Extensively Drug-Resistant New Delhi Metallo-β-Lactamase–Encoding Bacteria in the Environment, Dhaka, Bangladesh, 2012

Carriage of the New Delhi metallo-β-lactamase variant 1 (NDM-1) enables drug resistance to move between communities and hospitals. In Bangladesh, we found the blaNDM-1 gene in 62% of environmental waters and in fermentative and nonfermentative gram-negative bacteria. Escherichia coli sequence type (ST) 101 was most commonly found, reflecting a common global relationship between ST101 and NDM-1.

transferred to 0.5 ml of sterile water in an Eppendorf tube and resuspended. One µl of this bacterial suspension was then used for PCR reactions to detect the NDM-1 gene (blaNDM-1) and the CTX-M-15 gene  and the rest stored at 4C. Samples that were positive for blaNDM-1 in the initial PCR were then re-plated at several dilutions to ensure well-spaced colonies. From these plates at least twenty further well-spaced colonies were re-grown and individually investigated by PCR and sequencing for the presence of blaNDM-1. These represented a range of colonies of different color and morphology to ensure detection of as many different bacterial NDM-1 hosts at each site as possible.
Positive individual cultures were again streaked on brilliance agar plates to confirm purity and then speciated by Maldi-TOF using the Bruker MALDI Biotyper system, Mcroflex LT and Biotyper 3.0 software (Bruker Daltonics, Germany) and NDM presence re-confirmed by PCR. Isolates of each species were tested for MICs against several antibiotics (ETest, bioMérieux, Basingstoke, UK. Liofilchem, Roseto degli Abruzzi, Italy) on Mueller-Hinton plates (Becton Dickinson, Oxford, UK) and the results determined according to the manufacturer's instructions.

NDM Allele
Approximately 10 positive PCR products from each genus including duplicate examples from each individual species were sequenced to confirm the accuracy of the PCR reaction and also to determine the NDM variant present (these were amplified using primers NDMVF-TGGCTTTTGAAACTGTCGCACC and NDMVR-CTGTCACATCGAAATCGCGCGA designed up and downstream of the gene to ensure the entire gene was sequenced).

Gene Location
The location of the blaNDM-1 gene in each bacterial isolate was determined by pulsed field gel electrophoresis (PFGE) following S1 digestion of macro DNA in agarose plugs. The genomic location was detected using in-gel hybridization with a P 32 -labeled blaNDM-1 probe at 65C as described previously.

Additional Resistance Alleles
E. coli blaNDM-1 positive isolates (10) collected from a range of sites that were positive for these species were further investigated for additional resistance alleles that are often found associated with blaNDM-1 including the 16S ribosomal methylase genes armA, rmtB, rmtC, rmtF, ampC genes and blaCTX-M genes by multiplex PCR using published primers and conditions.

Common NDM Plasmid Types
Plasmid types that are commonly associated with blaNDM-1 were investigated by PCR in the above E. coli isolates using the custom designed primer pairs itemized below. These were designed to amplify plasmid backbone genes from NDM harboring plasmids available in the genetic data bases and amplified using relevant controls.

E. coli Phylogeny
The 53 blaNDM-1 positive E. coli isolates collected in this study were further investigated for phylogenetic grouping using the recently updated (2013) Clermont multiplex PCR method and also by the high resolution 2 loci clonal EXPEC typing method, using the described primers and conditions and applicable sequencing. Several examples of each different group determined by these methods were also subjected to full multi-locus sequence typing using the MLST E. coli typing system, primers and conditions described at the university of Warwick http://mlst.warwick.ac.uk/mlst/dbs/Ecoli.

Initial Species Identification
Initial species identification was by colony color on brilliance agar plates. Swabs from each site were plated out on individual plates. Clear colony types >100 were found on all plates, indicative of carbapenem resistant non-fermentative bacteria being found at all sites. Red colonies indicative of E. coli were found on 11 plates varying in number from 1 or 2 colonies to 50-80 per plate. Dark blue colonies suggestive of K. pneumoniae were found on 19 plates and varied in number from 1 or 2 to more than 100 per plate.

Comparison of E. coli Phylogeny Methods
The 2 locus clonal EXPEC typing method correctly identified all isolates belonging to ST101 and ST405 whereas the Clermont method was inaccurate for many of the strains analyzed placing ST648 and ST405 (phylogroup D) as B2 as well as half of the ST101 (phylogroup B1) as phylogroup E (Technical Appendix Table 2). Interestingly, all ST648 phylogroup D E. coli strains were fimH negative and fumC allele 4 (Technical Appendix Table 2). Only a single phylogroup B1 strain was present with this 2 locus designation in the database and so this system incorrectly identified this ST648 as phylogroup B1 instead of D.

Resistance Profile of blaNDM-1 Bacterial Species
Overall the bacterial isolates displayed antibiotic resistance profiles that are very similar to clinical isolates that carry blaNDM-1 (Technical Appendix Table 3). The E. coli and K. pneumoniae isolates were resistant to all β-lactams with the single exception of one K. pneumoniae strain that was sensitive to Aztreonam. They were also resistant to all clinical aminoglycosides and only sensitive to fosfomycin, tigecycline and colistin reflecting the additional presence of 16s RNA methylases in all strains classifying the majority of these isolates as XDR. In general, the MICs to meropenem were higher than those to imipenem, validating the inclusion of meropenem in the initial screening. The various Acinetobacter spp. and Citrobacters were generally less resistant, several being sensitive to aminoglycosides and occasionally ciprofloxacin, the Acinetobacters were additionally generally sensitive to rifampicin.