LETTER
Colistin is one of the few first-line options for treating complicated infections with certain multidrug-resistant bacteria (1). The emergence and global dissemination of mcr (mobile colistin resistance) genes have threatened the usefulness of colistin (1, 2). However, the spread of mcr in nonclinical communities remains understudied, especially in Lebanon, which is experiencing severe economic and medical crises and challenges in infrastructure and antimicrobial stewardship. In Lebanon, the overreliance on colistin in medicine and animal farming was recently documented along with the emergence of mcr-1 on poultry farms and in environmental matrices (3–9). Therefore, it was necessary to assess the occurrence of these genes in the community. For this purpose, 72 fecal samples were collected from discarded diapers of toddlers (≤2 years old) in reputable community daycares at five major locations/cities across Lebanon. The daycares were licensed to receive these age groups and followed the protocols mandated by the Lebanese Ministry of Public Health, which required parents to notify the daycare of any sickness or unusual symptoms experienced by the toddlers and resident staff to maintain records of any medication/illness experienced by the toddlers while at the daycare. Ethical approval was not required to conduct the study. In order not to bias the sampling, we instructed the daycare staff to collect fecal samples from healthy/nonsymptomatic, including nondiarrheic, diapers. The samples were suspended in buffered peptone water, and an aliquot (100 μl) was spread onto an Escherichia coli selective medium, RAPID’E. coli 2 agar (Bio-Rad, USA), supplemented with colistin (4 μg/ml) (Sigma-Aldrich, USA) (2–9). Thirteen (18%) samples yielded E. coli (violet to pink colonies). Twenty-four E. coli samples (one to four colonies per sample) were purified, and their identity was further confirmed using PCR analysis (2–10), which also showed that the isolates were mcr-1 positive and mcr-2 to mcr-8 negative. mcr-1 detection was confirmed by commercial sequencing of the amplicons. The MIC of colistin against the isolates ranged between 4 and 32 μg/ml (Table 1). Plasmids were extracted and successfully transformed into chemically competent E. coli JM109 using the heat shock method (2–9). The transformants were mcr-1 positive and colistin resistant (MIC ≥ 4 μg/ml), confirming that the gene was plasmid-borne. PCR-based replicon typing (11) showed that isolates harbored diverse plasmids, including IncI2, IncI1α, IncX1, IncX4, IncF2, and IncFIB, which are associated with worldwide dissemination of mcr-1 and the spread of other antibiotic resistance genes (12, 13). Using the disk diffusion assay, the isolates were shown to exhibit resistance to penicillin (100% of isolates), ampicillin (83.5%), amoxicillin-clavulanic acid (83.5%), cefepime (33.5%), cefotaxime (75%), cephalexin (87.5%), cefixime (71%), doripenem (8.5%), imipenem (4.2%), meropenem (21%), gentamicin (12.5%), kanamycin (29.2%), streptomycin (29.2%), tetracycline (42%), ciprofloxacin (12.5%), norfloxacin (8.5%), trimethoprim-sulfamethoxazole (50%), and chloramphenicol (25%). All the isolates were multidrug resistant (resistance to ≥3 classes of antibiotics). Five random isolates were selected for whole-genome sequencing (WGS) analysis. Using ResFinder (version 3.0) database (14), mcr-1.1 was detected in the genomes with 2 to 16 other acquired genes that encode resistance to important antibiotics (Table 1). Multilocus sequence type (MLST) analysis showed that three isolates belonged to ST57, ST69, and ST1011 (Table 1), which are associated with mcr-1-positive E. coli isolated from diverse sources, including clinical samples, chickens, and food (raw cheese) (15–19), perhaps indicating transmission to humans via the food chain and/or environment, which corroborates previous findings about the occurrence of mcr-1 in these matrices in Lebanon.
TABLE 1.
Antibiotic resistance profiles and sequence types of the colistin-resistant and mcr-1-positive E. coli isolated from fecal matter of toddlers in different locations in Lebanon
| Locationa | Toddler age (yr) |
E. coli
isolate codeb |
Colistin MIC (μg/ml) |
Antibiotic resistance profilec |
Intermediate resistance profile |
Additional acquired ABR genes detected by WGS analysisd |
Sequence type (ST)e |
|---|---|---|---|---|---|---|---|
| Beirut | ≤1 | Bei-2.1 | 4 | PEN-AMP-AMC-LEX | |||
| Bei-2.3 | 16 | PEN-TET | KAN | ||||
| Bei-2.4 | 8 | PEN-AMP-AMC-CTX-LEX- CFM-KAN-STR-SXT-CHL |
FEP-NOR | ||||
| Bei-2.5 | 4 | PEN-AMP-AMC-FEP-CTX- LEX-CFM-STR-TET-SXT |
KAN |
aph(3″)-Ib, blaCTX-M-15, blaTEM-1B, dfrA1, mdf(A), qnrS1, tet(B), tet(C), sul2 |
Unknown ST | ||
| Bei-6.1 | 4 | PEN-AMP-AMC-CTX-LEX- CFM-GEN-KAN-STR-TET- CIP-NOR-SXT-CHL |
FEP |
aac(3)-IId, aph(3″)-Ib, ant(3″)-Ia, aph(3′)-Ia, aph(6)-Id, blaCMY-15, blaTEM-1B, blaTEM-1C, dfrA1, floR, fosA6, mdf(A), tet(B), sul2 |
ST57 | ||
| Bei-6.2 | 16 | PEN-TET | AMP-KAN-STR | ||||
| Bei-6.3 | 8 | PEN-LEX-TET | STR | ||||
| Bei-6.4 | 4 | PEN-AMP-AMC-GEN-KAN- TET-CIP-NOR-SXT-CHL |
STR | ||||
| Saida | ≤1 | Sai-7.1 | 8 | PEN-AMP-AMC-CTX-LEX- CFM-MEM-KAN-TET-SXT |
FEP-DOR-STR-CIP | ||
| Sai-10.1 | 32 | PEN-AMP-AMC-FEP-CTX- LEX-CFM-DOR-MEM-IPM- SXT |
KAN-STR-TET | ||||
| Sai-12.1 | 16 | PEN-AMP-AMC-FEP-CTX- LEX-CFM-STR-SXT-CHL |
KAN | ||||
| ≤2 | Sai-69.1 | 16 | PEN-AMP-AMC-CTX-LEX- CFM-TET |
IPM-GEN-KAN-CIP | |||
| Sai-71.1 | 32 | PEN-AMP-FEP-CTX-LEX- GEN-KAN-STR-TET-CIP- SXT-CHL |
DOR-IPM-NOR |
aac(3)-IId, aadA2, aph(3″)-Ib, aph(6)-Id, blaCFE-1, blaCMY-2, blaCTX-M-3, blaTEM-141, dfrA12, erm(42), floR, fosA3, mdf(A), mph(A), tet(A), sul2 |
ST1011 | ||
| Tripoli | ≤1 | Tri-26.1 | 8 | PEN-AMC-FEP-CTX-LEX- KAN-SXT |
AMP-MEM-IPM- STR-TET-CHL |
blaCFE-1, mdf(A) | Unknown ST |
| Tri-26.2 | 4 | PEN-AMP-AMC-FEP-CTX- LEX-CFM-KAN-STR-TET |
MEM | ||||
| Tri-35.1 | 8 | PEN-AMP-AMC-CTX-LEX- CFM-SXT |
DOR-MEM-IPM-KAN | ||||
| Choueifat | ≤2 | Cho-42.1 | 4 | PEN-AMP-AMC-LEX-CFM- STR |
FEP-CTX-DOR-GEN | ||
| Cho-42.2 | 32 | PEN-AMP-AMC-CTX-LEX- CFM-SXT |
MEM-KAN | ||||
| Cho-42.3 | 16 | PEN-AMP-AMC-CTX-LEX- CFM-SXT |
|||||
| Cho-43.1 | 8 | PEN-AMP-AMC-CTX-LEX- CFM-CHL |
DOR-IPM-KAN- STR-TET |
||||
| Cho-44.1 | 16 | PEN-AMP-AMC-FEP-CTX- LEX-CFM-MEM |
DOR-IPM-KAN | ||||
| Cho-44.2 | 32 | PEN-AMP-AMC-FEP-CTX- LEX-CFM-DOR-MEM |
IPM-KAN | ||||
| Jounieh | ≤2 | Jou-50.1 | 16 | PEN-AMP-AMC-CTX-LEX- CFM |
DOR-MEM-IPM- KAN-STR-CIP |
blaCFE-1, blaCMY-2, mdf(A), qnrB19 | ST69 |
| Jou-50.2 | 16 | PEN-AMP-AMC-CTX-LEX- CFM-MEM |
FEP-DOR-IPM- KAN-STR-CIP |
Location refers to the city in Lebanon where the sampling occurred.
The isolate code indicates the location/city, diaper number, and isolate number. Therefore, Bei-2.1 is isolate number 1 from diaper number 2 that was sampled in Beirut.
Antibiotic resistance profiles were determined using the disk diffusion assay as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines (21). Ampicillin (AMP), amoxicillin plus clavulanic acid (AMC), cefepime (FEP), cefotaxime (CTX), cephalexin (LEX), cefixime (CFM), doripenem (DOR), imipenem (IPM), meropenem (MEM), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), ciprofloxacin (CIP), norfloxacin (NOR), trimethoprim-sulfamethoxazole (SXT), and chloramphenicol (CHL).
ABR, antibiotic resistance; WGS, whole-genome sequencing.
Unknown STs are those that could not be matched to previously known STs, potentially indicating that the unknown STs were novel and/or unavailable in the MLST database.
To our knowledge, this is the first study on mcr-mediated colistin resistance in toddlers in the Middle East and North Africa (MENA) region, which includes several countries with challenges in antimicrobial stewardship and catastrophic medical and humanitarian crises. Assessment of the available literature showed that the occurrence of mcr-1-positive E. coli in Lebanese toddlers ranks second after Bolivia (38.3%) (20). In conclusion, there is a paramount need to regulate the use of colistin, especially in agricultural practices, in order to restrict the spread of mcr in the Lebanese population.
Data availability.
Sequencing data reported in this study can be found under SRA accession numbers SRX7741060, SRX7741058, SRX7741057, SRX7741056, and SRX7741055.
ACKNOWLEDGMENTS
This study was supported by the AUB-URB program and CFS Seed grant.
We thank the Food Microbiology Laboratory (FAFS-AUB) personnel and M. Bou-Dargham and N. Hammad for their help in acquiring and processing the samples.
We declare that we have no conflicts of interest.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Sequencing data reported in this study can be found under SRA accession numbers SRX7741060, SRX7741058, SRX7741057, SRX7741056, and SRX7741055.