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.

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.

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.

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).

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.

Acquisition of Extended-Spectrum ß-Lactamase GES-6 Leading to Resistance to Ceftolozane-Tazobactam Combination in Pseudomonas aeruginosa.

A clinical Pseudomonas aeruginosa isolate resistant to all ß-lactams, including ceftolozane-tazobactam and carbapenems, was recovered. It belonged to sequence type 235 and produced the extended-spectrum ß-lactamase (ESBL) GES-6 differing from GES-1 by two amino acid substitutions (E104K and G170S). GES-6 possessed an increased hydrolytic activity toward carbapenems and to ceftolozane and a decreased susceptibility to ß-lactamase inhibitors compared to GES-1, except for avibactam. We show here that resistance to ceftolozane-tazobactam may occur through acquisition of a specific ESBL in P. aeruginosa but that ceftazidime-avibactam combination remains an effective alternative.

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.

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.

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.

Transposition of Tn1213 Encoding the PER-1 Extended-Spectrum ß-Lactamase.

PER-1 is an extended-spectrum ß-lactamase that is encoded by a gene located in composite transposon Tn1213 made by two distinct insertion sequences, namely, ISPa12 and ISPa13. In vitro mobilization performed in Escherichia coli shows that Tn1213 is functional and is able to mobilize the blaPER-1 gene, although at a very low frequency (ca. 1 × 10-9).

Screening and Characterization of Multidrug-Resistant Gram-Negative Bacteria from a Remote African Area, São Tomé and Príncipe.

The occurrence of resistance to last-resort antibiotics was evaluated among Enterobacteriaceae isolates recovered from hospitalized children in a remote African archipelago, São Tomé and Príncipe, where there is limited access to those antibiotics. Fifty patients were screened for colonization by carbapenem-, pan-aminoglycoside-, or polymyxin-resistant Enterobacteriaceae A total of 36 isolates (including 30 Escherichia coli and 4 Klebsiella pneumoniae) were recovered from 23 patients, including 26 isolates harboring the blaOXA-181 carbapenemase gene, a single isolate harboring the 16S rRNA methylase gene rmtB encoding pan-resistance to aminoglycosides, and 8 isolates coharboring both genes. A single isolate possessed the plasmid-borne colistin resistance gene mcr-1 A high clonal relationship was found for OXA-181-producing E. coli (4 clones), and conversely, three of the four OXA-181-producing K. pneumoniae isolates were clonally unrelated. This study overall showed a high prevalence of resistance to last-resort antibiotics in this country, where no epidemiological data were previously available.

Genetic and Functional Characterization of an MCR-3-Like Enzyme-Producing Escherichia coli Isolate Recovered from Swine in Brazil.

A collection of 126 pigs was screened for carriage of colistin-resistant Enterobacteriaceae in a farm in Minas Gerais, Brazil. Out of this collection, eight colistin-resistant Escherichia coli isolates were recovered, including one from Minas Gerais State producing a new MCR-3 variant (MCR-3.12). Analysis of the lipopolysaccharide revealed that MCR-3.12 had a function similar to that of MCR-1 and MCR-2 as a result of the addition of a phosphoethanolamine group to the lipid A moiety. Genetic analysis showed that the mcr-3.12 gene was carried by an IncA/C2 plasmid and was embedded in an original genetic environment. This study reports the occurrence of the MCR-3-like determinant in South America and is the first to demonstrate the functionality of this group of enzymes as a phosphoethanolamine transferase.

High Rate of MCR-1-Producing Escherichia coli and Klebsiella pneumoniae among Pigs, Portugal.

The mcr-1 (mobile colistin resistance 1) gene, which encodes phosphoethanolamine transferase, has been recently identified as a source of acquired resistance to polymyxins in Escherichia coli. Using the SuperPolymyxin selective medium, we prospectively screened 100 pigs at 2 farms in Portugal for polymyxin-resistant Enterobacteriaceae and recovered 98 plasmid-mediated MCR-1-producing isolates. Most isolates corresponded to nonclonally related E. coli belonging to many sequence types; we also found 2 Klebsiella pneumoniae sequence types. The mcr-1 gene was carried on IncHI2 or IncP plasmid backbones. Our finding of a high rate of MCR-1 producers on 2 pig farms in Portugal highlights the diffusion of that colistin-resistance determinant at the farm level. The fact that the pigs received colistin as metaphylaxis in their feed during the 6 weeks before sampling suggests selective pressure.

Moraxella Species as Potential Sources of MCR-Like Polymyxin Resistance Determinants.

Plasmid-mediated resistance to polymyxins mediated by the MCR-1/2 determinants has been reported in Enterobacteriaceae worldwide. Using PCR-based and cloning strategies, a series of Moraxella spp. were screened for mcr-like genes. Moraxella spp. that are mainly animal pathogens but may also be human pathogens were identified as potential reservoirs of mcr-like genes.

In Vitro Study of ISApl1-Mediated Mobilization of the Colistin Resistance Gene mcr-1.

The plasmid-mediated mcr-1 gene encodes a phosphoethanolamine transferase that confers resistance to polymyxins. The mcr-1 gene is associated with insertion sequence ISApl1 (IS30 family). In vitro mobilization assays demonstrated the functionality of the composite transposon structure ISApl1-mcr-1-ISApl1 Transposition generated a 2-bp duplication and occurred in AT-rich DNA regions. This is the first report demonstrating the mobility of the mcr-1 gene by transposition.

High Prevalence of Carbapenemase-Producing Enterobacteriaceae among Hospitalized Children in Luanda, Angola.

This study aimed to evaluate the prevalence of carbapenemase-producing Enterobacteriaceae in Luanda, Angola. A total of 157 rectal samples were collected from children visiting a pediatric hospital in Luanda in March 2015. Fifty-seven imipenem-nonsusceptible enterobacterial isolates were recovered, most of which were non-clonally related. The blaOXA-181 (50/57) and blaNDM-1 (7/57) carbapenemase genes were identified. Notably, OXA-181-producing Escherichia coli isolates rarely coproduced extended-spectrum ß-lactamases and consequently remained susceptible to broad-spectrum cephalosporins. The blaOXA-181 gene was always located on an IncX3 plasmid, while the blaNDM-1 gene was located on either IncFIA or IncA/C plasmids. The study identified a high prevalence of OXA-181 among hospitalized children in Angola.

Genetic Features of MCR-1-Producing Colistin-Resistant Escherichia coli Isolates in South Africa.

A series of colistin-resistant Escherichia coli clinical isolates was recovered from hospitalized and community patients in South Africa. Seven clonally unrelated isolates harbored the mcr-1 gene located on different plasmid backbones. Two distinct plasmids were fully sequenced, and identical 2,600-bp-long DNA sequences defining a mcr-1 cassette were identified. Promoter sequences responsible for the expression of mcr-1, deduced from the precise identification of the +1 transcription start site for mcr-1, were characterized.