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 finding 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 finding 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 finding 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 finding 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 first 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 finding 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 first identified 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 first 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 identified 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 genes are becoming largely distributed among Enterobacteriaceae, A. baumannii, P. aeruginosa and other Gram-negative bacilli. The prevalence of carbapenemase producer in each species in this study, higher frequency in A. baumannii (37.3 %) followed by K. pneumoniae (27.3%), P. aeruginosa (24.8 %) and E. coli (21.1%), which agree with many studies that reported A. baumannii and K. pneumonia were the most predominant carbapenemase producer strains [31,32]. The prevalence of carbapenemase producer varies from area to area. This variation could be attributed to differences in time of collection of isolates and differences in study populations and designs. A study in Turkey showed the most carbapenemase strains were K. pneumoniae (13.6%), Pseudomonas spp. (17.8%), A. baumannii (13.8%), S. maltophilia 7.5% and E. coli 2.8% [33]. 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%) [21], while in Tanzania, E. coli was the most prevalent species with carbapenemase producing (14%), followed by K. pneumoniae (10.57%), P. aeruginosa (10.13%), K. oxytoca (1.76%) and A. baumannii (1.3%) [19].
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 finding, 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 [37–39]. 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].