Prevalence and antibiotic susceptibility pattern of CTX-M type extended-spectrum β-lactamases among clinical isolates of gram-negative bacilli in Jimma, Ethiopia

Extended-spectrum β-lactamases (ESBLs) are a predominant cause of β-lactam resistance in Gram-negative bacilli (GNB) [1, 2]. Incidences of infections caused by ESBLs producing GNB are increasing in prevalence worldwide, both in the healthcare as well as community settings, posing significant therapeutic challenges [3–5]. ESBLs are most often a plasmid mediated heterogeneous group of β-lactamase enzymes, that confer resistance to a wide range of commonly used β-lactam antibiotics including third generation cephalosporins (e.g., ceftriaxone, cefotaxime and ceftazidime) as well as monobactams (aztreonam) [6]. TEM and SHV type ESBLs used to be the dominant ESBL genotypes [7]. However, in the past decade, the CTX-M type ESBLs have become the most widely distributed and globally dominant genotypes [8].

The CTX-M type enzymes are a group of class A ESBLs that in general exhibit much higher levels of activity against cefotaxime and ceftriaxone than ceftazidime [6, 9]. The presence of CTX-M type ESBLs is often associated with co-resistance phenotypes in particular to fluoroquinolones and aminoglycosides, in addition to tetracycline, and trimethoprim/sulfamethoxazole co-resistance, which is commonly observed among TEM and SHV type ESBLs [10, 11]. The group of CTX-M type ESBLs currently constitutes more than 170 allelic variants, which cluster into five major groups based on sequence homologies. The five CTX-M groups are: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25 [12]. Each group consists of a number of particular variants with dominant variants being restricted in distribution to specific geographic areas, while few others are globally distributed. CTX-M-14 and CTX-M-15 were the most commonly isolated variants worldwide [10, 13].

In Africa, CTX-M-15 (of the CTX-M-1 group) is the most frequently reported variant, although some other variants were also detected in the region [14, 15]. CTX-M type ESBLs have now spread and could be detected among many different bacterial strains of clinical importance. This is particularly true for Enterobacteriaceae revealing an ESBL phenotype such as Escherichia coli and Klebsiella pneumoniae, which often cause potentially serious infections in the hospital as well as community setting [13].

In Ethiopia, multiple studies have reported prevalence of ESBLs ranging from 25 to 38.5% among Enterobacteriaceae in clinical samples obtained from various hospitals, including Jimma University Specialized Hospital (JUSH) [16–19]. However, there is no data on the prevalence and antibiotic susceptibility patterns of CTX-M type ESBLs produced by GNB. Therefore, the aim of the present study was to determine the relative frequency and distribution of the bla_CTX-M genes, as well as the overall susceptibility patterns in ESBL producing clinical isolates of GNB in JUSH, southwest Ethiopia.

The present study is the first report describing the molecular epidemiology of ESBL-encoding genes in Ethiopia. We demonstrate a high level of prevalence of CTX-M-type ESBLs among all ESBL positive isolates at JUSH. In total, 95.8% of all ESBL genes detected were of CTX-M type, and almost unanimously CTX-M-1 group variant type 15 (97.1% of all CTX-M positive isolates). These findings are in accordance with the fact that the CTX-M type ESBLs are the most widely distributed and globally dominant ESBL genotypes to date [13, 27, 28]. Of the groups, CTX-M-1 was also described to be highly prevalent in Italy [29], India [30], Switzerland [31], Saudi-Arabia [32], Syria [33], Pakistan [34] and China [35].

Factors and mechanisms which contribute to the emergence and increasing prevalence of CTX-M ESBLs of all groups are complex and may involve both, plasmid dissemination as well as clonal spread of bacterial strains [36, 37]. In addition, the selective pressure exerted by the frequent use of wide spectrum cephalosporins may promote their epidemiological success [10, 28, 38]. Especially in Ethiopia, the widespread misuse and overuse of cephalosporins may contribute to the selection and spread of CTX-M gene carrying clones [39–41]. The frequency of the CTX-M genotype among the ESBL gene-positive Enterobacteriaceae isolates was also remarkably high (95.5%) compared to similar findings among clinical Enterobacteriaceae isolates with prevalence rates of 91% in Brazil [42], 80.3% in Germany [43] and 79% in Switzerland [31].

Other than E. coli (92.3% CTX-M-15) and K. pneumoniae (100% CTX-M-15), CTX-M were also detected among other members of ESBL producing Enterobacteriaceae (K. oxytoca, M. morganii, P. mirablis, P. stuartii, E. hermannii and E. cloacae) as well as non-fermenting GNB (P. aeruginosa and A. fecalis) in 87.5% (n = 21) and 100% (n = 4), respectively. Out of all screen positive isolates (112) 41 were found to be non-ESBL producers. Thereby, most (35/41) were lactose non-fermenting GNB with known extensive intrinsic resistance mechanisms. Other isolates may be resistant due to genes not tested within this study, or due to derepression of wild type β-lactamases or even permeability defects. Among screen positive Enterobacteriaceae isolates, 92% (67/73) were also positive for an ESBL gene tested within this study.

Although, this study was small, it indicates the dissemination of the CTX-M genes to other GNB besides Enterobacteriaceae in Jimma. Similar findings have been reported in Switzerland [31], Argentina [44], Netherlands [45] and Japan [13]. The frequency of ESBL gene positive Pseudomonas aeruginosa was low (21.4%, n = 3) when compared to other GNB. This is probably due to the fact that most resistance mechanisms in Pseudomonas aeruginosa are mediated by the overproduction of AmpC β-lactamases as well as acquired metallo-β-lactamases, decreased permeability and efflux pumps [46]. In addition, plasmid incompatibility and host range of ESBL encoding plasmids might also play a role in our setting [13]. The emergence and spread of CTX-M-producing isolates in the community, particularly among E. coli in urinary tract infections (UTI), were reported from China [47], Brazil [48] and the UK [49]. A trend in this direction can also be seen in our study, as all the outpatient urine isolates of E. coli (n = 2), K. pneumoniae (n = 2), M. morganii (n = 1), P. mirablis (n = 1) and E. cloacae (n = 1) with an ESBL gene were shown to carry CTX-M genes. However, the total sample size of outpatient isolates in the present study is small compared to the inpatient sample number.

The overall resistance pattern of the total CTX-M positive Enterobacteriaceae is very high for most antibiotics tested in the present study. The carbapenems (0% resistance) followed by amikacin (3% resistance) were found to have the highest susceptibility rates. However, all CTX-M-positive isolates identified in this study showed a MDR phenotype as well as remarkably high rates of co-resistance to fluoroquinolones, aminoglycosides, and trimethoprim/sulfamethoxazole. Only one E. coli isolate positive for an ESBL gene (CTX-M-15) was not resistant against third generation cephalosporins, while still maintaining an MDR phenotype. In this particular case, the CTX-M operon seems to be non-functional perhaps due to mutations. These findings are consistent with studies from Ghana [50], Lebanon [51] and India [52] which propose imipenem and amikacin as possible drugs for the management of infection caused by CTX-M-producing isolates. The results are also in accordance with findings of high prevalence of MDR phenotype (88.4%) among ESBL-producing E. coli and K. pneumoniae isolates in a previous phenotypic characterization of strains in JUSH [17]. Comparably high rates of co-resistance to non-β-lactam antibiotics were also reported from Brazil [42], South Korea [53] and Indian hospitals [54].

Surprisingly, colistin/polymyxin, which is not available in Ethiopia, showed resistance rates of above 10%. However, this rate has to be interpreted with caution, as the data based on VITEK® 2 testing system is unreliable for detecting colistin resistance [55], and results obtained by these methods may be overrated and require confirmation by ISO-standard broth microdilution method as nowadays recommended by EUCAST [56, 57]. As the respective recommendation was issued after completion of the study, it was not taken into consideration.

In the present study, only clinically relevant isolates of in- and outpatients were used, a screening upon admission, or screening of healthy controls was not performed. However, the high rates of ESBL positive organisms in outpatients without contact to the health care system within the last 3 months, argues for considerable ESBL carrier rates among the general population. Within the study population, mainly samples from internal medicine, pediatrics and ICU were ESBL positive and MDR, whereas in the surgical patient group many patients were found to harbor non-fermenters with MDR phenotype which are negative for the ESBL and carbapenemase genes tested within this study (see Additional file 2).

This conclusion is supported by a study conducted at black lion hospital in Addis Ababa (Ethiopia) reporting a high gastrointestinal colonization rate with ESBL producing Enterobacteriaceae among hospitalized patients [58]. It is well known, that many of the patients who develop health care-associated ESBL infections have preceding colonization of the gastrointestinal tract [59, 60]. A combination based on lack of hygiene and high colonization rates with ESBL positive organisms are likely to drive the ESBL rates in JUSH.

Within the sample group, other prominent resistance determinants were also investigated as part of the CT103 panel. Thereby, no KPC, NDM-1, VIM, IMP or Oxa48-like coding organism was detected. Previously, we could demonstrate the presence of NDM-1 in Acinetobacter baumannii in the area [61]. NDM-1 gene transfer to other isolates seems not to have occurred in relevant numbers. However, the presence of CTX-M-15 genes in different species in such high prevalence argues for horizontal gene transfer currently or in the past. The transfer might have occurred by plasmid exchange, which is especially common among Enterobacteriaceae, or by less frequent recombination events, e.g. involving IS elements. How recent or frequent such events have been cannot be elucidated given the methodology used, as the genes are found in numerous different species and isolates, it certainly cannot be explained simply by local clonal expansion of one strain.

This study demonstrates a remarkably high level of CTX-M genes in GNB isolated in JUSH. The most predominant group was CTX-M-1 allele 15 and a few percent CTX-M-9 allele 24 among all the ESBLs gene positive clinical isolates. In South Africa, CTX-M-2 and -3 group are most prevalent, and CTX-M-14 and -15 in Egypt [62]. Meropenem, imipenem, colistin and amikacin were found to have the highest in vitro efficacy against the CTX-M-producing isolates. The high level of resistance to β-lactam and non-β-lactam antibiotics as well as the trend of a MDR profile associated with the CTX-M genes are alarming and emphasize the need for diagnostic antimicrobial susceptibility testing for appropriate choice of antimicrobial therapy and limiting the spread of antimicrobial resistance in Ethiopia and in the region.