The expression of aminoglycoside resistance genes in integron cassettes is not controlled by riboswitches.

Regulation of gene expression is a key factor influencing the success of antimicrobial resistance determinants. A variety of determinants conferring resistance against aminoglycosides (Ag) are commonly found in clinically relevant bacteria, but whether their expression is regulated or not is controversial. The expression of several Ag resistance genes has been reported to be controlled by a riboswitch mechanism encoded in a conserved sequence. Yet this sequence corresponds to the integration site of an integron, a genetic platform that recruits genes of different functions, making the presence of such a riboswitch counterintuitive. We provide, for the first time, experimental evidence against the existence of such Ag-sensing riboswitch. We first tried to reproduce the induction of the well characterized aacA5 gene using its native genetic environment, but were unsuccessful. We then broadened our approach and analyzed the inducibility of all AgR genes encoded in integrons against a variety of antibiotics. We could not observe biologically relevant induction rates for any gene in the presence of several aminoglycosides. Instead, unrelated antibiotics produced mild but consistently higher increases in expression, that were the result of pleiotropic effects. Our findings rule out the riboswitch control of aminoglycoside resistance genes in integrons.

Hidden dissemination of carbapenem-susceptible OXA-48-producing Proteus mirabilis.

OBJECTIVES: To detect a potential hidden dissemination of the blaOXA-48 gene among Proteus mirabilis isolates obtained from a single centre. METHODS: P. mirabilis from diverse clinical samples presenting an ESBL phenotype or obtained from blood cultured from 2017 to 2019 were evaluated. Bacterial identification was performed using MALDI-TOF MS. MICs were determined using International Organization for Standardization (ISO) standard microdilution and interpreted following EUCAST guidelines. WGS was performed using both short- and long-read technologies and assemblies were done using Unicycler. Resistomes were assessed using the ResFinder database. SNPs were detected using the PATRIC bioinformatics platform. Cloning experiments were performed using the pCRII-TOPO cloning kit. RESULTS: Thirty-one out of 108 (28.7%) isolates were positive for blaOXA-48 and blaCTX-M-15. Twenty-nine out of 31 of the isolates were susceptible to temocillin, piperacillin/tazobactam, ertapenem and meropenem, whereas only 2/31 showed a resistance phenotype against these antibiotics. Both blaOXA-48 and blaCTX-M-15 genes were detected within the same chromosomally integrated new transposon in all isolates. The resistant isolates displayed a single mutation located in the putative promoter upstream of blaOXA-48. Cloning experiments confirmed that the mutation was responsible for the resistance phenotype. CONCLUSIONS: The presence of a chromosomal copy of blaOXA-48 did not confer resistance to carbapenems, but a single mutation in the promoter could lead to an increase in resistance. This study shows a hidden circulation of OXA-48-positive, but carbapenem- and piperacillin/tazobactam-susceptible, P. mirabilis isolates that can become resistant to ß-lactams after a single mutation.

Evidence for Pseudoxanthomonas mexicana as the recent origin of the blaAIM-1 carbapenemase gene.

OBJECTIVES: Elucidating the recent evolutionary history of clinically important antibiotic resistance genes may inform measures to delay the future emergence of additional resistance genes in clinics. This study investigated the recent origin of blaAIM-1, a metallo-ß-lactamase gene found in Pseudomonas aeruginosa, and the possible role of ISCR15 in its mobilisation and transfer into clinical species. METHODS: Comparative genomics were used to identify the recent origin of blaAIM. Mobilisation attempts were performed under different conditions by cloning ISCR15 and the blaAIM-1-like gene in Escherichia coli. RESULTS: Several blaAIM-1 homologues were identified in the Pseudoxanthomonas genus, with conserved synteny of the locus between species and absence of elements associated with mobility. The closest AIM-1 homologue (97.7% amino acid identity) was found in a Pseudoxanthomonas mexicana (P. mexicana) strain. Cloning the blaAIM-like gene in Escherichia coli resulted in high resistance towards carbapenems. While blaAIM-1 is surrounded by ISCR15 elements in clinical strains, in vitro experiments failed to demonstrate their role as mobilising elements. CONCLUSIONS: This study presents evidence that P. mexicana, an environmental species occasionally associated with infections, is the origin of the B3 metallo-ß-lactamase AIM-1. The presence of terIS, a plausible recognition site for ISCR15, in other parts of the P. mexicana genome suggests a more complex and yet not understood mobilisation mechanism.

Large-scale characterization of the macrolide resistome reveals high diversity and several new pathogen-associated genes.

Macrolides are broad-spectrum antibiotics used to treat a range of infections. Resistance to macrolides is often conferred by mobile resistance genes encoding Erm methyltransferases or Mph phosphotransferases. New erm and mph genes keep being discovered in clinical settings but their origins remain unknown, as is the type of macrolide resistance genes that will appear in the future. In this study, we used optimized hidden Markov models to characterize the macrolide resistome. Over 16 terabases of genomic and metagenomic data, representing a large taxonomic diversity (11 030 species) and diverse environments (1944 metagenomic samples), were searched for the presence of erm and mph genes. From this data, we predicted 28 340 macrolide resistance genes encoding 2892 unique protein sequences, which were clustered into 663 gene families (<70 % amino acid identity), of which 619 (94 %) were previously uncharacterized. This included six new resistance gene families, which were located on mobile genetic elements in pathogens. The function of ten predicted new resistance genes were experimentally validated in Escherichia coli using a growth assay. Among the ten tested genes, seven conferred increased resistance to erythromycin, with five genes additionally conferring increased resistance to azithromycin, showing that our models can be used to predict new functional resistance genes. Our analysis also showed that macrolide resistance genes have diverse origins and have transferred horizontally over large phylogenetic distances into human pathogens. This study expands the known macrolide resistome more than ten-fold, provides insights into its evolution, and demonstrates how computational screening can identify new resistance genes before they become a significant clinical problem.

The Class A Carbapenemases BKC-1 and GPC-1 Both Originate from the Bacterial Genus Shinella.

Comparative genomics identified the environmental bacterial genus Shinella as the most likely origin of the class A carbapenemases BKC-1 and GPC-1. Available sequences and PCR analyses of additional Shinella species revealed homologous ß-lactamases showing up to 85.4% and 93.3% amino acid identity to both enzymes, respectively. The genes conferred resistance to ß-lactams once expressed in Escherichia coliblaBKC-1 likely evolved from a putative ancestral Shinella gene with higher homology through duplication of a gene fragment.

ISEcp1-Mediated Transposition Leads to Fosfomycin and Broad-Spectrum Cephalosporin Resistance in Klebsiella pneumoniae.

A fosfomycin-resistant and carbapenemase (OXA-48)-producing Klebsiella pneumoniae isolate was recovered, and whole-genome sequencing revealed ISEcp1-blaCTX-M-14b tandemly inserted upstream of the chromosomally encoded lysR-fosA locus. Quantitative evaluation of the expression of lysR and fosA genes showed that this insertion brought a strong hybrid promoter leading to overexpression of the fosA gene, resulting in fosfomycin resistance. This work showed the concomitant acquisition of resistance to broad-spectrum cephalosporins and fosfomycin due to a single genetic event.

Characterization of FosL1, a Plasmid-Encoded Fosfomycin Resistance Protein Identified in Escherichia coli.

Fosfomycin is gaining renewed interest for treating urinary tract infections. Monitoring fosfomycin resistance is therefore important in order to detect the emergence of novel resistance mechanisms. Here, we used the Rapid Fosfomycin NP test to screen a collection of extended-spectrum-ß-lactamase-producing Escherichia coli isolates from Switzerland and found a fosfomycin-resistant isolate in which a novel plasmid-mediated fosfomycin resistance gene, named fosL1, was identified. The FosL1 protein is a putative glutathione S-transferase enzyme conferring high-level resistance to fosfomycin and sharing between 57% to 63% amino acid identity with other FosA-like family members. Genetic analyses showed that the fosL1 gene was embedded in a mobile insertion cassette and had likely been acquired by transposition through a Tn7-related mechanism. In silico analysis over GenBank databases identified the FosL1-encoding gene in addition to another variant (fosL1 and fosL2, respectively) in two Salmonella enterica isolates from the United States. Our study further highlights the necessity of monitoring fosfomycin resistance in Enterobacteriaceae to identify the emergence of novel mechanisms of resistance.

Eradication of a Multidrug-Resistant, Carbapenemase-Producing Klebsiella pneumoniae Isolate Following Oral and Intra-rectal Therapy With a Custom Made, Lytic Bacteriophage Preparation.

In July 2017, a patient presented colonization with a multidrug-resistant, carbapenemase (KPC-3)-producing Klebsiella pneumoniae isolate. A custom-made, lytic bacteriophage preparation was administered to the patient in December 2017, with subsequent eradication of the microorganism and without adverse effects.

Identification of FosA8, a Plasmid-Encoded Fosfomycin Resistance Determinant from Escherichia coli, and Its Origin in Leclercia adecarboxylata.

A plasmid-located fosfomycin resistance gene, fosA8, was identified from a CTX-M-15-producing Escherichia coli isolate recovered from urine. Identification of this gene was obtained by whole-genome sequencing. It encoded FosA8, which shares 79% and 78% amino acid identity with the most closely related FosA2 and FosA1 enzymes, respectively. The fosA8 gene was located on a transferable 50-kb plasmid of IncN type encoding high-level resistance to fosfomycin. In silico analysis and cloning experiments identified fosA8 analogues (99% identity) in the genome of Leclercia decarboxylata, which is an enterobacterial species with natural resistance to fosfomycin. This finding adds L. decarboxylata to the list of enterobacterial species that are a reservoir of fosA-like genes which have been captured from the chromosome of a progenitor and are then acquired by E. coli.

Characterization of PAN-1, a Carbapenem-Hydrolyzing Class B ß-Lactamase From the Environmental Gram-Negative Pseudobacteriovorax antillogorgiicola.

The gene encoding the metallo-ß-lactamase (MßL) PAN-1 was identified in the genome of the environmental Gram-negative species Pseudobacteriovorax antillogorgiicola. PAN-1 shares 57% amino-acid identity with the acquired MßL SPM-1, its closest relative. Kinetic parameters performed on purified PAN-1 showed it displayed a hydrolytic activity toward most ß-lactams including carbapenems but spared cefepime and aztreonam. These results further highlight that environmental bacterial species may be reservoirs of MßL encoding genes.

mcr-9, an Inducible Gene Encoding an Acquired Phosphoethanolamine Transferase in Escherichia coli, and Its Origin.

The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9 Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin.

Functional Characterization of a Miniature Inverted Transposable Element at the Origin of mcr-5 Gene Acquisition in Escherichia coli.

Plasmid-mediated colistin resistance of the mobile colistin resistance (MCR) type is a growing concern in Enterobacteriaceae since it has been described worldwide in humans and animals. Here, we identified a series of MCR-producing Escherichia coli isolates corresponding to two different clones (represented by isolates PS1 and PS8b) producing MCR-1 and MCR-5, respectively, obtained from pig fecal samples in France. Plasmid analysis showed that the plasmid carrying the mcr-1 gene (pPS1) possesses an IncHI2 backbone, whereas the mcr-5 gene was carried onto a 6,268-bp nontypeable non-self-conjugative plasmid (pPS8b). Detailed analysis of plasmid pPS8b revealed a 3,803-bp-long cassette containing the mcr-5 gene that was bracketed by two inverted-repeat (IR) sequences with 5-bp-long direct repeats at each extremity, similarly to an insertion sequence, but with the exception that no transposase gene was identified within this cassette. By performing in vitro transposition experiments, we showed that the mcr-5 cassette could be mobilized by the TnAs1 transposase provided in trans, displaying a mobilization mechanism similar to that of miniature inverted-repeat transposable elements (MITEs).

Colistin resistance in Parisian inpatient faecal Escherichia coli as the result of two distinct evolutionary pathways.

BACKGROUND: Beyond plasmid-encoded resistance (mcr genes) prevalence in strain collections, large epidemiological studies to estimate the human burden of colistin-resistant Escherichia coli gut carriage are lacking. OBJECTIVES: To evaluate the prevalence of colistin-resistant E. coli carriage in inpatients and decipher the molecular support of resistance and the genetic background of the strains. METHODS: During a 3 month period in 2017, we prospectively screened patients in six Parisian hospitals for rectal carriage of colistin-resistant E. coli using a selective medium, a biochemical confirmatory test and MIC determination. WGS of the resistant strains and their corresponding plasmids was performed. RESULTS: Among the 1217 screened patients, 153 colistin-resistant E. coli strains were isolated from 152 patients (12.5%). The mcr-1 gene was identified in only seven isolates (4.6%) on different plasmid scaffolds. The genetic background of these MCR-1 producers argued for an animal origin. Conversely, the remaining 146 colistin-resistant E. coli exhibited a phylogenetic distribution corresponding to human gut commensal/clinical population structure (B2 and D phylogroup predominance); 72.6% of those isolates harboured convergent mutations in the PmrA and PmrB proteins, constituting a two-component system shown to be associated with colistin resistance. CONCLUSIONS: We showed that the occurrence at a high rate of colistin resistance in human faecal E. coli is the result of two distinct evolutionary pathways, i.e. the occurrence of chromosomal mutations in an endogenous E. coli population and the rare acquisition of exogenous mcr-1-bearing strains probably of animal origin. The involved selective pressures need to be identified in order to develop preventative strategies.

ZHO-1, an intrinsic MBL from the environmental Gram-negative species Zhongshania aliphaticivorans.

OBJECTIVES: Our aim was to characterize the putative MBL of the environmental strain Zhongshania aliphaticivorans isolated from a marine environment. METHODS: The putative MBL was identified in silico using the NCBI database. The ß-lactamase gene was cloned into different Escherichia coli backgrounds. Kinetic parameters were determined using the purified enzyme. RESULTS: The enzyme named ZHO-1 shared 51% amino acid identity with the acquired class B carbapenemases IMP-1, KHM-1 and DIM-1. Expression of the blaZHO-1 gene in a susceptible E. coli resulted in a carbapenemase phenotype. Kinetic parameters determined from purified ZHO-1 enzyme showed that it had significant hydrolytic activity against most ß-lactams including penicillins, cephalosporins and carbapenems, with the exception of aztreonam and cefepime. CONCLUSIONS: This study adds to the knowledge regarding environmental species as a reservoir of possible clinically relevant MBLs.

Increased Resistance to Carbapenems in Proteus mirabilis Mediated by Amplification of the blaVIM-1-Carrying and IS26-Associated Class 1 Integron.

Objective: The aim of the study was to decipher the mechanisms and associated genetic determinants responsible for increased carbapenem resistance among Proteus mirabilis clinical isolates. Methods: The entire genetic structure surrounding the ß-lactam resistance genes was characterized by PCR, gene walking, and DNA sequencing. Results: A series of clinical P. mirabilis isolates were consecutively recovered from different patients at the Military hospital of Sofia, Bulgaria. They showed variable levels of resistance to carbapenems. All isolates produced the same carbapenemase VIM-1 that was chromosomally encoded. We showed that increased resistance to carbapenems was related to an increased number of blaVIM-1 gene copies. Conclusion: We showed here that increased carbapenem resistance in P. mirabilis may result from increased expression of the blaVIM-1 carbapenemase gene through multiplication of its copy number.