ABSTRACT

Stool samples from 38 travelers returning from India were screened for extended-spectrum cephalosporin- and carbapenem-resistant Enterobacteriaceae implementing standard selective plates. Twenty-six (76.3%) people were colonized with CTX-M or DHA producers, but none of the strains was colistin resistant and/or mcr-1 positive. Nevertheless, using overnight enrichment and CHROMagar Orientation plates supplemented with colistin, four people (10.5%) were found to be colonized with colistin-resistant Escherichia coli. One cephalosporin-susceptible sequence type 10 (ST10) strain carried a 4,211-bp ISApl1-mcr-1-ISApl1 element in an IncHI2 plasmid backbone.

TEXT

The emergence of colistin-resistant (COL-R-Ent) Enterobacteriaceae harboring the plasmid-mediated mcr-1 gene has raised serious concerns (1, 2). These strains (especially Escherichia coli) have been isolated worldwide from humans (1, 310), food-producing animals (4, 6, 1114), the food chain (7, 10, 1517), and the environment (4, 15). However, most of these studies searched for mcr-1 in previously stored extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESC-R-Ent) (8, 12, 13, 15, 17). This leads to a question about the actual prevalence of mcr-1-positive Enterobacteriaceae (mcr-1-Ent), especially because mcr-1 can be carried by plasmids not coharboring extended-spectrum β-lactamase (ESBL), plasmid-mediated AmpC (pAmpCs), and/or carbapenemase genes (5, 7, 12, 14, 18). Moreover, although it is known that the prevalence of intestinal colonization with ESC-R-Ent in returning travelers is very high (19), data regarding the mcr-1-Ent are needed. Only Arcilla et al. have explored this phenomenon, indicating that 0.9% of the ESBL producers isolated from stools of Dutch travelers co-possessed mcr-1 (8).
We analyzed the pre- and posttrip stool samples (both within 1 week) of 38 people living in Switzerland and traveling to India during January to August 2015. A questionnaire was also filled out at each sampling time indicating that in the 12 months before going to India, participants frequently visited other countries but never suffered diarrhea. On the other hand, after the journey to India (mean stay, 17.8 days), 39% of the travelers suffered from travelers' diarrhea and additional symptoms, although antibiotics were not taken (see Table S1 in the supplemental material).
To detect ESC-R-Ent, ∼20 μg of fresh stools was enriched overnight in 10 ml LB broth containing a 10-μg disk of cefuroxime. Then, 100 μl was plated on BLSE, ChromID ESBL (bioMérieux), and Supercarba selective plates (20). After overnight incubation, colonies were identified using the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker). Microdilution GNX2F panels (Trek Diagnostics) were used to obtain the antibiotic MICs. The CT103XL microarray was used to detect bla genes. Multilocus sequence typing (MLST) for E. coli and Klebsiella pneumoniae (Warwick and Pasteur schemes, respectively) was used to determine the sequence type (ST); the E. coli phylogenetic group was also determined (21). The remaining stool samples were stored at −80°C without cryoprotectant.
Based on this initial screening, three (7.9%) people had stool samples positive for ESBL-producing E. coli before traveling, whereas 29 (76.3%) of them returned from India colonized with ESC-R-Ent, such as 26 CTX-M- and 3 DHA-producing E. coli. No carbapenemase producers were detected (Table 1). Overall, E. coli strains found in returning travelers were of non-hyperepidemic STs and were mainly of phylogenetic groups A1 (n = 11) and B1 (n = 8). Notably, all of these ESC-R-Ent had polymyxin MICs of ≤0.5 μg/ml and did not contain mcr-1 as determined by PCR (1).
TABLE 1
TABLE 1 Extended-spectrum cephalosporin- and/or colistin-resistant Enterobacteriaceae detected in stool samples from 38 people before and after traveling to India during 2015a
a
Abbreviations used throughout: ID, identification code for the traveler; m, male; f, female; ND, not defined; ST, sequence type; CC, clonal complex; Phy, phylogenetic group; COL, colistin; PolB, polymyxin B; −, negative; +, positive; NA, not available or not applicable; wt, wild type (non-ESBL). Data highlighted in gray represent the colistin-resistant strains. All of these strains were detected with the CHROMagar Orientation plates containing colistin (4 μg/ml) plus vancomycin (8 μg/ml) with or without cefotaxime (2 μg/ml).
b
Based on the CheckPoints CT103XL microarray results.
c
MICs were obtained in microdilution with the GNX2F Trek panels; data in parentheses represent Etest results. For both methodologies, the MICs were obtained using colonies that grow on MacConkey agar plates without antibiotics.
d
Relatives or couples traveling together.
e
Relatives or couples traveling together.
f
Relatives or couples traveling together.
g
Friends traveling together.
h
Friends traveling together.
i
The new STs could not be assigned because whole-genome sequencing for the strains was not performed as requested by the curator of the E. coli MLST scheme (see Table S3 in the supplemental material).
To improve our ability to detect possible Col-R-Ent (including those non-ESC-R), stored stools were enriched in 10 ml LB broth with two 10-μg disks of colistin. After overnight incubation, 100 μl was spread on four agar plates: MacConkey agar without or with colistin (4 μg/ml) and CHROMagar Orientation plus colistin (4 μg/ml) and vancomycin (8 μg/ml) without or with cefotaxime (2 μg/ml). MacConkey plates yielded numerous undistinguishable colonies of species naturally resistant to polymyxins (e.g., Proteus spp. and Serratia marcescens). In contrast, thanks to the typical color appearance of the different Gram-negative organisms, the CHROMagar Orientation plates allowed us to clearly identify Col-R E. coli (Col-R-Ec) colonies.
As depicted in Table 1 (rows highlighted in gray), only one person was colonized with a Col-R-Ec strain before traveling, whereas four (10.5%) returning travelers had stools positive for Col-R-Ec (of which three coproduced ESBLs or pAmpCs). Remarkably, two (26-Ec and 100R-Ec) out of the overall five Col-R-Ec strains were non-ESC-R (see Table S2 in the supplemental material) and were identified only with the CHROMagar Orientation plates containing colistin but not cefotaxime. Notably, Col-R Klebsiella spp., Enterobacter cloacae, Enterobacter aerogenes, and Salmonella species strains were not found (2, 22). We cannot exclude that storing the stools at −80°C could influence the viability of Col-R strains.
Overall, one (2.6%) ST10/A1 E. coli strain (100R-Ec) was PCR positive for mcr-1 (Table 1). This strain had a colistin MIC of 12 μg/ml by Etest and was coresistant to quinolones, trimethoprim-sulfamethoxazole, and tetracyclines (see Table S2 in the supplemental material). The strain carried HI2, FII, FIB, and X1 plasmid replicon types (PBRT kit; Diatheva). Experiments with transformation into chemically competent DH5α E. coli failed to obtain mcr-1-carrying cells (23). Conjugation experiments using E. coli recipient strain JF33 (rifampin resistant) performed at 25 and 37°C and with selection on MacConkey plates containing colistin (2 μg/ml) and rifampin (50 μg/ml) were also unsuccessful.
To complete the bacterial typing of the mcr-1-positive strain and to identify the resistome and mobilome, raw genome data (MiSeq; Illumina) were screened by ResFinder-2.1 and PlasmidFinder-1.3 (24, 25). 100R-Ec carried resistance genes for aminoglycosides [aadA1, aadA2, and aph(3′)-Ia], β-lactams (blaTEM-1), polymyxins (mcr-1), quinolones (qnrS1), macrolides [mph(A)], phenicols (cmlA1), sulfonamides (sul3), tetracyclines [tet(A)], and trimethoprim (dfrA12 and dfrA14).
By PlasmidFinder, the presence of IncF, IncX1, and IncHI2 plasmids was confirmed in the 100R-Ec strain. The mcr-1 gene was identified at the end of a 26-kb contig (GenBank accession no. KX090925), flanked by two copies of ISApl1 in the opposite orientation forming a 4,211-bp ISApl1-mcr-1-ISApl1 element. The 22,133-bp sequences preceding the ISApl1-mcr-1-ISApl1 element shared >99% identity with DNA sequences of large IncHI2 plasmids, such as pKST313 from Salmonella enterica serotype Typhimurium (GenBank accession no. LN794248), pAPEC-O1-R from avian-pathogenic E. coli (GenBank accession no. DQ517526) (26, 27), and pSA26 carrying ISApl1-mcr-1 from an ST68 E. coli strain (GenBank accession no. KU743384). The mcr-1 element was inserted into the tra1 locus of the IncHI2 plasmid backbone immediately downstream of the conjugative protein gene trhY. The disruption of the tra region may explain the defective conjugation ability of this plasmid. Notably, the presence of mcr-1 between duplicated ISApl1 insertion elements has so far only been observed in one human KPC-2-producing E. coli isolate in Germany and two IncHI2 plasmids isolated in the United Kingdom (4, 10).
Taking into account that 100R-Ec was of ST10/A1, and due to the high identity of the mcr-1-containing element with plasmids found in bacteria from animal origin, we speculate that this Col-R E. coli strain was acquired during the trip in India via the food chain or from environmental sources. Remarkably, the traveler was cocolonized with an ST155 CTX-M-producing E. coli strain (100R-Ec-Drig). A follow-up screening of the stool samples 3 and 6 months after returning from the trip indicated that the person was no longer colonized with both E. coli strains.
The remaining mcr-1-negative but Col-R E. coli strains (26-Ec, 78R-Ec, 86R-Ec, and 88R-Ec) underwent conjugation experiments, but all failed to demonstrate a hypothetical plasmid-mediated mechanism of colistin resistance. We should note that such strains have been previously observed in humans and animals (8, 9, 28) and that they are Col-R, probably due to chromosomal mutations (10, 29). Notably, the stool samples from three of the travelers mentioned above were negative for Col-R Ent at the 3- and 6-month follow-up screenings. (ID-78 did not submit the stool samples for screening.) The risk factors determining the intestinal colonization with Col-R-Ec of people visiting India are unclear and should be investigated in the near future.
Our results indicate that the prevalence of intestinal mcr-1-Ent is probably underestimated because some isolates carrying only mcr-1 (like 100R-Ec) could be routinely undetected when the screening is targeting the ESC-R and/or carbapenem-resistant strains (5, 7, 8, 12, 14, 18). It is possible that the ISApl1-mcr-1-ISApl1 element present in 100R-Ec possesses the ability to transpose frequently, moving the mcr-1 gene into the IncHI2 scaffold but also promoting its transposition in different plasmids. Multifocal plasmid types (IncP, IncX4, and IncI2) carrying mcr-1, but not bla genes conferring resistance to extended-spectrum cephalosporins (ESCs), have been previously reported, and some of them demonstrated a great potential for dissemination by conjugation among diverse Gram-negative species of the gut flora (1, 4, 18). Such colonizing bacteria may subsequently develop into difficult-to-treat infections, as observed for the ESBL producers acquired by people traveling from low- to high-prevalence countries (3032).
The spread of non-ESC-R mcr-1-Ent could contribute to the silent expansion of this life-threatening resistance gene in both human and nonhuman settings. Specific and sensitive surveillance programs should be rapidly implemented to prevent unexpected outbreaks due to mcr-1-Ent.

ACKNOWLEDGMENTS

This work was supported by the Swiss National Science Foundation (SNF; grant no. 153377 to A.E.). The study was approved by the Ethikkommission Nordwest- und Zentralschweiz (EKNZ 239/12). Odette J. Bernasconi is a Ph.D. student (2015 to 2018) supported by the Hans Sigrist Foundation (Bern, Switzerland). João Pires is a Ph.D. student (2014 to 2017) supported by the SNF.
We thank Sara Kasraian and Alexandra Collaud for technical help.

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cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 60Number 8August 2016
Pages: 5080 - 5084
PubMed: 27297483

History

Received: 15 April 2016
Returned for modification: 19 May 2016
Accepted: 28 May 2016
Published online: 22 July 2016

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Authors

Odette J. Bernasconi
Institute for Infectious Diseases, University of Bern, Bern, Switzerland
Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland
Esther Kuenzli
Swiss Tropical and Public Health Institute, Basel, Switzerland
Division of Communicable Diseases, Institute for Social and Preventive Medicine, University of Zurich, Zurich, Switzerland
João Pires
Institute for Infectious Diseases, University of Bern, Bern, Switzerland
Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland
Regula Tinguely
Institute for Infectious Diseases, University of Bern, Bern, Switzerland
Alessandra Carattoli
Institute for Infectious Diseases, University of Bern, Bern, Switzerland
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy
Christoph Hatz
Swiss Tropical and Public Health Institute, Basel, Switzerland
Division of Communicable Diseases, Institute for Social and Preventive Medicine, University of Zurich, Zurich, Switzerland
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
Institute for Infectious Diseases, University of Bern, Bern, Switzerland

Notes

Address correspondence to Andrea Endimiani, [email protected].

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