Global scenario of the RmtE pan-aminoglycoside-resistance mechanism: emergence of the rmtE4 gene in South America associated with a hospital-related IncL plasmid.

Antimicrobial resistance (AMR) mechanisms, especially those conferring resistance to critically important antibiotics, are a great concern for public health. 16S rRNA methyltransferases (16S-RMTases) abolish the effectiveness of most clinically used aminoglycosides, but some of them are considered sporadic, such as RmtE. The main goals of this work were the genomic analysis of bacteria producing 16S-RMTases from a 'One Health' perspective in Venezuela, and the study of the epidemiological and evolutionary scenario of RmtE variants and their related mobile genetic elements (MGEs) worldwide. A total of 21 samples were collected in 2014 from different animal and environmental sources in the Cumaná region (Venezuela). Highly aminoglycoside-resistant Enterobacteriaceae isolates were selected, identified and screened for 16S-RMTase genes. Illumina and Nanopore whole-genome sequencing data were combined to obtain hybrid assemblies and analyse their sequence type, resistome, plasmidome and pan-genome. Genomic collections of rmtE variants and their associated MGEs were generated to perform epidemiological and phylogenetic analyses. A single 16S-RMTase, the novel RmtE4, was identified in five Klebsiella isolates from wastewater samples of Cumaná. This variant possessed three amino acid modifications with respect to RmtE1-3 (Asn152Asp, Val216Ile and Lys267Ile), representing the most genetic distant among all known and novel variants described in this work, and the second most prevalent. rmtE variants were globally spread, and their geographical distribution was determined by the associated MGEs and the carrying bacterial species. Thus, rmtE4 was found to be confined to Klebsiella isolates from South America, where it was closely related to ISVsa3 and an uncommon IncL plasmid related with hospital environments. This work uncovered the global scenario of RmtE and the existence of RmtE4, which could potentially emerge from South America. Surveillance and control measures should be developed based on these findings in order to prevent the dissemination of this AMR mechanism and preserve public health worldwide.

Population genomics and antimicrobial resistance dynamics of Escherichia coli in wastewater and river environments.

Aquatic environments are key niches for the emergence, evolution and dissemination of antimicrobial resistance. However, the population diversity and the genetic elements that drive the dynamics of resistant bacteria in different aquatic environments are still largely unknown. The aim of this study was to understand the population genomics and evolutionary events of Escherichia coli resistant to clinically important antibiotics including aminoglycosides, in anthropogenic and natural water ecosystems. Here we show that less different E. coli sequence types (STs) are identified in wastewater than in rivers, albeit more resistant to antibiotics, and with significantly more plasmids/cell (6.36 vs 3.72). However, the genomic diversity within E. coli STs in both aquatic environments is similar. Wastewater environments favor the selection of conserved chromosomal structures associated with diverse flexible plasmids, unraveling promiscuous interplasmidic resistance genes flux. On the contrary, the key driver for river E. coli adaptation is a mutable chromosome along with few plasmid types shared between diverse STs harboring a limited resistance gene content.

Highly Tigecycline-Resistant Klebsiella pneumoniae Sequence Type 11 (ST11) and ST147 Isolates from Companion Animals.

In this study, we characterized two tigecycline-resistant Klebsiella pneumoniae isolates from dog urine samples. The isolates were genetically unrelated, belonging to sequence type 11 (ST11) and ST147, both classically related to human isolates. To the best of our knowledge, this is the first identification of tigecycline-resistant isolates from animals. We unveil here the worrisome circulation among animals of bacterial clones resistant to this last-resort antibiotic.

Coexistence of mcr-1 and blaNDM-1 in Escherichia coli from Venezuela.

We studied the presence of the mobile colistin resistance gene mcr-1 in human, animal, and environmental Enterobacteriaceae samples from Cumana, Venezuela, that were collected in 2015. The mcr-1 gene was detected in 2/93 Escherichia coli isolates from swine (novel ST452) and human (ST19) samples that were resistant to colistin. Whole-genome sequencing and transformation experiments identified mcr-1 on an IncI2 plasmid. One of the isolates also bore the widely spread carbapenemase NDM-1. A One Health approach is necessary to further elucidate the flux of these high-risk genes.

RmtC and RmtF 16S rRNA Methyltransferase in NDM-1-Producing Pseudomonas aeruginosa.

We investigated 16S rRNA methyltransferases in 38 blaNDM-1-positive Pseudomonas aeruginosa isolates and found RmtC in 3 isolates, 1 of which also harbored RmtF. The isolates were clonally unrelated; rmtC and rmtF genes were located on a chromosome with the blaNDM-1 gene. Strategies are needed to limit the spread of such isolates.

Prevalence and molecular characterisation of New Delhi metallo-ß-lactamases NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India.

The growing prevalence of carbapenem resistance in Enterobacteriaceae worldwide is a major concern. New Delhi metallo-ß-lactamase (NDM)-mediated carbapenem resistance has been identified in Enterobacteriaceae from numerous countries including those of the Indian subcontinent. Currently, seven NDM ß-lactamase variants (NDM-1 to -7) have been identified. This study evaluated the detection and molecular characterisation of NDM variants in Enterobacteriaceae at a tertiary care hospital in India. A total of 464 isolates were tested; 57 (12.3%) were resistant or showed reduced susceptibility to imipenem and meropenem. All carbapenem-resistant isolates were blaNDM-positive by PCR, but 13 isolates bore variants that differed in sequence from blaNDM-1. NDM-5, NDM-6 and NDM-7 were identified in two, eight and three isolates, respectively. blaNDM variants were located on plasmids of >100kb with IncF, IncA/C and untypeable replicon types. Genes encoding the 16S rRNA methyltransferases RmtB, RmtC and ArmA as well as those for AmpC ß-lactamases were also located on the same plasmids as blaNDM in different combinations. The prevalence of NDM-5 to -7 variants was significantly higher in Escherichia coli (P=0.015) and they were more frequently isolated from the urology ward (P=0.037) than NDM-1. The mortality rate was comparable between patients infected with isolates positive for blaNDM-1 and blaNDM variants [25% (11/44) vs. 23% (3/13)]. Expression of blaNDM variants in E. coli using the same promoter showed that NDM-7 conferred higher resistance to imipenem. The diverse genotypic features of blaNDM indicate rapid evolution of NDM resulting from their wide spread in the Indian subcontinent.

Klebsiella pneumoniae sequence type 11 from companion animals bearing ArmA methyltransferase, DHA-1 ß-lactamase, and QnrB4.

Seven Klebsiella pneumoniae isolates from dogs and cats in Spain were found to be highly resistant to aminoglycosides, and ArmA methyltransferase was responsible for this phenotype. All isolates were typed by multilocus sequence typing (MLST) as ST11, a human epidemic clone reported worldwide and associated with, among others, OXA-48 and NDM carbapenemases. In the seven strains, armA was borne by an IncR plasmid, pB1025, of 50 kb. The isolates were found to coproduce DHA-1 and SHV-11 ß-lactamases, as well as the QnrB4 resistance determinant. This first report of the ArmA methyltransferase in pets illustrates their importance as a reservoir for human multidrug-resistant K. pneumoniae.

Association of the novel aminoglycoside resistance determinant RmtF with NDM carbapenemase in Enterobacteriaceae isolated in India and the UK.

OBJECTIVES: 16S rRNA methyltransferases are an emerging mechanism conferring high-level resistance to clinically relevant aminoglycosides and have been associated with important mechanisms such as NDM-1. We sought genes encoding these enzymes in isolates highly resistant (MIC >200 mg/L) to gentamicin and amikacin from an Indian hospital and we additionally screened for the novel RmtF enzyme in 132 UK isolates containing NDM. METHODS: All highly aminoglycoside-resistant isolates were screened for armA and rmtA-E by PCR, with cloning experiments performed for isolates negative for these genes. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to determine the methylation target of the novel RmtF methyltransferase. RmtF-bearing strains were characterized further, including susceptibility testing, PFGE, electroporation, PCR-based replicon typing and multilocus sequence typing of rmtF-bearing plasmids. RESULTS: High-level aminoglycoside resistance was detected in 140/1000 (14%) consecutive isolates of Enterobacteriaceae from India. ArmA, RmtB and RmtC were identified among 46%, 20% and 27% of these isolates, respectively. The novel rmtF gene was detected in 34 aminoglycoside-resistant isolates (overall prevalence 3.4%), most (59%) of which also possessed a bla(NDM) gene; rmtF was detected in 6 NDM producers from the UK. It was found on different plasmid backbones. Four and two isolates showed resistance to tigecycline and colistin, respectively. CONCLUSIONS: RmtF was often found in association with NDM in members of the Enterobacteriaceae and on diverse plasmids. It is of clinical concern that the RmtF- and NDM-positive strains identified here show additional resistance to tigecycline and colistin, current drugs of last resort for the treatment of serious bacterial infections.

Fitness cost and interference of Arm/Rmt aminoglycoside resistance with the RsmF housekeeping methyltransferases.

Arm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants of Escherichia coli, corresponding to the genotypes rsmF(+), ΔrsmF, rsmF(+) rmtC(+), and ΔrsmF rmtC(+). When analyzed for the antimicrobial resistance pattern, the ΔrsmF bacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility in E. coli. Competition experiments between the isogenic E. coli strains showed that, contrary to expectation, acquisition of rmtC does not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.