The plasmid-located
gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal
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
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
gene was cloned and produced in an
K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the
gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the
and
genes located downstream of
Genetic analysis showed that the
gene was carried by an IncHI2 plasmid.
analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from
spp. In particular,
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
genes may circulate silently and remain undetected unless induced by colistin.
Adaptation proceeds through the selection of mutations. The distribution of mutant fitness effect and the forces shaping this distribution are therefore keys to predict the evolutionary fate of ...organisms and their constituents such as enzymes. Here, by producing and sequencing a comprehensive collection of 10,000 mutants, we explore the mutational landscape of one enzyme involved in the spread of antibiotic resistance, the beta-lactamase TEM-1. We measured mutation impact on the enzyme activity through the estimation of amoxicillin minimum inhibitory concentration on a subset of 990 mutants carrying a unique missense mutation, representing 64% of possible amino acid changes in that protein reachable by point mutation. We established that mutation type, solvent accessibility of residues, and the predicted effect of mutations on protein stability primarily determined alone or in combination changes in minimum inhibitory concentration of mutants. Moreover, we were able to capture the drastic modification of the mutational landscape induced by a single stabilizing point mutation (M182T) by a simple model of protein stability. This work thereby provides an integrated framework to study mutation effects and a tool to understand/define better the epistatic interactions.
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.
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.
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.
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.
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.
Two extended-spectrum cephalosporin-resistant Neisseria gonorrhoeae isolates were discovered among 6,340 (0.03%) French isolates between 2010 and 2014. One isolate corresponded to the F89 ...multidrug-resistant N. gonorrhoeae isolate harboring a penA mosaic; whole-genome sequencing highlighted an additional R251H substitution in the ftsX gene recently involved in cephalosporin resistance. The other, ceftriaxone-resistant isolate (MIC, 0.25 mg/liter) harbored the PBP2 pattern XXXVI plus a P551S substitution and belonged to sequence type ST1579 (multilocus sequence typing MLST).
The quantitative characterization of mutational landscapes is a task of outstanding importance in evolutionary and medical biology: It is, for example, of central importance for our understanding of ...the phenotypic effect of mutations related to disease and antibiotic drug resistance. Here we develop a novel inference scheme for mutational landscapes, which is based on the statistical analysis of large alignments of homologs of the protein of interest. Our method is able to capture epistatic couplings between residues, and therefore to assess the dependence of mutational effects on the sequence context where they appear. Compared with recent large-scale mutagenesis data of the beta-lactamase TEM-1, a protein providing resistance against beta-lactam antibiotics, our method leads to an increase of about 40% in explicative power as compared with approaches neglecting epistasis. We find that the informative sequence context extends to residues at native distances of about 20 Å from the mutated site, reaching thus far beyond residues in direct physical contact.
•A retrospective single-centre study was undertaken including 147 samples from 92 patients.•The overall percentage agreement was 98% for detection of typical bacteria.•Significant thresholds in ...culture were compared with nucleic acid copy number.•Median turnaround time was significantly shorter for the FilmArray Pneumonia Panel Plus than for culture.
This study aimed to evaluate the performance of FilmArray Pneumonia Panel Plus (FA-PP) for the detection of typical bacterial pathogens in respiratory samples from patients hospitalized in intensive care units (ICUs).
FA-PP was implemented for clinical use in the microbiology laboratory in March 2020. A retrospective analysis on a consecutive cohort of adult patients hospitalized in ICUs between March 2020 and May 2020 was undertaken. The respiratory samples included sputum, blind bronchoalveolar lavage (BBAL) and protected specimen brush (PSB). Conventional culture and FA-PP were performed in parallel.
In total, 147 samples from 92 patients were analysed; 88% had coronavirus disease 2019 (COVID-19). At least one pathogen was detected in 46% (68/147) of samples by FA-PP and 39% (57/147) of samples by culture. The overall percentage agreement between FA-PP and culture results was 98% (93–100%). Bacteria with semi-quantitative FA-PP results ≥105 copies/mL for PSB samples, ≥106 copies/mL for BBAL samples and ≥107 copies/mL for sputum samples reached clinically significant thresholds for growth in 90%, 100% and 91% of cultures, respectively. FA-PP detected resistance markers, including mecA/C, blaCTX-M and blaVIM. The median turnaround time was significantly shorter for FA-PP than for culture.
FA-PP may constitute a faster approach to the diagnosis of bacterial pneumonia in patients hospitalized in ICUs.
The FilmArray Blood Culture Identification 2 panel (BCID2; bioMérieux) is a fully automated PCR-based assay for identifying bacteria, fungi, and bacterial resistance markers in positive blood ...cultures (BC) in about 1 h. In this multicenter study, we evaluated the performance of the BCID2 panel for pathogen detection in positive BC. Conventional culture and BCID2 were performed in parallel at four tertiary-care hospitals. We included 152 positive BC-130 monomicrobial and 22 polymicrobial cultures-in this analysis. The BCID2 assay correctly identified 90% (88/98) of Gram-negative and 89% (70/79) of Gram-positive bacteria. Five bacterial isolates targeted by the BCID2 panel and recovered from five positive BC, including three polymicrobial cultures, were missed by the BCID2 assay. Fifteen isolates were off-panel organisms, accounting for 8% (15/182) of the isolates obtained from BC. The mean positive percent agreement between the BCID2 assay and standard culture was 97% (95% confidence interval, 95 to 99%), with agreement ranging from 67% for Candida albicans to 100% for 17 targets included in the BCID2 panel. BCID2 also identified the
gene in seven BC, including one for which no extended-spectrum β-lactamase (ESBL)-producing isolate was obtained in culture. However, it failed to detect ESBL-encoding genes in three BC. Two of the 18
genes detected by the BCID2 were not confirmed. No carbapenemase,
, or MREJ targets were detected. The median turnaround time was significantly shorter for BCID2 than for culture. The BCID2 panel may facilitate faster pathogen identification in bloodstream infections.
Rapid molecular diagnosis combining the identification of pathogens and the detection of antibiotic resistance genes from positive blood cultures (BC) can improve the outcome for patients with bloodstream infections. The FilmArray BCID2 panel, an updated version of the original BCID, can detect 11 Gram-positive bacteria, 15 Gram-negative bacteria, 7 fungal pathogens, and 10 antimicrobial resistance genes directly from a positive BC. Here, we evaluated the real-life microbiological performance of the BCID2 assay in comparison to the results of standard methods used in routine practice at four tertiary care hospitals.
The genome of the Omicron variant of concern (VOC) contains more than 50 mutations, many of which have been associated with increased transmissibility, differing disease severity, and the potential ...to elute immune responses acquired after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination or infection with previous VOCs. Due to a better tropism for the upper respiratory tract, it was suggested that the detection of the Omicron variant could be preferred in saliva, compared to nasopharyngeal swabs (NPS). Our objective was to compare the SARS-CoV-2 levels in saliva fluid and NPS to estimated Ct values, according to the main SARS-CoV-2 variants circulating in France since the beginning of 2021. We analyzed 1,289 positive reverse transcription-polymerase chain reaction (RT-PCR) results during the three major waves: Alpha, Delta, and Omicron. NPS and saliva sampling were performed for 909 (71%) and 380 (29%) cases, respectively. The Ct values were significantly lower in the NPS samples than in the saliva samples for the three main VOCs. Still, the difference was less pronounced with the Omicron variant than for the Alpha and Delta variants. In contrast, in the saliva samples, Ct values were significantly lower for the Omicron variant than for the Delta (difference of -2.7 Ct) and the Alpha (difference of -3.0 Ct) variants, confirming a higher viral load in saliva. To conclude, the higher viral load in saliva was evidenced for the Omicron variant, compared to the Alpha and Delta variants, suggesting that established diagnostic methods might require revalidation with the emergence of novel variants.
Established methods for SARS-CoV-2 diagnostics might require revalidation with the emergence of novel variants. This is important for screening strategy programs and for the investigation of the characteristics of new variants in terms of tropism modification and increased viral burden leading to its spread. SARS-CoV-2 RT-PCR screening on saliva samples reported lower but acceptable performance, compared to nasopharyngeal samples. Due to a better tropism for the upper respiratory tract, it was suggested that the detection of the Omicron variant could be preferred in saliva, compared to nasopharyngeal swabs. Our study analyzed 1,289 positive RT-PCR results during the three major waves in France: Alpha, Delta, and Omicron. Our findings also showed a higher viral load in saliva for the Omicron variant, compared to the Alpha and Delta variants.
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