Detection and Characterization of Carbapenem resistant Gram-negative bacilli isolates recovered from hospitalized patients at Soba University Hospital, Sudan

Background: Antimicrobial resistance (AMR) poses a threat to global health security. Whilst over the past decade, there has been an increase in reports of nosocomial infections globally caused by carbapenem resistant Gram-negative bacilli (GNB), data from Africa have been scanty. We performed a study of carbapenem resistance genes among GNB isolated from patients treated in hospitals in Khartoum state, Sudan. Methods: A cross-sectional study was conducted at Soba University Hospital (SUH) and Institute of Endemic Diseases, University of Khartoum for the period October 2016 to February 2017. 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 the blaNDM, blaVIM, blaIMP, blaKPC, and blaOXA-48. In addition to blaCTXM, blaTEM and blaSHV. DNA sequencing and bioinformatics analysis were used to detect genes subtypes.


Introduction
The prevalence and distribution of antimicrobial resistant bacterial infections in the nosocomial setting in Africa is poorly de ned (1,2). 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 (3). ESBLs are one of the most common β-lactamases encoding resistance genes distributed among Gram negative bacilli through plasmids and transposons (4) and the novel βlactamases with direct carbapenem-hydrolyzing activity has contributed to an increased prevalence of carbapenem resistant Enterobacteriaceae (CRE), which is causing therapeutic failure worldwide (5). Carbapenemase enzymes include New Delhi Metallo-beta-lactamase ( NDM), veron integron metallobeta-lactamases ( VIM), imipenemase ( IMP), Klebsiella pneumoniae carbapenemases ( KPC), and oxacillinase-48 ( OXA-48 ) (6). 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 (7). Moreover resistance 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 (8).
Carbapenem resistance genes enhance the mechanism of antibiotic resistance among Enterobacteriaceae and non-lactose fermenting Gram-negative bacilli in consequence of the selective pressure assessed by inadequate use of carbapenem and third generation cephalosporins (9). Moreover plasmids coding for carbapenemases may carry co-resistance genes for other β-lactam and non β-lactam antibiotics (7).
Detection of carbapenemase producing isolates by clinical Microbiology laboratory is essential to provide appropriate therapy and update therapeutic guide-lines for the clinicians. Furthermore the use of molecular analysis to detect resistance genes provides evidence on clinically observed treatment failure. Whilst over the past decade, there has been an increase in reports of nosocomial infections globally caused by carbapenem resistant Gram-negative bacilli (GNB), data from Africa have been scanty and antimicrobial stewardship is not optimally practiced. We performed a study of carbapenem resistance genes among GNB isolated from patients treated in hospitals in Soba University Hospital Khartoum state.

Study design and clinical Isolates
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, suspected as carbapenemase producing based on carbapenem sensitivity testing zone inhibition (zone size less than 20 mm). These were isolated from cultures of varios clinical specimens: blood, urine, wound swabs, sputum, tips of catheters, and other body uids, between 1st October 2016 and 25th February 2017 from hospitalized patients in Soba University Hospital. Quality control strains [E. coli (ATCC #25922) and P. aeruginosa (ATCC #27853)] used in antimicrobial susceptibility testing and the biochemical test had been used for primary identi cation (10). Molecular identi cation (PCR) was used for all study isolates to con rm the biochemical identi cation using species speci c primers for Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii. A universal primer (16SrRNA) was used for identi cation of unknown species by biochemical tests.  (8). The purity and integrity of each PCR product was evaluated and the speci c ampli ed product was detected by comparing with standard DNA ladder.

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 (13) (http: //blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignment for highly similar sequences, were retrieved from NCBI using the MEGA version 7 software (14). Phylogenetic tree of bla NDM genes and their evolutionary relationship with those obtained from database were conducted using MEGA version 7 (14).

Statistical analysis
Data were analyzed using SPSS software version 20.0. Cross tabulation was used to present the relationships between data of antimicrobial sensitivity, phenotypic tests and resistant genes detection among the study isolates, qualitative data were performed through χ2test, and signi cance was set at p ≤ 0.05.

Demographic distribution
The demographic characteristics of patients under study and the frequency of GNB isolates according to age groups are shown in ( Figure.1). The most of the isolates were from neonates less than one year old in 42.5% followed by adult group between 13-80 years 38.3% and 19.5% for age group between 1-12 years. Males 53.4% (110/206) were predominant among admitted wards patients and females 46.6% (96/206).

Prevalence of carbapenemase producing Gram-negative bacilli based on phenotypic tests
Carbapenemase activity was detected in 171 (83%) of the 206 clinical isolates, which 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 isolates by phenotypic tests are shown in Table 3. This suggests that 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 (58.7%) of the 206 study isolates using PCR, one or more carbapenemase genes were detected in the isolates. blaNDM was the most commonly detected among the isolates, mainly in K. pneumonia, which was the species with the highest number of these genes. blaNDM was also detected more often in 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%). ESBL were detected among these isolates, it showed a high prevalence in 183 isolates (88.8%) as the following blaCTXM 127(61.6%), blaSHV 84(40.7%) and blaTEM 80 (38.8). The genes were unevenly distributed among the different study isolates. For more details, see Table 4.
The frequency of carbapenemase producer Gram-negative bacilli by type of specimens, hospital units and bacteria species isolates.
With regard to the distribution of Carbapenemase producer among hospital units, the most carbapenemase producing isolates associated with patients were found in the neonatal intensive care unit NICU 32(26%), followed by medicine wards 26

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 to be close to NDM-6 from India, New Zealand, and the United States as shown in Fig. 3.

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. Carbapenem resistant Enterobacteriaceae (CRE) are a considerable health problem worldwide and associated with increased mortality, therefore rapid detection of carbapenem resistance and adequate treatment of such cases is mandatory. This study was undertaken, to determine the prevalence of different types of carbapenemase producing bacteria among Gram-negative bacilli isolated from various hospitalized patients in Khartoum State. The accurate detection of carbapenemase producing microorganisms is challenging for laboratories, it not only requires phenotypic tests but also genotypic tests to detect of all genes associated with carbapenemase production. In the present study, among 206 isolates 171(83%) were positive by phenotypic analysis including isolates with resistance to carbapenem, whilst, genotypic analysis detected 121 (58.7%) positive isolates. This nding indicates that the studied carbapenem resistance is not only associated with enzyme encoding resistant genes but also due to other resistance mechanisms such as overproduction of ESBLs, porin loss or mutation (15,16).
The current situation according to this study, show that the prevalence of carbapenemase production 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 (unpublished data) and other done in 2013 by Ali reported the MBL was 37.7% among Pseudomonas spp. isolates in Khartoum state (unpublished data). This high frequency of MBL in Khartoum state is a result of the excessive use of meropenem in the treatment of patients associated with ESBL infections. This nding agrees with a study in Egypt, which reported that carbapenem resistance rate was 62.7% among Enterobacteriaceae (17). High rates of carbapenem resistance haves also been observed in Uganda in a study conducted by Okoche in 2015. He found 28.6% of isolates were produced carbapenemases (18). In Tanzania the prevalence of carbapenemase producing isolates was 35% (19), in South Africa it was found to be 68% (20). and in Nigeria 11.9% (21). Carbapenem resistance in low and middle income countries in Africa are likely to be increase as long as the use of antibiotics in these countries remains unrestricted and most people consume the antibiotics without a clinical prescription (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 isolates, for instance, blaNDM 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 (58.7%) of the study 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 that reported the blaNDM gene was observed between 31% and 55% of Carbapenemase resistant Enterobacteriaceae (24,25), and a study in South Africa published the most carbapenemase gene was blaNDM among K. pneumonia (20). BlaNDM-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 blaKPC genes are dominant in some countries such as Greece, Israel, and USA, while blaNDM genes are prevalent in isolates reported from the Far East, India, and Pakistan (16). Carbapenemase production in Turkey mostly occurs in blaOXA genes (23). OXA-48 was reported rst from Turkey, then followed by reports from Middle Asia and Europe (27). In this current study the genes were unevenly distributed among the different study bacterial isolates. In this study, the blaNDM gene was found in high prevalence (88.4%) compared to other genes, such as blaIMP (5.7%), blaOXA-48 (4.1%), blaVIM (1.6%) and blaKPC (0%). Our nding disagrees with many studies, for instance in the 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 prevalent gene (28,29). In this study blaKPC wasn't detected among the isolates, which that disagrees with global reports of high prevalence of blaKPC genes among international isolates (16,30).
The blaNDM-1 gene was rst identi ed in a clinical isolate of K. pneumoniae in New Delhi, India, and has since been disseminated around the world (31). NDM variants have been described, differing by several amino acid changes. A rst variant, blaNDM-2, has been described in an A. baumannii clinical isolate from an Egyptian patient in Germany, blaNDM-4, blaNDM-5 and blaNDM-6 have been detected from E. coli in India and blaNDM-7 from E.coli in France (31). In this study, 107 blaNDM producer isolates had been identi ed using PCR, the most common subtype 75 (70%) was blaNDM-1. Other subtypes of blaNDM were detected by sequencing including blaNDM-5, and blaNDM-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, wounds, and sputum as reported in many studies in Uganda (19), Tanzania (35), Nigeria (21), and India (36). In this study Carbapenem producers were more frequently isolated from blood (39%) followed by wounds (25%) and urine (22%). This is in line with a study in South Africa which reported blood was the most common specimen type (25%), followed by urine (22%) (20).
Many studies considered young patient 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). High rates of carbapenem resistant infections were observed 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 (37).
Carbapenem resistant Gram negative bacilli are usually resistant to other routinely used antimicrobial agents (38)(39)(40). The plasmids carrying carbapenemase genes like blaNDM-1 are diverse and can harbor a high number of additional resistance genes (e.g., ESBL-alleles) as well as other carbapenemase genes like blaOxa-48, blaVIM. These plasmids were considered as the source of multidrug resistance in one single bacterium (25,41). Moreover, mechanisms of resistance to β-lactam by producing ESBL, AmpC and carbapenemase were also noticed among the isolates that produce different combinations of the enzymes. In this study co-resistance of blaNDM with blaOXA-48, blaVIM and blaIMP were reported in few isolates. In connection to co-resistance with ESBL, blaCTXM, blaSHV and blaTEM was detected in high prevalence 87/107 (81.3%) of blaNDM positive isolates. Most of the isolates carried blaNDM with one ESBL gene (43.5%), blaNDM with two ESBL genes (39.2%) and blaNDM with three ESBL genes (17.3%). This agrees with various studies which have reported co-resistance among clinical isolates (42,43). These co-production genes among some isolates as observed in this study are indicative of the existence of multi-drug resistant pathogens, which are responsible for treatment failure and outbreaks of infections.
which impact on treatment outcomes and higher treatment costs (44).
Sudan is a large country which shares its borders with other seven countries. People move freely between these borders with the potential of passaging antibiotic resistance strains. The dynamic movements of the people will make it challenging to monitor AMRs in these countries especially at the boarders. These challenges may also represent an opportunity for a wider continental monitoring collaboration between the countries rather than country-speci c. Such approach will aid in universal and intergovernmental initiative to control and limit spread of AMRS.

Conclusion
The increasing numbers of carbapenemase producing bacilli isolated, particularly with the blaNDM carbapenemase genes among clinical isolates and belong to Indian lineage. Improved antibiotic stewardship and infection control measures, and close surveillance across all hospitals in Sudan is required. The relative distribution of distribution of Carbapenemase genes among GNB in nosocomial infections in Africa requires to be de ned.

Declarations
Ethics approval and consent to participate:     Sequences isolated in this study are designated by grey triangle.