We detected for the first time blaNDM-5 and blaOXA-181 in Escherichia coli isolates from hospitalized patients and healthy volunteers in Chad. These resistance genes were located on IncX3 and IncF plasmids. Despite the large diversity of E. coli clones, the identified resistant intestinal isolates belonged mainly to the same sequence type.
KEYWORDS: carbapenemases, Escherichia coli, clinical samples, fecal carriage prevalence, Chad
ABSTRACT
We detected for the first time blaNDM-5 and blaOXA-181 in Escherichia coli isolates from hospitalized patients and healthy volunteers in Chad. These resistance genes were located on IncX3 and IncF plasmids. Despite the large diversity of E. coli clones, the identified resistant intestinal isolates belonged mainly to the same sequence type.
INTRODUCTION
The rapid spread of carbapenemase-producing Enterobacteriaceae (CPE) represents a serious public health problem because carbapenems are the last antibiotics available to treat infections caused by multidrug-resistant (MDR) Gram-negative bacilli (1). To date, CPE emergence and dissemination have been described worldwide, but reports on CPE in Africa are rare, thus limiting our knowledge on the need of alternative strategies to treat patients infected by these bacteria (2). The few reports on CPE in Central Africa highlighted the presence of CPE isolates that produce NDM-4 in Cameroon, OXA-181 and NDM-1 in Angola, and NDM-7 in Gabon (2). Here, we determined the prevalence and genetic characteristics of CPE isolated from clinical and fecal carriage samples of hospitalized patients and healthy volunteers in Chad.
From January to the end of March 2017, 197 (inpatients, n = 133; outpatients, n = 64) nonduplicate Enterobacteriaceae and clinically significant bacteria were isolated from clinical samples processed in three main hospitals of N’Djamena, Chad. In the same period, 200 fecal samples (100 from patients hospitalized for more than 48 h and 100 from healthy volunteers in the community) were screened for CPE carriage using the selective medium ChromID Carba Smart plates (bioMérieux, Marcy-l’Etoile, France). Disk diffusion antimicrobial susceptibility testing (AST) of all clinical and fecal samples using the EUCAST guidelines and clinical breakpoints (http://www.eucast.org/) identified 18 potential CPE isolates (Table 1), and this finding was confirmed by PCR analysis. All 18 isolates were identified as Escherichia coli by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonics). The prevalence of carbapenemase-producing E. coli (CPEc) was 2.5% (5/197) in clinical samples, and 6.5% (13/200) in fecal carriage samples (n = 3 samples from healthy volunteers and n = 10 samples from patients; P < 0.001). These results are in agreement with previous literature data and confirm stronger selective antibiotic pressure and bacterial transmission in inpatients (3, 4). The presence of CPE in fecal samples from healthy volunteers is alarming, potentially increasing the risk of community-acquired CPE infections.
TABLE 1.
Characteristics and resistance genes of carbapenemase-producing E. coli isolates recovered from clinical and fecal carriage samples
| Source | Isolate | Carbapenem MICs (mg/liter) for:a
|
ST | PCR detection of antimicrobial resistance genesb
|
Resistance to non-beta-lactamsc | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ETP | IPM | MRM | DOR | Carbapenemase | ESBL | PMQR genes | 16S rRNA methylase | Other β-lactamases | ||||
| Clinical | 313 | 32 | 4 | 32 | 4 | 88 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | armA | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, AK, TET, SXT |
| 374 | 32 | 4 | 32 | 6 | 2 | OXA-181/NDM-5 | CTXM-15 | aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, AK, TET, SXT | ||
| 376 | 32 | 4 | 32 | 4 | 2 | OXA-181/NDM-5 | CTXM-15 | aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, AK, TET, SXT | ||
| 421 | 32 | 3 | 32 | 2 | 2 | NDM-5 | CTXM-15 | aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, AK, TET, SXT | ||
| 537 | 0.5 | 0.5 | 0.19 | 4 | 39 | OXA-181 | CTXM9 | qnrS | NA, OFX, CIP, LEV, TET, SXT | |||
| Carriage | 1 | 2 | 0.25 | 0.38 | 0.19 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, TET, SXT | |
| 2 | 2 | 0.38 | 0.25 | 0.25 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, TET, SXT | ||
| 3 | 1.25 | 0.25 | 0.25 | 0.25 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, TET, SXT | ||
| 4 | 1.25 | 0.25 | 0.38 | 0.91 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, TET, SXT | ||
| 5 | 3 | 0.38 | 0.5 | 0.25 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, TET, SXT | ||
| 6 | 0.12 | 0.25 | 0.03 | 0.03 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, TET, SXT | ||
| 7 | 0.01 | 2 | 0.12 | 0.19 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr, oqxAB | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, TET, SXT | ||
| 8 | 0.01 | 1.5 | 0.12 | 0.19 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, TET, SXT | ||
| 9 | 1 | 0.38 | 0.38 | 0.12 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, TET, SXT | ||
| 10 | 0.06 | 0.19 | 0.03 | 0.03 | 692 | OXA-181 | CTXM-15 | qnrS, qnrB, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, TET, SXT | ||
| 11 | 2 | 0.25 | 0.25 | 0.12 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, TET, SXT | ||
| 12 | 1 | 0.5 | 0.5 | 0.12 | 692 | OXA-181 | CTXM-15 | qnrS, aac(6’)-Ib-cr | TEM-1/OXA-1 | NA, OFX, CIP, LEV, CN, TOB, N, TET, SXT | ||
| 13 | 0.5 | 0.38 | 0.38 | 0.12 | 4 | OXA-181 | CTXM-15 | qnrS | TEM-1/OXA-1 | TET, SXT | ||
ETP, ertapenem; IPM, imipenem; MEM, meropenem; DOR, doripenem. The MICs of ertapenem, imipenem, meropenem, and doripenem were determined using the Etest method (bioMérieux), and the MIC of colistin was determined using the broth microdilution method.
ESBL, extended-spectrum β-lactamase; PMQR, plasmid-mediated quinolone resistance.
NA, nalidixic acid; OFX, ofloxacin; CIP, ciprofloxacin; LEV, levofloxacin; CN, gentamicin; TOB, tobramycin, N, netilmicin; AK, amikacin; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole.
Besides resistance to broad-spectrum cephalosporins, all 18 CPE isolates were also frequently resistant to ciprofloxacin (94%), gentamicin (89%), amikacin (22%), and trimethoprim-sulfamethoxazole (100%) and remained susceptible to fosfomycin, chloramphenicol, and colistin.
The presence of genes encoding antibiotic resistance determinants was assessed using multiplex PCR followed by sequencing (see primers and references in Table S1 in the supplemental material). Among the five clinical CPEc isolates, two carried the blaOXA-181 gene, one carried the blaNDM-5 gene, and two carried both genes. All 13 CPEc isolates from fecal carriage samples harbored the blaOXA-181 gene. Associated resistance determinants are reported in Table 1.
Genotyping data obtained by multilocus sequence typing (MLST) (http://bigsdb.web.pasteur.fr/), variable-number tandem-repeat typing (5), and the unweighted pair group method with arithmetic mean (UPGMA) hierarchical clustering allowed for the assessment of the genetic relationships among the 18 CPEc isolates. The dendrogram was elaborated based on the Nei’s distance using the Phylip and Populations packages and visualized with TreeDyn (with annotations). The dendrogram highlighted the presence of two clusters (I and II) and three unique profiles (Fig. 1). Cluster I included 12 CPEc isolates from fecal carriage samples (n = 10 from inpatients and n = 2 from healthy volunteers) that belonged to sequence type 692 (ST692) and harbored the blaOXA-181 gene. Cluster II was composed of three CPEc clinical isolates that belonged to ST2 and harbored the blaNDM-5 gene alone or with the blaOXA-181 gene. The CPEc isolates that belonged to ST4 (one fecal sample from a volunteer), ST39, and ST88 (one clinical sample each for ST39 and ST88) formed three unique profiles. The high number of CPEc isolates included in the two clusters (15/18; 83%) could reflect the importance of interhuman transmission of these multidrug-resistant bacteria in Chad. Analysis of the patients’ medical records suggested a probable nosocomial outbreak among the ten patients (samples 1 to 10) from cluster I (Fig. 1) because they were in the same hospital at the same time. Phylogroups were also determined based on the PCR method developed by Clermont et al. (6), and most strains (14/18, 78%) belonged to commensal clones B1 and C (Fig. 1).
FIG 1.
UPGMA dendrogram based on the variable-number tandem-repeat (VNTR) (9 loci) and MLST data for the 18 CPEc isolates. The relationships between patterns were assessed using the UPGMA hierarchical clustering method. ST, sequence type; NTP, nontypeable plasmid; HGRN, National Reference General Hospital; RH, Renaissance Hospital; MCH, Mother and Child Hospital; ICU, Intensive Care Unit.
Mating experiments performed using the azide-resistant E. coli strain J53 as the recipient were successful for most of the isolates. In the case of transfer failure, plasmid DNA was extracted using the GeneJet plasmid miniprep kit (7) and transferred by electroporation into E. coli DH10B (Invitrogen, Cergy-Pontoise, France) (8). Plasmids were characterized by PCR-based replicon typing and plasmid relaxase gene typing (PRaseT) (9). PRaseT showed that blaOXA-181 was located on IncX3 plasmids, and blaNDM-5, blaCTX-M-9, and blaCTX-M-15 were located on IncF plasmids in all clinical isolates. In most fecal isolates, the blaCTX-M-15 gene was located on a nontypeable plasmid, except for four isolates where it was located on an IncF, IncR, or Mobp5-3 plasmid (Fig. 1).
Overall, our results revealed that most CPEc isolates (12/18; 67%) were E. coli ST692 harboring the blaOXA-181 gene carried by the IncX3 plasmid, as already described in Burkina Faso (10). This suggests the circulation of this clone in sub-Saharan Africa; however, the factors that contribute to this success are still unknown.
We determined the complete sequences of all 17 IncX3 plasmids carrying blaOXA-181 using the Oxford Nanopore MinION platform (Oxford Nanopore Technologies). We obtained 51-kb plasmids, which were highly similar (99% nucleotide identity) to pOXA181_EC14828 (GenBank accession no. KP400525), a blaOXA-181-harboring plasmid first described in China in 2015 (11). The blaOXA-181 gene was found in a 14,107-bp region flanked by two IS26 elements in direct orientation, with duplicated nucleotides (GT) at both extremities. The PMQR gene qnrS1 was found downstream of blaOXA-181 and was the only other resistance gene found on these plasmids (apart from a truncated ere gene). Similar genetic environments were described in plasmids from other countries in Africa, in Europe, and in Asia (4, 10–16).
NDM-5, an NDM variant, was first identified in 2011 in an E. coli isolate from a patient in the United Kingdom who traveled to the Indian subcontinent (17) and, since then, has been increasingly detected worldwide. Interestingly, two E. coli clones recovered from clinical samples in this study were previously found to coharbor blaOXA-181 and blaNDM-5 genes in Egypt and Myanmar (18, 19). The high frequency of population movements between Chad, Egypt, and China for medical treatment and economic relationships could promote the dissemination of carbapenemase-encoding genes.
This study shows the alarming CPE circulation in community and hospital environments in Chad. The transmission of MDR bacteria among inpatients could lead to therapeutic deadlocks associated with high mortality rates, especially in Chad where AST is expensive and limited. Our results call for urgent public health efforts to set up educational campaigns on strict infection prevention/control measures targeting the Chadian population and health care professionals and better surveillance to limit the spread of MDR bacteria.
Supplementary Material
ACKNOWLEDGMENTS
We thank the GenSeq technical facilities of ISEM, Institut des Sciences de l’Evolution de Montpellier and labex Centre Méditerranéen de l’Environnement et de la Biodiversité where some of the sample preparations were performed. We thank Elisabetta Andermarcher for assistance in preparing and editing the manuscript.
We declare no competing interests.
This work was supported by a grant from the Islamic Development Bank (IDB). The IDB was not involved in the study design, collection, analysis, interpretation of data, or in writing the manuscript.
O.O.M., C.C., M.H., H.J.-P., and S.G. conceived and designed the experiments. O.O.M., F.C., M.L., K.G., A.D., and C.S. performed the experiments. O.O.M., F.C., M.L., Y.D., A.T., J.B., D.D., C.C., A.-L.B., and S.G. contributed to the writing of the manuscript. All authors read and approved the final manuscript.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00646-19.
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