Genetic Environments of Plasmid-Mediated blaCTXM-15 beta-lactamase gene in Enterobacteriaceae from Africa

The most widely distributed blaCTX-M gene on a global scale is blaCTX-M-15. The dissemination has been associated with clonal spread and different types of mobile genetic elements. This study aimed to review and describe the genetic environments of blaCTX-M-15 gene detected from Enterobacteriaceae in published literature from Africa. A literature search for relevant articles was done through PubMed, and Google Scholars electronic databases, 43 articles from 17 African countries were included in the review based on the eligibility criteria. Insertion sequences were reported as part of the genetic environment of blaCTX-M-15 gene in 32 studies, integrons in 13 studies, and plasmids in 23 studies. In this review, five insertion sequences including ISEcp1, IS26, orf447, IS903, and IS3 have been detected associated with the genetic environment of blaCTX-M-15 in Africa. Seven different genetic patterns were seen in blaCTX-M-15 genetic environment. Insertion sequence ISEcp1 was commonly located upstream of the end of the blaCTX-M-15 gene while insertion sequence orf477 was located downstream. In some studies, ISEcp1 was truncated upstream of blaCTX-M-15 by insertion sequences IS26 and IS3. Class 1 integron (Intl1) was most reported to be associated with blaCTX-M-15 (13 studies), with Intl1/dfrA17–aadA5 being the most common gene cassette array. IncFIA-FIB-FII multi-replicons and IncHI2 replicon types were the most common plasmid replicon types that horizontally transfer blaCTX-M-15 gene. Aminoglycoside modifying enzymes, and plasmid-mediated quinolone resistance genes were commonly collocated with blaCTX-M-15 gene on plasmids. This review revealed the predominant role of ISEcp1, Intl1and IncF plasmid in the mobilization and continental dissemination of the blaCTX-M-15 gene in Africa.


Introduction
The most widely distributed bla CTX-M gene on a global scale is bla CTX-M-15 , especially in the enterobacterial species such as Escherichia coli, Klebsiella spp. and Salmonella enterica [1,2]. The global dissemination of the bla CTX-M-15 gene has been associated with the clonal spread of E. coli O25: H4-ST131 strains and different types of mobile genetic elements (MGEs) such as insertion sequences, transposons, integrons, phage elements, and conjugative plasmids [1][2][3]. Of these MGEs, insertion sequences (IS) are of special concern because this mobile element can facilitate the independent transposition with insertion mutation and genetic rearrangements in Enterobacteriaceae [4][5][6]. Several types of IS elements have been recognized; however, ISEcp1, IS26, orf447 and ISCR1 have been frequently found to be responsible for the mobilization and expression of different antimicrobial resistance genes [7]. ISEcp1 is the most frequently reported IS type [7]. ISEcp1 is a member of the IS1380 family and was first identified on the plasmid pST01 in E. coli strain 79 but has now been globally disseminated in association with different bla CTX-M phylogenetic clusters [8].
The roles of ISEcp1 and other MGEs in the genetic environments of bla CTX-M genes have been well described [7,9,10]. ISEcp1 is commonly located upstream of the bla CTX-M-15 gene

Materials and Methods
The literature search was conducted in the PubMed, AJOL and Google Scholar electronic databases between June 2018 and January 2019 for the purpose of this narrative and non-systematic review. The following terms were used for the literature search: bla CTX-M-15 gene AND Africa OR bla CTX-M-15 AND genetic environment AND Africa. A literature search was also conducted based on studies reporting the detection of bla CTX-M-15 from each African country, e.g., bla CTX-M-15 AND Nigeria, bla CTX-M-15 AND Egypt, bla CTX-M- 15 and Kenya, etc. The reference lists of all eligible articles were further reviewed and used to carry out a supplementary literature search. The articles were further screened after the removal of duplicates by titles and abstracts for their relevance to the study objectives and purpose. The primary outcomes of interest were to describe the genetic environment of bla CTX-M-15 in Enterobacteriaceae from different African countries.
For studies to be included in the qualitative description, the studies must have reported the genetic environment of the bla CTXM-15 resistance gene with special reference to the associated insertion sequences. The data were abstracted onto an Excel (Microsoft Office Excel 2010) spreadsheet. For each eligible study, data extracted included: first author details, year of publication, country from which the study was conducted, sources of the samples (animal, human or environment), enterobacterial species in which the bla CTX-M-15 gene was detected, insertion sequences associated with the genetic environment, additional data on other mobile genetic elements including type of integron and associated gene cassette arrays, plasmid and associated replicon types, as well as additional antimicrobial resistance genes associated with the bla CTX-M-15 gene on different plasmids.

Discussion
This review was carried out to describe the genetic environments of the internationally disseminated bla CTX-M-15 gene in Enterobacteriaceae from Africa. Most of the studies in this review were from human clinical settings, which suggests that bla CTX-M-15 -producing Enterobacteriaceae are a challenge to healthcare facilities in Africa. The bla CTX-M-15 gene has been associated with the pandemic-initiating E. coli O25: H4 ST131 clone that causes both community and human healthcare infections globally [2]. Review of the genetic environments of bla CTX-M-15 in Enterobacteriaceae revealed five ISs including ISEcp1, IS26, orf447, IS903, and IS3 which had been detected in Africa. With the exception of a novel IS3 type that was reported from Angola [16], all the other ISs have been reported from other parts of the world to be associated with the genetic environment of different AMR genes in general [7,8,55]. From all the studies reviewed, ISEcp1 was typically located upstream of bla CTX-M-15 gene. This IS often encodes a transposase that facilitates the mobilization of bla CTX-M-15 gene among integrons, transposons, plasmids, and chromosomes, as well as provides promoters that can activate the weakly expressed state of bla CTX-M-15 [56][57][58]. This IS has been reported to contribute to the global dissemination of bla CTX-M-15 genes in association with other MGEs [1,59,60]. The ISEcp1/ bla CTX-M-15 genetic association observed in this review has previously been reported from other parts of the world including India, France, Turkey, Poland, Canada, the United Kingdom, Spain, and China [8][9][10]55,56,58,[61][62][63][64]. This IS element has also been commonly found associated with other bla CTX-M genes and other beta-lactamase resistance genes [10,59]. Of the genetic environments associated with bla CTX-M genes, ISEcp1 is one of the most commonly detected IS elements in the genetic environment of bla CTX-M genes, suggesting a possible co-evolutionary relationship between the ISEcp1 and bla CTX-M genes [9,52,61,65].
The insertion site of ISEcp1 was different from study to study in this review; this may be due to the variation in bacterial strains, IS promoter types, and other factors associated with genetic environments of the bla CTX-M-15 gene. Three studies provided information on the promoter regions in this review; the −35 and −10 putative promoter regions (48 bp) were reported in five studies, while V and W sequences (127 bp) were in one study. In all cases, these promoter regions are important in the transcription, mobilization, and expression of the bla CTX-M-15 gene as previously described [7,9,10]. IS26 was another IS described in Africa. However, this IS element was located upstream of bla CTX-M-15 , disrupting ISEcp1 elements in all studies reporting the presence of IS26 and ISEcp1 in the genetic environment of bla CTX-M-15 . IS26 has also been reported from other parts of the world to be associated with bla CTX-M genes alone without ISEcp1 [64] or associated with bla CTX-M genes together with and located upstream of ISEcp1 [55,56,66], or located truncating ISEcp1 [55,64] in genetic arrangements with bla CTX-M genes similar to the findings of this review. In all these genetic arrangements involving IS26, the IS was suggested to be associated with transposition and stabilization of the ISEcp1/ bla CTX-M-15 complex on plasmids [63,67].
The genetic environment downstream of the bla CTX-M-15 revealed flanking of the bla CTX-M-15 gene by two different types of insertion sequences, orf447 and IS903. Both IS elements are the major IS elements commonly reported downstream of bla CTX-M [8,68,69]. However, based on this review, orf447 is the major IS element downstream of bla CTX-M-15 gene in Africa. In this review, seven different genetic patterns were observed; four of the five genetic patterns have previously been reported. ISEcp1-bla CTX-M-15 -orf477 genetic pattern has been reported from European and Indian strains of Enterobacteriaceae [55,61,66]; ISEcp1 bla CTX-M-15 has been reported from Spain, Canada, India, and Poland [64,[70][71][72][73]; ISEcp1-IS26-bla CTX-M-15 -orf447 has also been reported from France [55,74]; while the ISEcp1-IS3 bla CTX-M-15 pattern was reported to be novel from Angola [16]. Other genetic patterns have been reported in the genetic environments of other types of bla CTX-M and other beta-lactamase genes [8,61,75]. These genetic patterns from Africa reveal how the genetic environment of bla CTX-M-15 is consistent with what has been reported on global scales. Additionally, immigration, global migration, and traveling for tourism purposes could also contribute to these global genetic patterns of bla CTX-M-15 . Similar genetic environments of bla CTX-M-15 reported in this review and other novel genetic patterns have previously been reported from travelers returning to the United Kingdom from the Middle East, Africa, and Asian countries, which suggests the possible overseas acquisition of these genetic patterns [66].
Class 1 integrons were more commonly associated with bla CTX-M-15 compared to class 2 integrons; this is consistent with previous reports elsewhere [8,76]. Class 1 integrons are often associated with IS elements such as ISEcp1 and ISCR1. These integrons are often located adjacent to ISEcp1 and ISCR1 and function in the mobilization and transposition of bla CTX-M-15 genes [8]. In addition, some AMR genes associated with bla CTX-M-15 are captured within the conserved regions of the class 1 integrons. AMR genes were harbored within the cassette arrays of class 1 integron in different studies in this review. Antimicrobial resistance genes including dfrA17, dfrA5, dfrA1, aadA5, aadA2, aadA1 and catA1 were observed within the conserved region of the class 1 integrons, and these genes often confer multi-drug resistance to trimethoprim, aminoglycosides, and chloramphenicol. Conjugative plasmids are essential for the evolution and global dissemination of the bla CTX-M-15 gene. Similar to this review, several studies have found that the narrow-host range plasmid IncF is the predominant plasmid group that harbors the bla CTX-M-15 gene [77]. The IncF plasmid is mainly restricted to Enterobacteriaceae with support mechanisms such as lower fitness cost, transferability properties, plasmid addiction, and stability systems that favor: (i) the higher prevalence of bla CTX-M-15 in Enterobacteriaceae compared to other Gramnegative bacteria; and (ii) global dissemination of bla CTX-M-15 in association with other mobile genetic elements [11,59,78]. The IncFII-FIA-FIB multi-replicon plasmids were more commonly associated with bla CTX-M-15 in this review and have been widely distributed in the Enterobacteriaceae, especially E. coli, globally [79,80]. This replicon group could be maintained and propagated between enterobacterial species and from host to host without antimicrobial selective pressure [59,77]. This may provide some explanation to the rapid and global spread of the bla CTX-M-15 gene. Another important finding of this review was the presence of other antimicrobial resistance associated with bla CTX-M-15 often co-located on the same plasmid. Different AMR genes commonly co-exist on plasmids, therefore facilitating the co-dissemination of resistance genes and greater survival fitness of bacteria under antimicrobial selective pressure [78]. Antimicrobial resistance genes including the narrow-spectrum bla OXA-1 and bla TEM-1 beta-lactamases, aminoglycoside-modifying enzymes (aac-(6 )-lb-cr), tetracycline resistance genes (tetA and tetB), sulfonamide resistance genes (sul2 and sul3) and plasmid-mediated quinolone resistance genes (qnrA, qnrB and qnrS) were found to be consistently associated with bla CTX-M-15 from different studies in the review. These AMR genes have previously been reported to be co-located on IncFII-FIA-FIB plasmid replicons in association with bla CTX-M-15 -producing E. coli O25:H4-ST131 [81,82], conferring multi-drug resistance to different antimicrobial classes, complicating the genetic environments, and facilitating the global spread of bla CTX-M-15 in Enterobacteriaceae. In addition to the contribution of clonal spread of some bacteria of Enterobacteriaceae, especially E. coli and Klebsiella spp., the association of bla CTX-M-15 with mobile genetic elements such as insertion sequences, integrons, and conjugative plasmids may explain its global dominance and dissemination. This review has showed the diversity and the complexity of the genetic environments of bla CTX-M-15 beta-lactamase gene in Enterobacteriaceae from Africa. We recognize that a limited number of articles were included in this review, which was a limitation of this review. This is partly due to limited published articles on this subject matter in Africa. Our focus was to provide a narrative review that can serve as baseline literature for a future comprehensive and systematic review and indicate the need for more research on this internationally disseminated beta-lactamase resistance gene.