Dissemination of carbapenemase-producing Enterobacteriaceae harbouring blaNDM or blaIMI in local market foods of Yangon, Myanmar

The spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a serious threat to clinical practice and public health. These bacteria are present both in clinical settings and non-clinical environments. The presence of CPE in food stuffs has been reported, but sporadically so. Here, we screened for CPE in meat, seafood, and vegetable samples from local markets of Yangon, Myanmar. We obtained 27 CPE isolates from 93 food samples and identified 13 as Escherichia coli, six as Klebsiella pneumoniae, seven as Enterobacter cloacae complex, and one as Serratia marcescens. All except the E. cloacae complex harboured the carbapenemase genes blaNDM-1 or blaNDM-5, while all Enterobacter isolates carried the carbapenemase gene blaIMI-1. The blaIMI-1 gene was located in putative mobile elements EcloIMEX-2, -3, or -8. Using multi-locus sequence typing, E. coli, K. pneumoniae, and E. cloacae complex isolates were classified into 10, six, and five different sequence types, respectively. Our results demonstrate that diverse organisms with various carbapenemase genes are widespread in the market foods in Yangon, highlighting the need for promoting proper food hygiene and effective measures to prevent further dissemination.

Resistant isolates possessed multiple genes encoding aminoglycoside modification enzymes, chromosomal point mutation in quinolone-resistance-determining regions of gyrA and parC, as well as plasmid-mediated quinolone-resistance determinants. Notably, we found the 16S rRNA methylase gene rmtB in nine isolates resistant to gentamycin and amikacin. In contrast, all isolates except one were susceptible to fosfomycin and colistin. The only isolate resistant to the latter antibiotic was classified as Serratia, known to be intrinsically colistin-resistant 11 . Multilocus sequence typing (MLST) classified 13 E. coli isolates into 10 sequence types (STs) and six K. pneumoniae isolates each into a different ST (Fig. 1). Further evidence of isolate diversity includes bla NDM presence in differently sized plasmids or chromosomes (Fig. 1).
carbapenemase-producing E. cloacae complex possesses bla IMI-1 . Using the carbapenem-inactivation method (CIM), we identified seven carbapenemase-positive isolates of Enterobacter cloacae complex that are negative for bla NDM , bla KPC , bla IMP , or bla OXA-48 according to a PCR-dipstick test. Instead, whole genome sequencing revealed that they all possessed bla IMI-1 , a chromosomally encoded Ambler class A serine β-lactamase gene. In accordance with an earlier report 12 , bla IMI-1 presence conferred high resistance to imipenem and meropenem, but not to extended-spectrum cephalosporin and oxacephem antibiotics, such as ceftazidime and moxalactam (Fig. 2). These bacteria were also susceptible to gentamycin, minocycline, and levofloxacin. Five isolates out of seven were resistant to fosfomycin, and three were resistant to colistin, a last-resort antibiotic for treating CPE infections.
These six isolates were newly assigned to five STs representing previously undetected populations. We used whole-genome sequencing data of these isolates and those from the GenBank database to generate a core-genome single-nucleotide-polymorphism-based phylogenetic tree of the E. cloacae complex (Fig. 3). This tree grouped the newly assigned isolates into three clusters (G, M, and N) 13 . Each isolate carried bla IMI-1 between setB and yeiP; the gene was encoded on an integrative mobile element designated as EcloIMEX 14 . We identified three mobile elements, EcloIMEX-2 (GenBank accession number KR057494.1), -3 (KU870977.1), and -8 (MG547711.1). Of these, EcloIMEX-3 was found in phylogenetically different isolates, F038 and F054V.

Discussion
This study found diverse CPE in local market foods with higher isolation rates than previously reported (2.4%) 8 . Vegetables were the most contaminated, exhibiting the highest CPE-positive rate (28.1%) and containing approximately 60% of isolates identified. Following vegetables, meat samples had high isolation rates (25.0%). Carbapenem-resistant E. coli and K. pneumoniae isolates exhibited multidrug resistance because they harboured bla NDM-1 or bla NDM-5 , along with other antimicrobial-resistance determinants. Worryingly, most isolates possessed bla NDM-5 , which confers higher resistance against carbapenems than bla NDM-1 15 . Furthermore, we found evidence that E. coli isolates harbouring bla NDM have clinical links; five of the 10 STs identified had been previously detected in a tertiary-care hospital of Yangon 10 . In contrast, NDM-producing K. pneumoniae isolates belonged to STs that have not been detected thus far from Yangon clinical specimens, although four (ST15, 16, 39, 395) have been found in clinical isolates from other countries 16,17 . Notably, we identified isolate F069 in chicken and showed that it was resistant to all tested antibiotics except colistin. This strain belongs to ST395, a constituent of worldwide epidemic clonal group 258 18 .
Isolation of IMI-type carbapenemase is relatively rare. Here, we provided valuable empirical evidence of diverse IMI-producing E. cloacae complexes existing outside hospitals, confirming previous long-term clinical  www.nature.com/scientificreports www.nature.com/scientificreports/ observations that suggested that these bacteria are present in local communities 14,19 . Moreover, we confirmed the presence of IMI-type carbapenemase in Myanmar, suspected ever since bla IMI-2 was detected in a Dutch individual travelling to the country 20 .
We linked bla IMI-1 to a putative mobile element EcloIMEX in all isolates containing the gene. Among the three EcloIMEX detected, EcloIMEX-3 was found in genetically different group N (F038) and G (F054V) isolates, suggesting independent acquisition of the element. Thus, EcloIMEX is probably responsible for mobilizing bla IMI -type genes to E. cloacae complex 14,19 .
We isolated a bla IMI-1 -harbouring E. coloacae complex from fish and water spinach, which is usually cultivated in water-rich regions, corroborating previous reports that associated these bacteria with aquatic environments 6,21,22 . Of note, we isolated two water spinach isolates of the same ST and EcloIMEX type from different districts, suggesting the existence of a shared reservoir of bla IMI-1 dissemination. Other sources are also likely, given the presence of bla IMI-1 -harbouring isolates in other vegetables, as well as chicken and mutton, across all four surveyed districts.
Overall, our results demonstrate widespread dissemination of bla IMI-1 -harbouring E. coloacae complex in Yangon. These organisms are susceptible to several common antibiotics, meaning that multiple treatment options are available in the event of infection such as reported previously 23,24 . However, they may sporadically become more resistant to drugs through acquiring multidrug-resistance plasmids in local communities. We therefore strongly recommend vigorous countermeasures to prevent pathogen spread through daily consumption of food stuffs in regions heavily contaminated by CPE and other multidrug-resistant microbes.
In conclusion, this study provided evidence that local market foods in Yangon, Myanmar frequently harboured diverse CPE isolates, some with highly drug-resistant phenotypes. We note two important limitations of our study: the small sample size and the possible existence of multiple sources for CPE spread. These factors prevented us from tracking exact CPE dissemination profiles in market food samples. Thus, further study is necessary to fully understand the extent of CPE dissemination in this region and to identify the major source(s) of their spread. Such knowledge will enable the development of effective countermeasures against contamination of food with CPE. Nevertheless, our research highlights the immediate need to take corrective action against the current situation of CPE contamination in food. www.nature.com/scientificreports www.nature.com/scientificreports/ Methods isolation of carbapenemase-producing organisms from market food. Food sampling and bacterial isolation were conducted as previously described 25 . From November to December 2017, chicken, pork, beef, fish, clam, and assorted vegetables were collected from eight street stalls in Yangon. Samples were placed in an ice box and transported to the laboratory at the Department of Medical Research, Yangon, where they were either examined immediately or stored at 4 °C until further analysis. For sample processing, 25 g of flesh was mixed with 225 mL buffered peptone water in a stomacher bag and incubated at 35 °C for 18 ± 2 h. Subsequently, a loopful of enrichment culture was streaked onto CHROMagar ECC (CHROMagar, Paris, France), supplemented with 0.25 μg/mL meropenem and 70 μg/mL ZnSO 4 26 , then cultured at 35 °C for 24 ± 2 h.

Bacterial identification, antimicrobial-susceptibility tests, and PCR for carbapenemase genes.
Bacteria were identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI Biotyper; Bruker Daltonics, Germany). Antimicrobial susceptibility was measured using a MicroScan WalkAway 96 Plus (Beckman Coulter, Brea, CA, USA) and dry plate (Eiken Chemical, Tokyo, Japan), then classified based on the Clinical and Laboratory Standards Institute Guidelines (M100-S24). For colistin, resistance was defined according to the European Committee on Antimicrobial Susceptibility Testing criteria. Carbapenem-resistant Enterobacteriaceae isolates were screened for carbapenemase production using CIM 27 . In addition, PCR-dipstick chromatography was performed to detect four major carbapenemase genes (bla NDM , bla KPC , bla IMP , and bla OXA-48 ) 28 .
Whole-genome sequencing and plasmid analysis. Bacterial isolates were cultured overnight in brain-heart infusion broth (BD Bacto, Franklin Lakes, NJ, USA) containing 0.25 μg/mL meropenem. Genomic DNA was then prepared using a PowerSoil DNA isolation kit (Qiagen, Valencia, CA, USA). Library preparation was performed as described previously 9 or using KAPA Hyper Plus Kits (KAPA Biosystems, Woburn, MA, USA). Paired-end sequencing with 250 bp reads was performed in MiSeq (Illumina, San Diego, CA, USA). Reads were then de novo-assembled using CLC Genomics Workbench 11.0.1 (CLC Bio, Aarhus, Denmark) for subsequent analyses.
Multilocus sequence typing for E. coli and K. pneumoniae were performed using EnteroBase (http:// enterobase.warwick.ac.uk/) and the Institute Pasteur's MLST database (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html), respectively. Existing methods 29 were followed for MLST of the E. cloacae complex. Resistance-gene identification was performed in ResFinder 2.1 30 . Evolutionary relatedness of isolates was assessed using CSI Phylogeny 1.4 31 , and iTOL was used to construct the phylogenetic tree 32 . To classify EcloIMEX, local BLAST searches were performed on sequences around bla IMI-1 , which contain setB and yeiP genes.
To determine the bla NDM -harbouring plasmid size, pulsed-field electrophoresis (PFGE) plugs prepared from isolates were treated with S1 nuclease (Takara Bio, Shiga, Japan) and subjected to PFGE in the CHEF Mapper XA system (Bio-Rad, Hercules, CA, USA). Separated DNA was then transferred to a nylon membrane and probed with a digoxigenin-labelled (Roche Diagnostics, Basel, Switzerland), bla NDM -specific DNA probe for Southern hybridization.

Data Availability
The sequence data were submitted to the DDBJ/GenBank/ENA database under BioProject number PRJDB5126.