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CMY-1/MOX-family AmpC β-lactamases MOX-1, MOX-2 and MOX-9 were mobilized independently from three Aeromonas species

Abstract Objectives To investigate the origin of CMY-1/MOX-family β-lactamases. Methods Publicly available genome assemblies were screened for CMY-1/MOX genes. The loci of CMY-1/MOX genes were compared with respect to synteny and nucleotide identity, and subjected to phylogenetic analysis. Results The chromosomal ampC genes of several Aeromonas species were highly similar to known mobile CMY-1/MOX variants. Annotation and sequence comparison revealed nucleotide identities >98% and conserved syntenies between MOX-1-, MOX-2- and MOX-9-associated mobile sequences and the chromosomal Aeromonas sanarellii, Aeromonas caviae and Aeromonas media ampC loci. Furthermore, the phylogenetic analysis showed that MOX-1, MOX-2 and MOX-9 formed three distinct monophyletic groups with the chromosomal ampC genes of A. sanarellii, A. caviae and A. media, respectively. Conclusions Our findings show that three CMY-1/MOX-family β-lactamases were mobilized independently from three Aeromonas species and hence shine new light on the evolution and emergence of mobile antibiotic resistance genes.


Introduction
Plasmid-encoded AmpC b-lactamases frequently cause b-lactam resistance in clinical strains. Their evolution has been thoroughly investigated and origins have been proposed for several known mobile AmpC enzymes, which occur in the chromosomes of species from several bacterial genera, such as Enterobacter, Citrobacter, Morganella and Aeromonas. [1][2][3] ISs are in several cases likely to be responsible for their mobilization through transposition of parts of the chromosomal ampC loci to mobile genetic elements (MGEs). 4 The origins of some AmpC b-lactamases, including the CMY-1/ MOX-family b-lactamases, are still unclear. CMY-1/MOX-family enzymes hydrolyse extended-spectrum cephalosporins and cephamycins, and, in contrast to other AmpC b-lactamases, several members of this family also provide increased resistance to moxalactam. 5,6 Although CMY-1/MOX enzymes are suspected to be derived from the chromosomal ampC gene of Aeromonas species, 6,7 critical evidence is lacking. Several variants of the CMY-1/ MOX-family b-lactamases have been reported on plasmids in Gram-negative pathogens from Europe and East Asia (CMY-1/ MOX-1/CMY-8/CMY-9/CMY-10/CMY-11/CMY-19, Japan/Korea/ Taiwan, Klebsiella pneumoniae, Escherichia coli, Serratia marcescens and Klebsiella aerogenes; MOX-2, Greece, K. pneumoniae; MOX-4, China, Aeromonas caviae; and MOX-9, Italy, Citrobacter freundii), whereas information on mobility is lacking for others (MOX-3/-5/-6/-7, Spain, Aeromonas spp. and A. caviae; and MOX-8, Thailand, A. caviae). Some CMY-1/MOX enzymes (CMY-1/MOX-1) are highly prevalent in East Asia and compromise the efficacy of blactam treatment, 8 whereas other variants (MOX-2 to MOX-9) are detected less frequently. Understanding from which species resistance genes were mobilized might help us to understand the events that led to their spread into the clinics. This may be valuable for future mitigation efforts, aiming to delay the arrival of novel antibiotic resistance genes at the clinics. The aim of this study was to identify the origin of the mobile CMY-1/MOX b-lactamases using a comparative genomics approach.

Materials and methods
All available CMY-1/MOX amino acid sequences were downloaded from the Comprehensive Antibiotic Resistance Gene Database (CARD). 9 CMY-1/MOX V C The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com sequences were clustered at 95% identity threshold using USEARCH v8.0.1445. 10 All plasmid sequences and genome assemblies were downloaded from the NCBI nucleotide and assembly databases and searched for CMY-1/MOX sequences using DIAMOND 11 blastx with an 85% identity cut-off. The ampC nucleotide sequences of Aeromonas enteropelogenes, Aeromonas hydrophila and Aeromonas sobria were also downloaded from the nucleotide database (accession no.s EU046614.1, NC_008570.1 and NG_047381.1). From each contig containing a CMY-1/MOX gene, 10 kbp upstream and downstream of the gene were extracted and annotated using PROKKA. 12 Proteins denoted as 'hypothetical' by PROKKA were searched against the UniProtKB protein sequence database using DIAMOND (50% id). To identify ISs or transposase-like genes at the extracted CMY-1/MOX loci, the sequences were searched against the ISFinder database. 13 A multiple sequence alignment of the identified CMY-1/MOX genes and Aeromonas ampC genes was computed using MAFFT 14 (-adjustdirectionaccuratelymaxiterate 1000 -globalpair). Phylogenetic analysis was conducted using RAxML 15 (raxmlHPC, 1000 times rapid bootstrapping) with the GTRCAT model. Unique mobile MOX-1, MOX-2 and MOX-9 sequences were included, as well as the CMY-1/MOX Aeromonas ampC genes that reflected the within-species diversity of the ampC locus (meaning that if, for example, several A. caviae ampC genes had different nucleotide similarities to the respective mobile CMY-1/MOX locus, a sequence representing each specific similarity towards the most similar mobile CMY-1/MOX locus was included). The extracted loci were compared at the nucleotide level using the NCBI online blastn 16 suite. Global average nucleotide identity (gANI) and alignment fraction (AF; fraction of orthologous genes between two genomes) as measures of genetic relatedness were calculated using ANIcalculator_v1. 17
The blastx search identified 35 CMY-1/MOX-encoding assembled (contigs or scaffolds) and plasmid sequences: A. caviae In the phylogenetic analysis, MOX-1, MOX-2 and MOX-9 each formed a monophyletic group, supported by bootstrap values .99, with the ampC gene of only one Aeromonas species (Figure 1). Previously suggested CMY-1/MOX-origins, the ampC genes from A. hydrophila and A. sobria, formed single branches. Annotation of the 21 kbp MOX loci showed that in all species except for aeromonads, CMY-1/MOX genes were encoded on MGEs and associated with ISCR1 or ISKpn9, as previously reported. 6,18 The CMY-1/MOX loci consisted of similar genes in all aeromonads (as shown in Figure 2).
Comparison of gene synteny showed that the mobile MOX-1 loci from Serratia, Pseudomonas, Klebsiella and Escherichia spp. matched the gene arrangement of the chromosomal A. sanarellii ampC locus, which differed from other Aeromonas ampC loci. The A. sanarellii synteny did not match with the gene arrangements at the mobile MOX-2 locus (Figure 2), which instead matched the A. hydrophila, A. caviae and A. media synteny.
Nucleotide sequence alignment revealed that the mobile MOX-1 loci from K. aerogenes and E. coli were 98% identical to the A. sanarellii ampC locus, over a length of 3980 bp. This region included the ampC/MOX-1 gene and the genes yqgF, yqgE-like, gshB and rsmED. The alignment included all intergenic regions, with the exception of 89 bp between gshB and rsmED. The ISCR1 upstream of MOX-1 in K. aerogenes was absent in A. sanarellii.  Ebmeyer et al.
The other MOX-1-containing mobile sequences from Klebsiella spp., Pseudomonas spp. and S. marcescens were 97% similar over 2649 bp, but lacked the gshB and rsmE genes. The mobile MOX-2 locus from the K. pneumoniae plasmid was only 90% similar to the A. sanarellii ampC locus, and the alignment over 1160 bp included only the ampC gene. The mobile C. freundii sequence containing MOX-9 was only 80% similar to the A. sanarellii ampC locus over 1123 bp, including only the ampC gene.
Alignment of the Aeromonas ampC loci against the mobile MOX-2 locus showed identities from 91% to 97% over 1629 bp for all A. caviae isolates and 99% over 1629 bp for one A. hydrophila (Aero_hydr_BWH65_V1) isolate. The alignment included the ampC gene, 109 bp of the mdtL efflux pump encoded downstream of the A. hydrophila ampC gene and the intergenic region of 293 bp. ISKpn9, the IS associated with mobile MOX-2, was absent at the A. hydrophila (Aero_hydr_BWH65_V1) and A. caviae ampC loci. The gANI between Aero_hydr_BWH65_V1 and the A. caviae isolates ASM95970v1, ASM81347v1 and ASM173021 was .98% and AF was 0.89-0.93, whereas gANI between Aero_hydr_BWH65_V1 and A. hydrophila ASM1480v1 was only 88% and AF was 0.83. Hence, Aero_hydr_BWH65_V1 originates from a misclassified A. caviae isolate.
The Aero_hydr_BWH65_V1 sequence was only 82% identical over 1100 bp to the mobile MOX-9 locus, with the alignment only including the ampC gene. Aligning the Aeromonas ampC loci against the mobile C. freundii MOX-9 sequence revealed 98%-99% nucleotide identity over 1285 bp to all four A. media ampC loci. The alignment included the A. media ampC gene and the 138 bp located between MOX-9 and ISKnp9, the IS associated with MOX-9 in the C. freundii mobile context. The ISKpn9 on the MOX-9encoding C. freundii and the MOX-2-encoding K. pneumoniae MGEs were 99% similar, whereas MOX-9 and MOX-2 were only 82% similar. No similarity was found in the regions between ISKpn9 and the MOX genes on these mobile elements.

Discussion
We have identified A. sanarellii, A. caviae and A. media as the origins of MOX-1, MOX-2 and MOX-9, respectively, based on phylogenetic analysis of the nucleotide sequences of both mobile CMY-1/MOX and Aeromonas ampC genes, conserved synteny and nucleotide identities 98% between chromosomal and mobile CMY-1/MOX loci. The IS/ISCR sequences associated with mobile CMY-1/MOX variants were absent from the Aeromonas ampC loci.
Nucleotide identities of .98% between mobile and non-mobile genes and their genetic environments conform to the hypothesis that the mobilizations and fixations of these genes are evolutionarily recent events that happened during the antibiotic era. Aeromonas species are primarily associated with aquatic environments, but are known to cause gastroenteritis or wound infections in humans after ingestion/contact with contaminated water, possibly triggering antibiotic use. As IS/ISCR1 can both mobilize 19 and increase expression of adjacent genes, 20 insertion next to the Aeromonas ampC may increase its responsiveness to antibiotic selection pressure. Our findings are hence consistent with the hypothesis that MOX-1, MOX-2 and MOX-9 were mobilized, selected for and transferred to species of the human microbiome during Aeromonas infections treated with b-lactams. Having said that, we cannot exclude that the critical horizontal transfer events took place in, for example, aquaculture settings or other aquatic environments where aeromonads thrive.