
Prevalence and distribution of Carbapenemase genes among gram negative bacilli Carbapenemase genes were detected in 121 (70.7%) from 171 positive carbapenemase producing isolates using PCR, one or more carbapenemase genes were detected in the strains. blaNDM was detected in the highest rate among the isolates mainly in K. pneumonia, which was the species with the highest number of these genes. blaNDM was also detected higher in other strains A. baumannii, P. aeruginosa and E. coli. The most prevalent gene was blaNDM 107(88.4%), followed by blaIMP 7 (5.7%), blaOXA-48 5(4.1%), blaVIM 2 (1.6%) and blaKPC 0 (0%). And ESBL were detected among these strains, it showed a high prevalence in 183 isolates (88.8%) as the following CTXM 127(61.6%), SHV 84(40.7%) and TEM 80(38.8). The genes were unevenly distributed among the different study isolates for more details table 4.
The frequency of carbapenemase producer Gram-negative bacilli by type of specimens and hospital units Carbapenemase producers were more frequent distributed among clinical isolates from blood (36%) and then followed by wounds (24%), urine (21%), body uids (7%) and catheter tips and sputum (6%) for both.

Molecular characterization of NDM genes
Out of 107 NDM genes detected 75 (70 %) were NDM-1, other subtypes of NDM were identi ed by sequencing including NDM-5, and NDM-6. Figure 2 Bioinformatics analysis of NDM genes Fourteen samples were successfully sequenced by Macrogen Company. All sample showed 97-100% similarity with NDM genes from the NCBI database with accession number MF379688 and MG764089.

Multiple sequence alignment
The Nucleotide sequences of NDM Deoxyribonucleic acid (DNA) sequences were compared against DNA databank using BLASTp. Fourteen different NDM beta-lactamases genes were compared against the NDM in the database, (http://blast.ncbi.nlm.nih.gov/Blast.cgi), they have produced a signi cant alignment to NDM-1 beta-lactamase of Klebsiella pneumoniae (gb|MK425054|), and the strains were shown 97% to 100% identity.

Phylogenetic tree
The phylogenetic analysis of the NDM proteins sequences revealed that the NDM-1 and NDM-5 were related to the same NDM lineage as the Indian and Bangladeshi isolates. The NDM-6 gene was found as closed to NDM-6 from India, New Zealand, and the United States as shown in Figure 2.

Discussion
Carbapenems have become the drugs of choice for the treatment of severe nosocomial infections caused by Gram-negative bacilli; however, carbapenemase producing Gram-negative bacilli have been reported worldwide. CRE is a considerable health problem worldwide and associated with increased mortality. The rapid detection of carbapenem resistance and adequate treatment of such cases is therefore mandatory. This study was therefore undertaken, to determine the prevalence of different types of carbapenemase producing bacteria among Gram-negative bacilli isolated from various hospitalized patients in Khartoum State. Accurate detection of carbapenemase producing microorganisms is a challenge for the laboratories, requiring not only phenotypic tests but also genotypic tests for all genes associated with carbapenemase production. In the present study, among 206 isolates 171(83%) were positive by phenotypic analysis including strains with resistance to carbapenem. Furthermore, genotypic analysis detected 121 (70.7%) positive strains. This nding indicates that the studied resistance is not only associated with enzyme encoding genes but also due to other resistance mechanisms such as overproduction of ESBLs, porin loss or mutations [13,14].
The current situation according to this study, show that the prevalence of carbapenemase producing among different gram negative isolates is increasing up to (83%). This nding is higher than the incidence in a previous study conducted in Khartoum state in 2017 which showed the prevalence was 56% by phenotypic tests [15] and other done in 2013 by Ali reported the MBL was 37.7% among Pseudomonas spp. isolates in Khartoum state [16]. This high frequency of MBL in Khartoum state is a result of excessive use of third generation cephalosporins, in addition to selective of ESBL with prevalence 88%, make the treatment option of those patients was meropenem and excessive use of it lead to release of carbapenem resistance genes. This nding agrees with a study in Egypt reported carbapenem resistance rate was 62.7% among Enterobacteriaceae [17]. As well, carbapenem resistance has been observed in Africa in high rate study conducted by Okoche et a.,l in 2015 in Uganda. He found 28.6% of strains were carbapenemase producer [18]. In Tanzania the prevalence of carbapenemase producer was 35% [19] and higher incidence 68% In South Africa [20]. Low prevalence was observed in Nigeria 11.9% [21]. This nding in the poor populations in Africa may be as a result of unrestricted use of antibiotics in these countries where most people consume the antibiotics without prescription by a clinician [22].
Carbapenemase genes have been recognized during the past ten years, and these genes are associated with mobile genetic elements that allow their rapid circulation among bacterial strains, for instance, NDM type have a potential for rapid spread within the country and to other countries [23]. In this study, carbapenemase genes were detected by using PCR in 121 (70.7%) of the resistant isolates. The most prevalent gene among the isolates was blaNDM (88.4%) mainly in K. pneumonia and other gram negative bacilli including A. baumannii, P. aeruginosa and E. coli, this agrees with studies in India reported the NDM gene was observed between 31% and 55% of Carbapenemase resistance Enterobacteriaceae [24,25], and study in South Africa published the most carbapenemase gene was NDM among K. pneumonia (20). As well, NDM-1 was reported as the most common carbapenemase gene in Saudi Arabia and other Middle Eastern countries [26].
Carbapenemase genes are reported to be more frequent in some regions. For example KPC genes are dominant in some countries such as Greece, Israel, and USA, while NDM genes are prevalent in isolates reported from the Far East, India, and Pakistan [14]. Carbapenemase production in Turkey mostly occurs in OXA type genes (23). OXA-48 was reported rst from Turkey, subsequently followed by reports from Middle Asia and Europe as well [27]. In this current study the genes were unevenly distributed among the different study isolates. In comparison with other carbapenem resistant genes were detected in low prevalence comparing with NDM gene blaIMP (5.7%), blaOXA-48 (4.1%), blaVIM (1.6%) and blaKPC (0%). This nding disagrees with many studies, in Okoche study, the most common gene was blaVIM 1(0.7%), and blaNDM-1 (2.6%) was the lowest gene [18], while Mushi reported IMP types were the most predominant at (21.6%) in his study (19). Other studies reported blaOXA-48 was the most prevalence gene [15,28]. In our study KPC wasn't detected among the isolates that disagree with international reports of high prevalence of KPC genes [14,29].
The blaNDM-1 was rst identi ed in a clinical isolate of K. pneumoniae in New Delhi, India, and suddenly got disseminated around the world [30]. NDM variants have been described, differing by several amino acid changes. A rst variant, NDM-2, has been described in an A. baumannii clinical isolate from Egyptian patient in Germany, NDM-4, NDM-5, NDM-6 have been detected from E. coli in India and NDM-7 from E.coli in France [30]. In this study, 107 NDM producer strains had been identi ed using PCR, the most subtype 75 (70 %) were NDM-1other subtypes of NDM were detected by sequencing including NDM-5, and NDM-6 among different Gram negative bacilli including K. pneumoniae, E. coli, A. baumannii, P. aeruginosa and Enterobacter spp.
Carbapenemase-encoding genes had been commonly associated with bacteria isolated from blood, urine, wound swabs, and sputum as reported in many studies in Uganda [19], Tanzania [34], Nigeria [21], and India [35]. In this study Carbapenem producer were more frequently isolated from blood (39%) followed by wound (25%) and urine (22%) this is in line with study in South Africa which reported blood was the most common specimen type (25%), followed by urine (22%) [20].
Many studies considered young age as a risk factor for CRE infection which agrees with current nding, that carbapenemase-producing Gram negative bacilli were most frequent in neonate age group isolated from nursery and pediatric wards (26% and 18% respectively). Besides, carbapenemase producers were observed in high rate among elderly patients from medicine (22%) and ICU (12%), which agrees with another study that found that CRE to be more frequently isolated in the elderly [36].
Carbapenem resistance gram negative bacilli are usually resistant to other routinely used antimicrobial agents [37][38][39]. The Plasmids carrying carbapenemase genes like NDM-1 are diverse and can harbor a high number of additional resistance genes (e.g., ESBL-alleles) as well as other carbapenemase genes like Oxa-48 types, VIM types, and so forth, as the source of multidrug resistance in one single bacterium [25,40]. Moreover mechanisms of resistance to β-lactam by producing ESBL, AmpC and carbapenemase were also noticed as some of the isolates produce different combinations of the enzymes. In our study co-resistance of NDM with OXA-48, VIM and IMP were reported in few strains. Co-resistance with ESBL (CTXM, SHV and TEM) was detected in high prevalence 87/107 (81.3%) of NDM positive isolates. Most of the strains carried NDM with one ESBL gene in (43.5%), NDM with two ESBL genes in (39.2%) and NDM with three ESBL genes in (17.3%). That with harmony with various studies reported co-resistance among clinical strains [41,42]. These co-production genes among some isolates as observed in this study are indicative of the existence of multi-drug resistant bacteria pathogens. That may be responsible for treatment failure and outbreaks of infections caused by resistant organisms. Resulting in more hospitalization and higher treatment costs as well as disease complications [43].

Conclusion
The prevalence of carbapenemase producing bacilli has been increasing in our setting which worries microbiologists as well as clinicians to prescribing carbapenems. NDM was found to be the most prevalent carbapenemase genes among clinical isolates and belong to Indian lineage. That we need for implementation of drastic infection control measures and regular surveillance to prevent further spread of these resistant organisms among the hospital isolates.In addition,screening for carbapenemase production should be performed in any Gram-negative isolates with any slight decrease in susceptibility to carbapenems. Figure 1 Antimicrobial Resistance pattern among different Gram-negative bacilli isolated from patients treated at Khartoum state hospitals.

Figure 2
Phylogenetic tree of the 14 NDM isolated from different Gram-negative bacilli: Phylogenetic tree of the 14 NDM genes, Sequences were analysed using MEGA7, the neighbor-joining method, and bootstrap analysis (1,000 replicates) based on the ClustalW algorithm. The scale bar indicates 0.1 nucleotide substitutions per site. Reference sequences shown as: accession number, gene subtype, country. Sequences isolated in this study are designated by grey triangle.

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