The emergence and global dissemination of the mobile colistin resistance (mcr) genes have threatened the use of colistin (polymyxin E), a last-resort antibiotic, in the treatment of complicated infections caused by certain multidrug-resistant (MDR) bacteria. In the USA, colistin has been neither marketed nor approved by the US Food and Drug Administration (FDA) for use in food animals, which is considered a main driver of the emergence and spread of mcr in other countries [1]. However, mcr can be carried on transmissible and robust plasmids with potentially other important antibiotic resistance and survival-associated genes that might allow for the selection and persistence of mcr-harbouring plasmids even in the absence of wide colistin use in US agriculture. Furthermore, mcr genes have been detected in some animal and human samples in the USA [2]. Notably, mcr-1 was reported in Escherichia coli isolated from a paediatric patient in Connecticut, four patients in Michigan, a patient in New York City and another in New Jersey [2]. Moreover, mcr-3.1- and mcr-9-carrying Salmonella enterica were identified in the faeces of two patients from the South Eastern USA and Washington State, respectively [2]. In the majority of these cases, acquisition of mcr-carrying bacteria by the patients was mainly attributed to travel abroad [2]. Taken together, it is conceivable that mcr is being introduced, becoming established and spreading in the USA unnoticeably [2]. However, studies on mcr in the USA remain limited, especially in potential ‘hotspot’ matrices associated with humans and agriculture.
Here we report the detection of a plasmid-borne mcr-9.1 gene in Morganella morganii isolated from a sewage influent sample collected from a wastewater treatment plant in Georgia. During a surveillance initiative, a composite sewage sample (1 L) was collected in a sterile Nalgene® bottle, transferred to the laboratory on ice and processed within 1 h of collection. An aliquot (100 μL) was spread onto RAPID’E. coli 2 agar (Bio-Rad, USA) supplemented with colistin (4 μg/mL) (Sigma-Aldrich, USA) [3]. Following incubation at 37°C, colonies were further purified and tested for colistin minimum inhibitory concentration (MIC) using the broth microdilution method as described by the Clinical and Laboratory Standards Institute (CLSI) [3]. An isolate showed high resistance to colistin (MIC > 640 μg/mL) (Table 1). Further analysis using the disk diffusion assay [3] showed that the isolate also exhibited resistance to other important antibiotics, including ampicillin, amoxicillin/clavulanic acid, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, ciprofloxacin, trimethoprim/sulfamethoxazole and chloramphenicol, indicating that the isolate was MDR.
To further characterise the strain, whole-genome sequencing (WGS) analysis was performed [3]. Briefly, genomic DNA was isolated and quantified using a QIAamp® DNA Mini Kit (QIAGEN, USA) and Qubit® Double-Stranded DNA (dsDNA) Broad-Range (BR) Assay Kit (Invitrogen, USA), respectively [3]. Sample libraries were prepared and quantified using a Nextera XT DNA Library Preparation Kit and Qubit dsDNA High-Sensitivity (HS) Broad-Range (BR) Assay Kit (Invitrogen), respectively [3]. Libraries were then diluted, denatured and loaded into a MiSeq reagent cartridge (MiSeq reagent kit v.2, 300 cycles) [3]. Sequencing was then performed using a MiSeq sequencer (Illumina Inc., USA) and a paired-end sequencing strategy (2 × 150 bp). Trimmomatic v.0.36 was used to remove low-quality reads. Additionally, the leading three and trailing three nucleotides were removed from the reads, and a four-nucleotide sliding window was used to also remove nucleotides from the 3′ ends when the average Phred score dropped below 20. Reads shorter than 75 bp were discarded. Draft genome sequences were assembled from the trimmed and filtered reads using the ‘–careful’ option in SPAdes v.3.9.0 [3]. Contigs shorter than 200 bp were discarded and the quality of the draft genome was evaluated with QUAST v.4.5 [3]. WGS analysis identified the isolate as M. morganii carrying multiple genes associated with antibiotic resistance (19 and 22 genes were detected using ResFinder v.3.0 and CARD databases, respectively), including mcr-9.1 (Table 1), corroborating that the strain was MDR. The WGS files were deposited in GenBank under BioSample no. SAMN20521926 and accession no. JAIHMK000000000.1. The mcr-9.1 gene was also detected using a gene-specific PCR analysis [4], while the identity of the isolate was further confirmed using a species-specific PCR analysis [5]. Notably, M. morganii is known to be intrinsically resistant to colistin and other antibiotics. This corroborated the high colistin MIC observed for this isolate and its resistance to ampicillin, amoxicillin/clavulanic acid and erythromycin (Table 1) and emphasised the identity as M. morganii. Further analysis showed that mcr-9.1 was transmissible to chemically competent E. coli JM109 using the heat-shock method [3]. Transformants were shown to be mcr-9.1-positive by PCR and colistin-resistant (MIC = 4 μg/mL), indicating that the gene was plasmid-borne, potentially transmissible and conferred colistin resistance. Furthermore, the transformants were not resistant to any of the other tested antibiotics, indicating that the associated genetic determinants of resistance were not on the mcr-9.1-carrying plasmid. PCR-based replicon typing (PBRT) [3] analysis showed that the plasmid in the transformants belonged to IncFII type. Additionally, WGS analysis with the PlasmidFinder v.1.3 database [3] showed that the M. morganii isolate carried two different IncF plasmids (IncFIB and IncFII) (Table 1), while the MOB-suite software confirmed that mcr-9.1 was located on the same contig (∼2402 bp) with plasmid sequences. Taken together, mcr-9.1 in the M. morganii strain appears to be carried on an IncFII plasmid. This is important because IncF plasmids (∼50–200 kb in size) have been associated with clinical strains of Enterobacteriaceae and the worldwide emergence and dissemination of important antibiotic resistance determinants…
