First Description of IncX3 Plasmids Carrying bla_OXA-181 in Escherichia coli Clinical Isolates in Burkina Faso

LETTER

Carbapenemase-producing Enterobacteriaceae (CPE) have been increasingly reported worldwide. The few studies available on CPE epidemiology in West and East Africa highlight 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) (1). Although bla_OXA-48 genes are widely spread in North Africa, bla_OXA-48 derivatives have been rarely reported in Africa. Indeed, bla_OXA-163 was detected only twice in Egypt and bla_OXA-181 (a point mutant analogue of OXA-48) only once in South Africa (1). Here, we describe the first four cases of Escherichia coli carrying the bla_OXA-181 gene in Burkina Faso.

Four E. coli strains (Table 1) were isolated from four patients in two hospitals in Ouagadougou, Burkina Faso. Carbapenem MICs, determined using the Etest (bioMérieux), were 1 to 1.5 mg/liter, 0.125 to 0.75 mg/liter, and 0.25 to 0.5 mg/liter for ertapenem, doripenem, and imipenem, respectively (Table 1). Three patients received antibiotics before strain isolation (Table 1). None of the patients reported recent travel outside Burkina Faso. Multiplex PCR and DNA sequencing targeting the most prevalent extended-spectrum-β-lactamase (ESBL)- and carbapenemase-encoding genes (2, 3) revealed the presence of bla_CTX-M-15 and of bla_OXA-181 in the four isolates. No other carbapenemase-encoding gene (corresponding to NDM, VIM, IMP, and KPC) was detected. Multilocus sequence typing (MLST) (http://bigsdb.web.pasteur.fr/) showed that the four strains belonged to new sequence type (ST) ST692, which is described here for the first time. Enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) (4) patterns (see Fig. S1 in the supplemental material) and the variable-number tandem-repeat (VNTR) (5) profile determined on the basis of analysis of 7 polymorphic loci (6-1-5-8-3-5-1; see Table 1) confirmed the genetic links among the four E. coli strains. However, the review of medical records indicated that the four patients were hospitalized in different structures (hospitals and wards). Although there was no relationship or housing shared between the patients, these data support the hypothesis of infections by the same multidrug-resistant clone circulating in these hospitals or in the general community.

TABLE 1.

Clinical and microbiological characteristics of the four bla_OXA-181-producing E. coli isolates and their respective transformants and of E. coli J53^a

Characteristic	Result
	EC187	EC187 (T)	EC292	EC292 (T)	EC309	EC309 (T)	EC327	EC327 (T)	E. coli J53
Patient	F, 12 yrs old		M, 2 yrs old		F, 65 yrs old		F, 21 yrs old
Origin	Urine		Suppuration		Suppuration		Urine
Clinical symptom or diagnosis	Dysuria		Abdominal pain		Peritonitis		Unknown
Use of antibiotics in the previous 3 mo	None reported		CFM, CRO, GE		AMC, GE		CRO, GE
MLST	ST692		ST692		ST692		ST692
VNTRb	6-1-5-8-3-5-1		6-1-5-8-3-5-1		6-1-5-8-3-5-1		6-1-5-8-3-5-1
MIC (mg/liter)
Ertapenem	1	0.5	1.5	0.5	1.5	0.5	1.5	0.38	0.06
Doripenem	0.125	ND	0.25	ND	0.25	ND	0.75	ND	ND
Imipenem	0.25	0.5	0.5	0.75	0.38	0.5	0.38	0.5	0.25
Associated resistance
ESBL	CTX-M-15	None	CTX-M-15	None	CTX-M-15	None	CTX-M-15	None
Non-β-lactam resistance	CIP, GE, SXT, TE	ND	CIP, GE, SXT, TE	ND	CIP, GE, SXT, TE	ND	CIP, GE, SXT, TE	ND

^a(T), transformant; F, female; M, male; ND, not determined; AMX, amoxicillin; AMC, amoxicillin-clavulanic acid (co-amoxiclav); CFM, cefixime; CIP, ciprofloxacin; CRO, ceftriaxone; GE, gentamicin; SXT, sulfamethoxazole-trimethoprim; TE, tetracycline.

^bData represent CNV1, CNV2, CNV3, CNV4, CNV7, CNV14, and CNV15.

Plasmid DNA was extracted by alkaline lysis and subsequently analyzed by gel electrophoresis as previously described (6). Comparative analysis was carried out using reference plasmids RP4 (54 kb), pCFF04 (85 kb), and pIP173 (126.8 kb) and showed two different plasmids of ca. 120 kb and ca. 54 kb, respectively, in each strain.

Mating experiments performed using azide-resistant E. coli strain J53 as a recipient under various conditions were unsuccessful. Plasmid DNA was extracted using a QIAprep Spin Miniprep kit (Qiagen) and transferred by electroporation into E. coli DH10B (Invitrogen, Cergy-Pontoise, France). The bla_OXA-181-carrying transformants showed ertapenem and imipenem MICs of 0.38 to 0.5 mg/liter and 0.5 to 0.75 mg/liter, respectively. Analysis by plasmid relaxase gene typing (7) and PCR-based replicon typing (8) identified IncX3-type relaxase and ColE-type replication initiation genes, respectively, in the transformants. Alkaline lysis of transformants and subsequent electrophoresis showed that these genes were carried by the ca. 54-kb plasmid. As a bla_OXA-181-carrying IncX3 plasmid was recently identified in E. coli in China (9), PCR mapping was carried out in the four strains and their respective transformants with primers designed using plasmid pOXA181_EC14828 as the template (GenBank accession number KP400525). All primers used for PCR mapping are reported in Table S1 in the supplemental material. PCR mapping gave similar results in all four E. coli strains. DNA regions surrounding the bla_OXA-181 gene are detailed in Fig. 1 and showed that bla_OXA-181 was part of the Tn2013 transposon, as previously described (10). The same genetic context was recovered in all six bla_OXA-181-surrounding sequences available in the GenBank database (GenBank accession numbers KP400525, AB972272, JN205800, NZ_JRKW01000020, JQ996150, and KT005457) (11, 12). The repA1 gene (encoding a ColE-type replication initiation protein) was downstream of Tn2013. This replicase gene was also found on plasmids pKP3-A (JN205800) and pMR3-OXA181 (NZ_JRKW01000020) that belong to the ColE and IncN incompatibility groups, respectively. This suggests that bla_OXA-181 might have come from a ColE-type scaffold. Fluoroquinolone resistance gene qnrS1 was also detected downstream of bla_OXA-181 (Fig. 1). An IncX3-specific backbone was recovered at the 5′ extremity of bla_OXA-181-surrounding regions and included, in addition to the repB replicase gene, the parA partition gene (13) and the umuD gene involved in SOS mutagenesis (14). Large-scale PCR mapping targeting various plasmid regions, including transfer, replication, and partition systems, was also performed and covered a total of 29,569 bp, which amounts to ca. 55% coverage compared to the estimated size of the plasmid (Fig. 1; see also Table S1 in the supplemental material). All PCR products displayed 100% identity to those encoded by the respective regions of plasmid pOXA181_EC14828 (Fig. 1).

FIG 1.

Genetic map of the four plasmids harboring bla_OXA-181 described in this report. Purple arrows represent the replicase genes. Light-gray arrows represent genes encoding hypothetical proteins. Yellow arrows represent genes encoding partition systems. Dark-gray arrows represent accessory genes. Green arrows represent transposase-encoding genes and insertion sequences. Red arrows represent antimicrobial resistance genes. Blue arrows represent genes implicated in plasmid transfer. The genetic context of bla_OXA-181 is visually extended at the bottom. Plasmid pOXA181_EC14828 was harbored by an E. coli isolate in China (GenBank accession no. KP400525) and was used as a model to map the four bla_OXA-181-carrying plasmids described in this report. Thin black lines represent the 25 oligonucleotide pairs used for PCR mapping in all four plasmids. All amplicons were fully sequenced and displayed 100% identity to those of plasmid pOXA181_EC14828.

Since the first description in Indian hospitals in 2011, OXA-181-positive Enterobacteriaceae have been reported worldwide (1, 11). Their emergence in West Africa in IncX3 plasmids is of particular concern because these plasmids mediate the spread of carbapenemases in Enterobacteriaceae (15, 16). Moreover, a recent study found an IncX3 plasmid harboring bla_OXA-181 in a Klebsiella variicola isolate in fresh vegetables imported to Switzerland from Asia (12). This plasmid, named pKS22 (KT005457), is highly similar to pOXA181_EC14828 (100% coverage and 99% identity) and therefore to the four IncX3 plasmids described in our report. The presence of highly similar IncX3 plasmids in Asia, Africa, and Europe might suggest the epidemic potential of the members of this plasmid lineage and their role in worldwide dissemination of OXA-181.

Funding Statement

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.