Detection of mcr-9-harbouring ESBL-producing Salmonella Newport isolated from an outbreak in a large-animal teaching hospital in the USA

Sir,

Since the first detection of mobile colistin resistance (mcr) in Escherichia coli, several studies have reported mcr-1 and its variants.^1–5 Nine additional mcr genes (mcr-2 to mcr-10) have been reported.⁶ Between 2003 and 2004 a nosocomial outbreak caused by Salmonella enterica serotype Newport strains was reported in a veterinary teaching hospital in Pennsylvania in the USA.⁷ Thirty-one Salmonella Newport strains were isolated from the infected animals. By using multiplex PCR,⁸ a retrospective study was conducted to detect the presence of the mcr genes in the outbreak isolates. Here, we report the detection of mcr-9-harbouring ESBL-producing Salmonella Newport ST45 from a horse from an outbreak in large animals in the USA. Studies involving mcr-carrying, ESBL-producing and colistin-resistant Salmonella in large animals are incomplete and we should draw particular attention to this public health crisis.

The outbreak index strain 0307-213 (CP012599.1) was found to be positive for mcr-9. We determined the MICs of 15 important antimicrobial drugs using a broth microdilution method. Quality control strains E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used and the results were assessed according to CLSI 2019 (and according to EUCAST for colistin). The MIC of colistin was 4 mg/L. Additionally, 0307-213 was also resistant to ampicillin (MIC >256 mg/L), chloramphenicol (MIC = 64 mg/L), tetracycline (MIC = 64 mg/L), sulfamethoxazole/trimethoprim (MIC = 64 mg/L), kanamycin (MIC = 64 mg/L), gentamicin (MIC = 32 mg/L), ceftriaxone (MIC >32 mg/L), cefoxitin (MIC = 64 mg/L), chloramphenicol (MIC = 64 mg/L), ciprofloxacin (MIC = 1 mg/L) and ceftiofur (MIC = 32 mg/L). In contrast, 0307-213 remained susceptible to meropenem, imipenem and tigecycline. A conjugation assay was performed, with E. coli J53 as the recipient strain and Salmonella Newport 0307-213 as the donor. Transconjugants were selected in Mueller–Hinton agar containing colistin at a concentration of 2 mg/L. Transconjugants carrying mcr-9 were successfully confirmed by PCR. The colistin MIC for the donor strain 0307-213 was higher (4 mg/L) than that for the E. coli J53 transconjugant (1 mg/L) and E. coli J53 recipient (0.125 mg/L). Furthermore, the E. coli J53 transconjugant also exhibited resistance to gentamicin (MIC = 32 mg/L) and was susceptible to all other antibiotics.

WGS was conducted using Pacbio RSII (with an average coverage of 100× and an average read length of 5.2 kbp) and the Illumina MiSeq platform (with an average coverage of 150× and an average read length of 184 bp). The assembled genome was further evaluated using the fastq-mcf tool (http://code.google.com/p/ea-utils).

Hybrid assembly of Illumina and PacBio data was performed using SPAdes hybrid assembler version 3.5.0 with default parameters (http://cab.spbu.ru/software/spades/). Center for Genomic Epidemiology web tools (https://cge.cbs.dtu.dk/services/) were used to detect the MLST type, antimicrobial resistance and virulence genes, the plasmid type and intrinsic chromosomal mutations known to be associated with antimicrobial resistance. Serotyping was conducted by both the White-Kauffmann-Le Minor scheme and in silico analysis using the SeqSero web tool (https://github.com/denglab/SeqSero). Both a serum agglutination test and WGS analysis confirmed the serotype to be Newport, belonging to ST45 and carrying mcr-9. Additionally, this strain carried genes encoding resistance to the following antimicrobials: β-lactams (including bla_CMY-2, bla_SHV-12 and bla_TEM-1B), aminoglycosides [i.e. aph(4)-Ia, aac(6′)-Iaa, aadA2, aac(3)-IV, aph(6)-Id, aph(3″)-Ia and aph(3″)-Ib], macrolides [i.e. mdf(A)], phenicols (i.e. catA2, floR and fexA), sulfamethoxazole (i.e. sul1 and sul2), trimethoprim (i.e. dfrA19) and tetracyclines [i.e. tet(A) and tet(D)]. In addition, qnrB2 and a point mutation in parC (T57S) (associated with quinolone resistance) were also found. These results supported a previous evaluation of bla resistance genes in this strain.⁹ Also, we detected typical Salmonella Pathogenic Island-1 and -2, in addition to fim and other fimbrial colonization factors, which play a crucial role in the full virulence of this index strain.

For further analysis of the mcr-9-carrying plasmid, plasmid contigs were extracted from whole-genome sequences using the PLACNETw web tool (https://castillo.dicom.unican.es/upload/). Analysis showed that the mcr-9 gene was from a plasmid of 192 620 bp (Figure 1a). This plasmid revealed sequence identity to a typical IncHI2-type plasmid. Additionally, we found that this plasmid carried an additional aph(4)-Ia.

Figure 1.

Genomic characterization of mcr-9-carrying Salmonella Newport 0307-213 and accompanying plasmid and countrywide phylogenomic investigation. (a) Sequence comparison of reconstructed mcr-9-positive plasmid from a whole-genome sequence of Salmonella Newport 0307-213 with S. enterica 4,[5],12:i:- MK191844 and E. coli MT077886, which were isolated from swine faecal samples, with additional Enterobacter CP012170 and Citrobacter CP022696 from human faecal samples. (b) Genetic comparison of mcr-9 gene context between 0307-213, S. enterica 4,[5],12:i:- MK191844, E. coli MT077886, Enterobacter CP012170 and Citrobacter CP022696. Blue indicates 90–100% identity, red indicates mcr-9, green indicates ISs and violet indicates wbuC. (c) Global phylogenomic analysis of all available Salmonella Newport genomes in the NCBI database (n = 475) that were from the USA; strain ATCC 27869 served as a reference genome. The orange colour and the red arrow indicate the clade that contains the mcr-9-carrying Salmonella Newport 0307-21 (highlighted in red) examined in this study. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

IncHI2 plasmids were found to be the predominant replicon type carrying mcr-9.⁹ Using BLAST to search the NCBI database, several bacterial isolates were found to have plasmids that were 99.9–100% identical to the sequence of the strain 0307-213 plasmid (99% coverage). These bacteria were all Enterobacteriaceae, namely two swine faecal isolates, Salmonella Typhimurium 4,[5],12:i:- MK191844.1 and E. coli MT077886.1, which were recovered between 2014 and 2015, and two human faecal isolates, Enterobacter hormaechei CP012170.1 isolated in 2009 and Citrobacter farmeri CP022696.1 isolated in 2015. Notably, most of these mcr-9-carrying Enterobacteriaceae strains originated from the USA, with the exception of C. farmeri CP022696.1, which was from Australia (Figure 1a).

The plasmid-located mcr-9 gene was inserted in a gene cassette consisting of IS903B-like/mcr-9/wbuC/IS26 (Figure 1b). The wbuC gene is frequently associated with an mcr-9-containing gene cassette that is typically flanked by insertion elements (ISs), such as IS903B and IS26, in strain 0307-213 (Figure 1b). The qseC/qseB genes are also frequently associated with the mcr-9 gene cassette, where they might participate in mcr-9 expression to confer colistin resistance.¹⁰ Curiously, these genes were not present in the mcr-9 gene cassette of strain 0307-213, but were found in the chromosome with ygiW (CP012599.1).

A BLAST search for the IS903B-like/mcr-9/wbuC/IS26 cassette in the NCBI database showed that it has been a recurrent structure since its discovery, with more than 89 of 100 isolates having 100% coverage and 99.7–100% identity. These isolates were mostly Salmonella spp. and Enterobacter spp., two human pathogens, indicating a significant public health threat, due to the rapid dissemination of mcr-9 by plasmids among Enterobacteriaceae isolates.

To track the possible origin of this outbreak due to Salmonella Newport 0307-213, we conducted a phylogenomic analysis by using available Salmonella Newport genomes that were reported from the USA (n = 475) (Table S1, available as Supplementary data at JAC Online) and strain ATCC 27869 was used as the genome reference (Figure 1c). SNP variant calling was performed using Snippy v4.4.4 (https://github.com/tseemann/snippy) and the phylogenomic tree was constructed using the maximum likelihood method with the RAxML model. We found that Salmonella Newport 0307-213 (SAMN03744303) independently clustered together with the other three isolates of bovine origin in one clade, with the same ST (ST45) (Figure 1c). In this clade, we found one isolate (SAMN03744301) obtained from the same outbreak and another isolate from Minnesota in 2006 (SAMN02699857), in addition to SAMN02403369 from Virginia in 2006. Even though these isolates harboured similar MDR genetic elements, only 0307-213 harboured an identical IncHI2 plasmid with mcr-9. This phylogenomic investigation provided a strong indication of a recent acquisition of colistin resistance via horizontal gene transfer in a Salmonella Newport strain.

In this study, we have reported the detection and genomic characterization of a highly virulent ESBL-producing Salmonella Newport with plasmid-mediated colistin resistance. This strain was isolated during an outbreak that locked down the University of Pennsylvania Large-Animal Teaching Hospital during 2003–04.⁷ To the best of our knowledge, this is the first report of a Salmonella Newport isolate harbouring mcr-9. This mcr-9 gene was located on an IncHI2 plasmid, which is predominant among mcr-9-carrying Enterobacteriaceae. The occurrence of mcr-9 flanked by mobile elements and accompanying conjugative plasmids highlights an urgent need for improved and enhanced surveillance to monitor the dissemination of colistin resistance between animal and human pathogens.

Data availability

Raw sequencing reads of 0307-213 have been deposited in the NCBI database under accession number SAMN03744303 and the plasmid sequence under accession number MW244439.

Funding

This work was supported by the National Program on Key Research Project of China (2017YFC1600103, 2018YFD0500501 and 2019YFE0103900) as well as the European Union’s Horizon 2020 Research and Innovation Program under Grant Agreement No. 861917–SAFFI, Zhejiang Provincial Key R&D Program of China (2020C02032), Zhejiang Provincial Natural Science Foundation of China (LR19C180001), Opening Fund of Key Laboratory of Microorganism Technology and Bioinformatics Research of Zhejiang Province (2017E10010), NIH grant AI117135 and USDA grant 2013-67015-21285.

Transparency declarations

None to declare.

Supplementary data

Table S1 is available as Supplementary data at JAC Online.