Detection and Characterisation of Carbapenem Resistant Gram-negative Bacilli Isolates Recovered From Hospitalised 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). This study aimed to detect and characterize carbapenem resistance Gram negative bacteria isolated from hospitalized patients in Soba University Hospital (SUH) in Khartoum State, Sudan Results: A total of 206 GNB isolates from different clinical specimens were obtained from hospitalised patients between October 2016 to February 2017. Of 206 isolates, 171 (83%) were confirmed resistant phenotypically and 121 (58.7%) isolates were positive for the presence of one or more carbapenemase genes. New Delhi metallo-β-lactamase (NDM) types were the most predominant genes, blaNDM 107(52%). Others included blaIMP 7 (3.4%), blaOXA-48 5(2.4%), blaVIM 2 (0.9%) and blaKPC 0 (0%). Co-resistance genes with NDM producing GNB were detected in 87 (81.3%) of all blaNDM positive isolates. A significant association between phenotypic and genotypic resistance was observed (P < 0.001). NDM1 was the most frequent subtype observed in 75 (70 %) isolates which clusters to the Indian lineage.Conclusions: The frequency of carbapenemase producing bacilli was found to be improperly high in SUH. NDM was found to be the most prevalent carbapenemase gene among clinical isolates. Close surveillance across all hospitals in Sudan is required. The relative distribution of carbapenemase genes among GNB in nosocomial infections in Africa needs to be defined.


Background
The prevalence and distribution of antimicrobial resistant bacterial infections in the nosocomial settings in Africa is poorly de ned [1,2]. Carbapenems have been considered as a robust antibiotic to treat extended spectrum β-lactamase resistant bacteria (ESBL) in the past ten years and are widely prescribed for treatment of multidrug-resistant Gram negative bacilli in systemic infections [3]. The genes encoding ESBL are one of the most commonly distributed among Gram negative bacilli through plasmids and transposons [4]. The novel β-lactamases with direct carbapenem-hydrolysing activity have contributed to an increased prevalence of carbapenem resistant Enterobacteriaceae (CRE), which is causing therapeutic failure worldwide [4]. CRE, carbapenem-resistant Pseudomonas aeruginosa, and carbapenemresistant Acinetobacter baumannii were rated in the critical and the highest priority pathogen ranks by World Health Organisation (WHO) in 2017 [5].
Carbapenem resistance genes enhance the mechanism of antibiotic resistance among Enterobacteriaceae and non-lactose fermenting Gram-negative bacilli in response to inappropriate of use carbapenem and third generation cephalosporins [9]. These plasmids encoding for carbapenemases may also carry co-resistance genes for resistance to other β-lactam and non β-lactam antibiotics [7]. Detection of carbapenemase producing isolates by clinical microbiology laboratories is essential to provide targeted therapy, antimicrobial stewardship and update local antibiotic guide-lines for clinicians. Furthermore, the use of molecular analysis to detect resistance genes provides con rmation of 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. This study aimed to detect and characterize carbapenem resistance GNB isolated from patients treated at Soba University Hospital in Khartoum state, Sudan.

Demographic distribution
The demographic characteristics of the inpatients and the frequency of GNB isolates according to age groups are shown in Figure 1. Most of the isolates were from pediatric patients less than one year old (42.5%), followed by age group 13-80 years (38%) and the remainder of paediatric patients age group 1-12 years (19.5%). Males 53.4% (110/206) were predominant among inpatients with females at 46.6% (96/206).

Antimicrobial susceptibility
The antibiotic resistance pattern is shown in Figure 2. Out of 206 isolates tested, the highest percentage resistance was 98% and 93.5%, for ampicillin and cephalexin respectively, followed by amoxicillin 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(52%), followed by blaIMP 7 (3.4%), blaOXA-48 5(2.4%), blaVIM 2 (0.9%) and blaKPC 0 (0%). ESBL were detected among these isolates with high prevalence in 183 isolates (88.8%) as the following; blaCTXM 126(61.6%), blaSHV 84(40.7%) and blaTEM 78 (37.8). The genes were unevenly distributed among the different study isolates and more details are given in Table 2.

Bioinformatics analysis of blaNDM genes
A subset samples were analysed to con rm the presumed most prevalent (NDM) gene type. Fourteen samples were sequenced and all showed 97-100% similarity with blaNDM genes from the NCBI database with accession number MF379688 and MG764089.
Multiple sequence alignment: The nucleotide sequence of NDM Deoxyribonucleic acid (DNA) sequences were compared against the DNA databank using BLASTp. Fourteen NDM beta-lactamase genes were compared against those NDM genes recorded in the database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The alignment of the NDM genes in the isolates were shown to have identical similarities within range of 97-100% with those available in the database.When multiple sequence alignment of NDM proteins was undertaken using

Phylogenetic tree
The phylogenetic analysis of the NDM protein sequences revealed that 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 Figure 3.

Discussion
Carbapenems have become the drug 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 globally and are 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 hospitalised patients at Soba University Hospital, Khartoum State, Sudan. The accurate detection of carbapenemase producing microorganisms is challenging for laboratories and requires phenotypic and genotypic tests to detect all genes associated with carbapenemase production. Of 206 isolates, 171(83%) were positive by phenotypic analysis, including isolates with resistance to carbapenem.
Genotypic analysis detected 121 (58.7%) positive isolates. The 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 which were detected among 183 (88.8%) of the study isolates, in addition to porin loss or mutation [10,11].
The current situation according to this study shows that the prevalence of carbapenemase production among different Gram negative isolates is increasing (up to 83%). This nding is higher than the incidence observed in a previous study conducted in Khartoum State in 2017 which showed the prevalence was 56% by phenotypic tests (unpublished data). Other studies completed in reported the MBL as 37.7% among Pseudomonasspp. isolated 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. our results are in agreement with a study in Egypt, which reported that carbapenem resistance was 62.7% among Enterobacteriaceae [12]. High rates of carbapenem resistance have also been observed in Uganda in a study conducted by Okoche in 2015.
Okoche found 28.6% of isolates were carbapenemase producers [13]. In Tanzania, the prevalence of carbapenemase producing isolates was 35% [14]. In South Africa, it was found to be 68% [15] and in Nigeria 11.9% [16]. Carbapenem resistance in low and middle income countries (LMICs) in Africa is likely to increase as result of unrestricted usage of antibiotics in LMICS as the majority of the population consume antibiotics without a clinical prescription [17].
Carbapenemase genes have been recently recognised and these genes are associated with mobile genetic elements that allow their rapid circulation among bacterial isolates. For instance, blaNDM have potential for rapid spread within Turkey and to other countries [18]. In this study, carbapenemase genes were detected using PCR in 121 (58.7%) of the sampled 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 is in keeping with studies in India, South Africa, Saudia Arabia and other Middle East countries [15,19,20,21]. A study in India, reported the blaNDM gene was observed between 31% and 55% of carbapenemase resistant Enterobacteriaceae [19,20]. In South Africa, blaNDM was the highest carbapenemase gene among K. pneumonia [15]. BlaNDM-1 was reported as the most common carbapenemase gene in Saudi Arabia and other Middle Eastern countries [21].
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 [11]. Carbapenemase production in Turkey mostly occurs in blaOXA genes [18]. OXA-48 was rst reported from Turkey, followed by reports from Middle Asia and Europe [22]. In the current study, the genes were unevenly distributed among the different bacterial isolates. The blaNDM gene was found in high prevalence (52%) compared to other genes, such as blaIMP (3.4%), blaOXA-48 (2.4%), blaVIM (0.9%) and blaKPC (0%). Our nding, however, differs with several studies. For instance in the Okoche study, the most common gene was blaVIM (10.7%), and blaNDM-1 (2.6%) was the lowest gene [13], while Mushi reported IMP types were the most predominant at 21.6% in his study [14]. Other studies reported blaOXA-48 was the most prevalent gene [23,24]. In this study, blaKPC was not detected among the isolates which is not in keeping with global reports of high prevalence of blaKPC genes among international isolates [11,25].
The blaNDM-1 gene was rst identi ed in a clinical isolate of K. pneumoniae in New Delhi, India in 2010, and has since been disseminated across the globe [26]. 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 [26]. 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 producers are becoming highly distributed among Enterobacteriaceae, A. baumannii, P. aeruginosa and other Gram-negative bacilli. The prevalence of carbapenemase production in each species in this study was highest in K. pneumoniae (41.5%) followed by P.  [29]. In Nigeria, the highest prevalence of carbapenemase producers was in P. mirabilis (16.0%), then P. aeruginosa, K.pneumoniae (13.3% each) and E. coli (11.5%) [16], while in Tanzania E. coli was the most prevalent species with carbapenemase production (14%) followed by; K. pneumoniae (10.57%), P. aeruginosa (10.13%), K. oxytoca (1.76%) and A. baumannii (1.3%) [14]. The prevalence of carbapenemase producing isolates varies from hospital to hospital. This variation could be attributed to differences in collection time of isolates, study designs and target populations.
Carbapenemase encoding genes have been commonly associated with bacteria isolated from blood, urine, wounds and sputum as reported in many studies in Uganda [13], Tanzania [14], Nigeria [16] and India [30]. In this nding, carbapenem producers were more frequently isolated from blood (39%) followed by wounds (25%) and urine (22%). This is compatible with a study in South Africa which reported blood was the most common specimen type (25%), followed by urine (22%) [15].
Young patient age has long been considered as a risk factor for Carbapenem resistant Enterobacteriaceae (CRE) infection which agrees with current nding that carbapenemase producing Gram negative bacilli were most frequent in children less than one year of age, located in the nursery and pediatric wards with 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 CRE to be more frequently isolated in the elderly (31).
Carbapenem resistant Gram negative bacilli are usually resistant to other routinely used antimicrobial agents [32][33][34]. 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 [20,35]. Moreover, mechanisms of resistance to β-lactam antibiotics by producing ESBL, AmpC and carbapenemase were also noticed among the isolates that produce different combinations of the enzymes. At the present 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 [36,37]. 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. These multidrug resistant pathogens impact on treatment outcomes and result in higher treatment costs [38].
Sudan is a large country which shares its borders with seven other countries. People move freely between these borders with the potential passage of antibiotic resistance strains. The dynamic movements of people will make it challenging to monitor AMR in these countries especially at the borders. These challenges may also represent an opportunity for wider continental monitoring and collaboration between countries rather than country speci c. Such an approach will aid in universal and intergovernmental initiatives to control and limit the spread of the AMR.

Conclusions
The frequency of carbapenemase producing bacilli was found to be improperly high in SUH. blaNDM was found to be the most prevalent carbapenemase gene among clinical isolates. Improved antibiotic stewardship and infection control measures, and close surveillance across all hospitals in Sudan is required. The relative distribution of carbapenemase genes among GNB in nosocomial infections in Africa needs to be investigated.  [40] and molecular identi cation using PCR [8] was used for all study isolates with universal primer (16SrRNA). For species speci c isolates identi ed on biochemical testing, species speci c primers for Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii were used for con rmation Table S1. All isolates were stored in 20% glycerol at -20 °C until use.

Subculture and disk diffusion susceptibility testing
Selection of antimicrobial panels and interpretation of disk diffusion for each bacteria was completed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [39].
Phenotypic screening and con rmatory test for carbapenemase Bacterial isolates were screened for carbapenemase production according to CLSI guidelines (CLSI, 2017). In this method, meropenem and imipenem discs (10 µg, each) (Mast Diagnostic, UK) were used.
Isolates that showed intermediate or resistant to imipenem or meropenem were considered as suspected carbapenemase producers. Phenotypic con rmatory tests for carbapenemases production were applied by boronic acid synergy test for class A β-lactamases, the EDTA synergy for metallo-β-lactamase and the Modi ed Hodge Test (MHT) for Enterobactercea to detect KPC and OXA-48 producers in addition to temocillin sensitivity [41].
The purity and integrity of each PCR product was evaluated and the ampli ed product was con rmed with reference to standard DNA ladder.

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

Bioinformatics analysis
Firstly we ensured the ambiguous sites were clari ed and determined the overall quality of the sequences by reviewing nucleotide chromatogram by using Finch TV software version 1.4.0 (http://www.geospiza.com/Products/ nchtv.shtml). Thereafter, nucleotide sequences of the NDM genes identi ed were searched for sequence similarity using nucleotide BLAST [42] (http: //blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignment for highly similar sequences, was retrieved from NCBI using the MEGA version 7 software [43]. Phylogenetic tree of bla NDM genes and their evolutionary relationship with those obtained from the NCBI database were completed using MEGA version 7 [43].

Statistical analysis
Data were analysed using SPSS software version 20.0. Cross tabulation was used to present the relationships between data of antimicrobial sensitivity, phenotypic tests and resistant gene detection among the study isolates, qualitative data were performed through χ 2 test and signi cance was set at p≤ 0.05. Availability of data and materials