The Emergence of Carbapenem- and Colistin-Resistant Enterobacteria in Senegal

Antibiotic resistance is a public health problem. The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a concern, particularly in Senegal. (1) Methods: Between January 2019 and July 2022, 240 isolates of enterobacteria resistant to third-generation cephalosporins and imipenem from biological samples from Fann Hospital (Dakar) and Hôpital Paix (Ziguinchor) were selected. The isolates were identified by MALDI-TOF mass spectrometry, and susceptibility tests were performed by the disk diffusion method. Antibiotic-resistance genes for class A beta-lactamases, carbapenemases, and plasmid resistance to colistin resistance (mcr-1–8) were screened by RT-PCR. (2) Results: The 240 enterobacteria were composed of: Escherichia coli (60.83%), Klebsiella pneumoniae (21.67%), Enterobacter cloacae (13.75%), Citrobacter freundii (2.08%), Serratia marcescens (0.83%), Klebsiella aerogenes (0.42%), and Proteus mirabilis (0.42%). Class A beta-lactamase genes were found in 229 isolates (70.41% blaTEM, 37.5% blaSHV, 83.75% blaCTX-A, and 0.42% blaCTX-B). The carbapenemase genes blaOXA-48 and blaNDM were found in 25 isolates, including 14 isolates with blaOXA-48, 13 isolates with blaNDM, and 2 isolates with both genes simultaneously. The mcr-8 gene was found in one isolate of E. cloacae. (3) Conclusions: The epidemiology of antibiotic-resistance genes in enterobacteria in Senegal shows the emergence of CPEs. This phenomenon is worrying, and rigorous surveillance is necessary to avoid further spread.


Introduction
Antibiotic resistance in enterobacterales is a public health problem that represents a threat to humans, animals, and the environment [1]. Multidrug-resistant enterobacterales infections result in longer hospital stays, higher patient care costs, and increased mortality [2]. Some of these infections are of nosocomial origin, thus complicating patient management [3,4]. Bacteria such as ESBL-producing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa are frequently isolated in hospital-acquired infections [5]. Antimicrobial resistance is one of the major health issues of this century and has a global impact on human health [6]. The global spread of antibiotic resistance and its mechanisms are constantly evolving and are often linked to countries' antibiotics strategies [6,7]. Limited data are available to assess the level of antimicrobial resistance currently observed in developing countries (DCs), particularly in West Africa [8][9][10]. For instance, in Senegal, antibiotics are often prescribed empirically [11]. Overuse of thirdgeneration cephalosporins (C3G) has favored the emergence of resistance toward these antibiotics [12,13]. As a result, carbapenems have been used since 2008 to combat ESBLproducing Enterobacteriaceae [14][15][16].
Although, colistin is mainly used in veterinary medicine [17], it has become increasingly used in some countries as a last-line therapeutic treatment against carbapenemresistant isolates [18]. The introduction of these antibiotics into the clinical setting has led to the appearance and emergence of bacteria which are resistant to them [10,17,19].
The main mechanism of beta-lactam resistance in enterobacterales is enzymatic inactivation caused by proteins, known as beta-lactamases, encoded by antibiotic resistance genes [20]. The widespread of ESBL genes across a wide variety of species is mainly due to the presence of these genes on plasmids that facilitate their dissemination [21]. This also applies to the spread of carbapenemases as well as the plasmid-mediated colistin resistance genes "mcr". Since 2009, a transferable colistin resistance mechanism (plasmidmediated mcr gene) has been described worldwide [22,23]. Colistin resistance can also be chromosomal and is mainly mediated in Enterobacteriaceae via modification of the bacterial outer membrane by alteration of lipopolysaccharide chains (LPSs) [24]. This resistance also involves the secondary resistome, as inactivation of a gene not essential to bacterial multiplication and not involved in colistin resistance (dedA) restores its sensitivity [25,26]. In Senegal and Benin, TEM, SHV, OXA-1 and CTX-M-15 beta-lactamases are well disseminated [27][28][29]. Available clinical studies on the epidemiology of Carbapenemase-producing Enterobacteriaceae (CPE) in West and East Africa show the presence of carbapenemase genes in Cameroon (bla NDM-4 ), Kenya (bla NDM-1 ), Sierra Leone (bla VIM and bla DIM-1 ), Senegal (bla OXA-48 ), and Tanzania (bla KPC , bla IMP , bla OXA-48 , bla VIM, and bla NDM ) [14]. OXA-48 is the major carbapenemase produced by enterobacteria and the most common enzyme that has emerged in all Mediterranean countries as well as in Africa [30,31]. The first carbapenemase gene described in Senegal was OXA-48 in 2008, but there are probably some that predate this, as few regular epidemiological studies have taken place in Senegal [15,32]. Recently, the emergence of New Delhi metallo-β-lactamase (NDM-type) carbapenemases in Senegal and in the rest of Africa among Acinetobacter baumannii isolates has been reported [14,33].
The objective of this study was to update the epidemiology of class A beta-lactamases, carbapenemases, and plasmid-mediated colistin resistance in enterobacterales in two remote regions of Senegal: Dakar (west of Senegal) and Ziguinchor (south of Senegal). To do so, we performed a phenotypic and molecular study of antibiotic resistance and determined the genes associated with this resistance.

Selection of Isolates
Between January 2019 and July 2022, isolates of enterobacteria at Fann Hospital (Dakar, western Senegal) and Paix Hospital (Ziguinchor, southern Senegal) were selected. The selection was based on the results of an antibiogram performed on Mueller-Hinton agar with a champagne cork image and resistance to C3G (ceftriaxone, ceftazidime) and/or to imipenem. The selected enterobacteria were isolated from clinical samples received from the diagnostic laboratory as part of routine bacteriological testing. These samples came from patients who were hospitalized or who presented for a bacteriological check-up. At Fann Hospital, the samples were from the neurology, neurosurgery, infectious diseases, and otorhinolaryngology departments, whereas at Paix Hospital, the departments involved were urology, infectious diseases, otorhinolaryngology, and pediatrics.

Distribution of Class A Beta-Lactamase Genes
The class A beta-lactamase genes responsible were found in 229 isolates. The blaCTX-A, blaCTX-B, blaSHV, and blaTEM genes were found in 83.75%, 0.42%, 37.5%, and 70.41% of enterobacteria isolates, respectively ( Table 2). These genes were found in isolates from both hospitals ( Figure 2). The blaCTX-A gene was found in 84.2% of E. coli, 96.1% of K. pneumoniae, and 66.6% of E. cloacae. The blaNDM gene was found in 3.4% of E. coli, 7.7% of K. pneumoniae, and 12.1% of E. cloacae. The distribution of genes by year is detailed in Supplementary Table S1.

Distribution of Class A Beta-Lactamase Genes
The class A beta-lactamase genes responsible were found in 229 isolates. The bla CTX-A , bla CTX-B , bla SHV , and bla TEM genes were found in 83.75%, 0.42%, 37.5%, and 70.41% of enterobacteria isolates, respectively ( Table 2). These genes were found in isolates from both hospitals ( Figure 2). The bla CTX-A gene was found in 84.2% of E. coli, 96.1% of K. pneumoniae, and 66.6% of E. cloacae. The bla NDM gene was found in 3.4% of E. coli, 7.7% of K. pneumoniae, and 12.1% of E. cloacae. The distribution of genes by year is detailed in Supplementary Table S1.

Carbapenem Resistance Genes
The blaOXA-48 and blaNDM genes were found in 25 isolates (14 strains with blaOXA-4 13 strains with blaNDM) and with those two strains (K. pneumoniae and E. cloacae) w produced double carbapenemase. The blaOXA-48 gene was found in six isolates at Paix pital in Ziguinchor and in eight isolates at Fann Hospital in Dakar (Figure 2). The b gene was found in five isolates at Paix Hospital and in eight isolates at Fann Hospital blaOXA-48 gene was mainly observed in E. coli (3.4%), E. cloacae (15.1%), K. pneumoniae (5 and C. freundii (20%), while the blaNDM gene was mainly detected in E. coli (3.4%), E. cl (12.1%), and K. pneumoniae (7.7%) ( Table 2). The isolates with the blaOXA-48 and blaNDM g came from the neurology, neurosurgery, and infectious disease departments of Fann pital, while the isolates with the blaOXA-48 and blaNDM genes came from the urology, i tious disease, and otorhinolaryngology departments of Paix Hospital. The blaOXA-23, b 24, blaOXA-58, blaKPC, blaVIM, and blaIMP genes were not found in our isolates.

Plasmid Colistin Resistance Genes
The mcr-8 gene was found in an isolate of E. cloacae isolated from a patient hos ized in the infectious diseases department of Fann Hospital in Dakar (Figure 2, Tab None of our isolates harbored the mcr-1, mcr-2, mcr-3, mcr-4, or mcr-5 genes.

Discussion
In Senegal, the epidemiology of antibiotic resistance genes in clinically isolate terobacteria is poorly understood, and information is often outdated [27,32,43]. Ther pressing need, therefore, to strengthen and update this epidemiological data. Our s focused on a variety of enterobacteria which were resistant to C3G and/or imipenem all tested isolates, resistance to C3G and C4G was 88.3% and 79.9% in Dakar, respecti and 89.5% and 81.4% in Ziguinchor, respectively. Across the country, E. coli resistan ceftriaxone was 81.5%, and cefepime resistance was 75.2%. Class A beta-lactamase g such as blaTEM and/or blaSHV were found in 66% of the isolates, whereas blaCTX-A genes found in 84% of the E. coli isolates. The expression of the blaCTX-A genes confers high l of resistance toward C3G (cefotaxime, ceftriaxone, and ceftazidime) and aztreonam A previous study carried out between 2009 and 2010 at Fann Hospital (Dakar, Sen reported the detection of blaTEM, blaSHV, blaOXA-1, and blaCTX-M-15 genes, with a predomin

Carbapenem Resistance Genes
The bla OXA-48 and bla NDM genes were found in 25 isolates (14 strains with bla OXA-48 and 13 strains with bla NDM ) and with those two strains (K. pneumoniae and E. cloacae) which produced double carbapenemase. The bla OXA-48 gene was found in six isolates at Paix Hospital in Ziguinchor and in eight isolates at Fann Hospital in Dakar (Figure 2). The bla NDM gene was found in five isolates at Paix Hospital and in eight isolates at Fann Hospital. The bla OXA-48 gene was mainly observed in E. coli (3.4%), E. cloacae (15.1%), K. pneumoniae (5.7%), and C. freundii (20%), while the bla NDM gene was mainly detected in E. coli (3.4%), E. cloacae (12.1%), and K. pneumoniae (7.7%) ( Table 2). The isolates with the bla OXA-48 and bla NDM genes came from the neurology, neurosurgery, and infectious disease departments of Fann Hospital, while the isolates with the bla OXA-48 and bla NDM genes came from the urology, infectious disease, and otorhinolaryngology departments of Paix Hospital. The bla OXA-23 , bla OXA-24 , bla OXA-58 , bla KPC , bla VIM , and bla IMP genes were not found in our isolates.

Plasmid Colistin Resistance Genes
The mcr-8 gene was found in an isolate of E. cloacae isolated from a patient hospitalized in the infectious diseases department of Fann Hospital in Dakar (Figure 2, Table 2). None of our isolates harbored the mcr-1, mcr-2, mcr-3, mcr-4, or mcr-5 genes.

Discussion
In Senegal, the epidemiology of antibiotic resistance genes in clinically isolated enterobacteria is poorly understood, and information is often outdated [27,32,43]. There is a pressing need, therefore, to strengthen and update this epidemiological data. Our study focused on a variety of enterobacteria which were resistant to C3G and/or imipenem. For all tested isolates, resistance to C3G and C4G was 88.3% and 79.9% in Dakar, respectively, and 89.5% and 81.4% in Ziguinchor, respectively. Across the country, E. coli resistance to ceftriaxone was 81.5%, and cefepime resistance was 75.2%. Class A beta-lactamase genes such as bla TEM and/or bla SHV were found in 66% of the isolates, whereas bla CTX-A genes were found in 84% of the E. coli isolates. The expression of the bla CTX-A genes confers high levels of resistance toward C3G (cefotaxime, ceftriaxone, and ceftazidime) and aztreonam [44]. A previous study carried out between 2009 and 2010 at Fann Hospital (Dakar, Senegal) reported the detection of bla TEM , bla SHV , bla OXA-1 , and bla CTX-M-15 genes, with a predomi-nance of the bla CTX-M-15 gene in E. coli isolates [27]. The prevalence of class A beta-lactamase genes found in our study (bla TEM , bla SHV , bla CTX-A , and bla CTX-B in E. coli) is comparable to the results of the latter study and demonstrates the persistence and establishment of this antibiotic resistance genotype in this hospital over ten years. Similarly, in a study conducted in Benin, these same genotypes were found in enterobacterales, thus demonstrating their distribution in West Africa [28,29]. Apart from isolates of clinical origin, strains isolated from animals and the environment also harbored bla CTX-M genes [45], suggesting a synergy of action between the three sectors (the "One Health" concept). Indeed, most ESBL genes are carried by plasmids and, thus, are easily transferable between different species of enterobacteria [21]. The bla SHV , bla TEM , bla CTX-A , and bla CTX-B genes have also been described in other enterobacteria such as K. pneumoniae and E. cloacae isolates. Other studies carried out in Senegal found the bla CTX-M-15 gene in Morganella morganii and the bla CTX-M group 1 and bla CTX-M group 9 genes in other enterobacterial species (E. coli, K. pneumoniae, E. cloacae, and C. koseri) isolated from urine, thus demonstrating the diversity of clinical enterobacteria isolates carrying these antibiotic resistance genes [43,46]. The genotypic results in Senegal confirm the presence as well as the circulation of CTX-M ESBL genes in E. coli between the west and the south of the country. Our study therefore shows the evolution of ESBL epidemiology in this country. This is with the emergence of new ESBL-carrying isolates such as C. freundii, E. aerogenes, P. mirabilis, and S. marcescens, as well as the appearance of carbapenemases, as is the case in other parts of the world [47].
The carbapenemase genes (bla OXA-48 and bla NDM ) were found in E. coli, K. pneumoniae and E. cloacae species, resulting in resistance to imipenem and/or ertapenem. This resistance is low in Dakar and Ziguinchor, where ertapenem resistance is only 9.7% and 8.1%, respectively, and imipenem resistance is 3.9% and 1.2%, respectively. Previous studies conducted in two Senegal hospitals, in Dakar, reported the detection of the bla OXA-48 gene in 11 isolates of enterobacteria between 2008 and 2009. On the other hand, in Saint Louis, bla OXA-48 was detected in 49 isolates of K. pneumoniae isolated from urine in 2016 [15,19,32]. bla OXA-48 is mainly found in K. pneumoniae and in E. coli, although other enterobacterial species can express this carbapenemase. In our study, in addition to these two species, C. freundii and E. cloacae isolates also harbored this carbapenemase gene. In the 14 isolates carrying the bla OXA-48 gene, we found resistance to ceftriaxone and cefepime and sensitivity to imipenem, and nine showed resistance to ertapenem. The OXA-48 gene has been described as the phantom threat, due to its discrete phenotype in the absence of co-resistance mechanisms [48]. In addition to bla OXA-48 , we observed the bla NDM gene in 13 isolates of enterobacteria, including three isolates of K. pneumoniae, three isolates of E. cloacae, and four isolates of E. coli. Both carbapenemase genes were found in isolates from the Paix (Ziguinchor) as well as Fann hospitals (Dakar), showing a homogeneous epidemiology between the west and the south of the country. The NDM enzyme is one of the most common carbapenemases in enterobacterales that is also observed in A. baumannii isolates [49][50][51]. In Senegal, NDM was first described in 2016 in an isolate of A. baumannii, and the literature has not yet reported its detection in enterobacteria [14,15]. Our results show that the NDM gene is present in both enterobacterial isolates isolated in western and southern Senegal. Elsewhere in Africa, carbapenemases are more commonly described in Pseudomonas aeruginosa and in A. baumannii, with the NDM enzyme being less frequently observed than OXA-48 [14]. The few available studies on the epidemiology of CPEs in West and East Africa report the identification of carbapenemases in Cameroon (NDM-4), Kenya (NDM-1), Sierra Leone (VIM and DIM-1), Senegal (OXA-48), and Tanzania (KPC, IMP, OXA-48, VIM, and NDM) [14].
Besides carbapenemase genes, colistin plasmid-mediated resistance genes deserve special attention. Indeed, colistin is much more widely used in veterinary medicine [17,52]. In human medicine, it represents a therapeutic option of last resort for the treatment of severe infections caused by multidrug-resistant enterobacteria [53][54][55]. In our study, we found an E. cloacae isolate with the mcr-8 gene, which was sensitive to colistin (MIC = 0.25 mg/L) and another E. cloacae isolate which was heteroresistant to colistin. This heteroresistance is indeed natural in some isolates of E. cloacae [56]. The isolate with the mcr-8 gene was isolated from a patient (a vegetable farmer) hospitalized in the infectious diseases department (Fann Hospital) for bacteremia. The mcr-8 gene was first described in China in an isolate of K. pneumoniae of animal origin [57]. This gene is often detected in isolates of poultry meat origin in veterinary settings. In Senegal, a low prevalence of colistin resistance was observed in 93 E. coli (2.2%) isolated from poultry, with neither the mcr-1 nor mcr-2 gene being detected [17]. Furthermore, in Africa, mcr genes are rarely clinically described. In Algeria, the mcr-8 gene was found in clinically isolated K. pneumoniae [41]. In France, the mcr-1 gene has been described in an E. cloacae isolate [58]. In Senegal, resistance to colistin is rarely described clinically, as this molecule is not yet prescribed for use in humans. In fact, in countries with limited resources, such as Senegal, it is important to educate on and raise awareness of the correct use of antibiotics in order to combat the spread of antibiotic-resistant strains [59].

Conclusions
Although studies and epidemiological surveillance on antibiotic resistance remain limited in Senegal, this work has uncovered a new epidemiological aspect of antibioticresistant isolates in the country. There is a "persistence" of ESBL-producing E. coli isolates and a spread of ESBL as well as carbapenemase genes such as OXA-48 and NDM to other enterobacterial species. Even if colistin resistance appears to be anecdotal, it is important to monitor this emergence. In view of this, it has become of paramount importance to set up a regular surveillance system to prevent the large-scale dissemination of antibiotic-resistant bacteria, especially CPE.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens12080974/s1. Table S1: Distribution of strains isolated and antibiotic resistance genes detected by year; Table S2: Distribution of antibiotic resistance by bacterial species.