Transcriptional Landscape of a blaKPC-2 Plasmid and Response to Imipenem Exposure in Escherichia coli TOP10

The diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFIIK plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFIIK-IncFIB blaKPC−2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance (blaKPC−2, blaTEM and aph(3′)-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of E. coli (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the SoxRS/OxyR oxydative stress regulons, the Fur regulon (for iron uptake machinery), and IscR regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase mepM and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on E. coli (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of E. coli to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments

Transcriptional Landscape of a bla KPC-2 Plasmid and Response to Imipenem Exposure in Escherichia coli TOP10

International audienceThe diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFII K plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFII K-IncFIB bla KPC−2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance (bla KPC−2 , bla TEM and aph(3 ′)-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of E. coli (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the SoxRS/OxyR oxydative stress regulons, the Fur regulon (for iron uptake machinery), and IscR regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase mepM and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on E. coli (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of E. coli to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments

Concomitant carriage of KPC-producing and non-KPC-producing Klebsiella pneumoniae ST512 within a single patient

International audienceBackground: KPC-producing Klebsiella pneumoniae of clonal group 258 are prominent in healthcare settings in many regions of the world. The blaKPC gene is mostly carried by a multireplicon IncFIIk-IncFI plasmid suspected to be highly compatible and stable in this genetic background. Here, we analysed the genetic diversity of an ST512 K. pneumoniae population in a single patient.Methods: Twelve K. pneumoniae isolates (n = 5 from urine samples and n = 7 from rectal swabs) were recovered from one patient over a 2 month period. Antimicrobial susceptibility testing, plasmid extraction and WGS were performed on all isolates. The first K. pneumoniae isolate, D1, was used as a reference for phylogenetic analysis.Results: Antimicrobial susceptibility testing, plasmid analysis and WGS revealed concomitant carriage of carbapenem-resistant and carbapenem-susceptible K. pneumoniae isolates of ST512, with the absence of the entire blaKPC-carrying plasmid in the susceptible population. Furthermore, 14 other genetic events occurred within the genome, including 3 chromosomal deletions (of 71 kb, 33 kb and 11 bp), 2 different insertions of ISKpn26 and 9 SNPs. Interestingly, most of the events occurred in the same chromosomal region that has been deleted independently several times, probably after homologous recombination involving 259 bp repeated sequences.Conclusions: Our study revealed (to the best of our knowledge) the first case of in vivo blaKPC-carrying plasmid curing and a wide within-patient genetic diversity of a single K. pneumoniae ST512 clone over a short period of carriage. This within-patient diversity must be taken into account when characterizing transmission chains using WGS during nosocomial outbreaks

Stepwise evolution and convergent recombination underlie the global dissemination of carbapenemase-producing Escherichia coli

International audienceBackground: Carbapenem-resistant Enterobacteriaceae are considered by WHO as "critical" priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates remains largely unknown as well as factors contributing to the acquisition of carbapenemase genes. Methods: We first analyzed the whole-genome sequence and the evolution of the E. coli sequence type (ST) 410 and its disseminated clade expressing the carbapenemase OXA-181. We reconstructed the phylogeny of 19 E. coli ST enriched in CP-Ec and corresponding to a total of 2026 non-redundant isolates. Using the EpiCs software, we determined the significance of the association between specific mutations and the acquisition of a carbapenemase gene and the most probable order of events. The impact of the identified mutations was assessed experimentally by genetic manipulations and phenotypic testing. Results: In 13 of the studied STs, acquisition of carbapenemase genes occurred in multidrug-resistant lineages characterized by a combination of mutations in ftsI encoding the penicillin-binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele related to that from ST38 inducing reduced susceptibility to diverse β-lactams spread across the species by recombination. We showed that these mutations precede in most cases the acquisition of a carbapenemase gene. The ompC allele from ST38 might have contributed to the selection of CP-Ec disseminated lineages within this ST. On the other hand, in the pandemic ST131 lineage, CP-Ec were not associated with mutations in ompC or ftsI and show no signs of dissemination

Herpes Simplex Virus 1 Replication, Ocular Disease, and Reactivations from Latency Are Restricted Unilaterally after Inoculation of Virus into the Lip

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Rapid detection of CTX-M-type ESBLs and carbapenemases directly from biological samples using the BL-DetecTool

International audienceAbstract Background Lateral flow immunoassays (LFIA) have shown their usefulness for detecting CTX-M- and carbapenemase-producing Enterobacterales (CPEs) in bacterial cultures. Here, we have developed and validated the BL-DetecTool to detect CTX-M enzymes and carbapenemases directly from clinical samples. Methods The BL-DetecTool is an LFIA that integrates an easy sample preparation device named SPID (Sampling, Processing, Incubation and Detection). It was evaluated in three University hospitals on urine, blood culture (BC) and rectal swab (RS) specimens either of clinical origin or on spiked samples. RS evaluation was done directly and after a 24 h enrichment step. Results The CTX-M BL-DetecTool was tested on 485 samples (154 BC, 150 urines, and 181 RS) and revealed a sensitivity and specificity of 97.04% (95% CI 92.59%–99.19%) and 99.43% (95% CI 97.95%–99.93%), respectively. Similarly, the Carba5 BL-DetecTool was tested on 382 samples (145 BC, 116 urines, and 121 RS) and revealed a sensitivity and specificity of 95.3% (95% CI 89.43%–98.47%) and 100% (95% CI 98.67%–100%), respectively. While with the Carba5 BL-DetecTool five false negatives were observed, mostly in RS samples, with the CTX-M BL-DetecTool, in addition to four false-negatives, two false-positives were also observed. Direct testing of RS samples revealed a sensitivity of 78% and 86% for CTX-M and carbapenemase detection, respectively. Conclusions BL-DetecTool showed excellent biological performance, was easy-to-use, rapid, and could be implemented in any microbiology laboratory around the world, without additional equipment, no need for electricity, nor trained personnel. It offers an attractive alternative to costly molecular methods

Behavior of PML-NB-associated proteins in HSV-1 infected cultured primary mouse TG neurons.

<p>(A) Immuno-FISH detection of neuronal or viral markers (green) and HSV-1 genomes (red) in RC-containing neurons. (i) neurofilaments, (ii) ICP4, (iii) ICP27, (iv) total HSV-1 proteins. (B) Immuno-FISH detection of PML-NB components (green) and HSV-1 genomes (red) in RC-containing neurons. (C) Quantification of infected neurons showing co-localization of HSV-1 genomes with PML-NB components. Fifty to 80 neurons were counted for each labeling. (D) Quantification of RC-containing infected neurons showing or not PML aggregates. Scale bars represent 10 μm.</p

Type 1 IFNα induces an ML-like pattern in infected cultured primary mouse TG neurons.

<p>(A) Immuno-FISH for the detection of PML and HSV-1 genomes in cells not treated (i) or treated (ii) with IFNα (1000 IU/mL). (B) Double DNA FISH for the detection of centromeric minor satellite sequences and HSV-1 genomes in cells treated with IFNα (1000 IU/mL). (C) Quantification of ML-like and RC patterns in neurons (three independent experiments) infected with <i>in</i>1374, <i>in</i>1330, or <i>ts</i>K for 24 h in the absence or presence of IFNα (1000 IU/mL). Results show means (± SD). (D) Quantification of ML-like and RC patterns in neurons from C57BL/6 wt or IFNAR KO mice, and infected by <i>in</i>1374 for 24 h in the absence or presence of IFNα (1000 IU/mL). Means of two independent experiments are shown.</p

vDCP-NBs are formed in the absence of functional ICP4 and ICP0 in cultured primary mouse TG neurons, and can be associated with viral transcription.

<p>(A) Neurons were infected with <i>in</i>1374 at 32°C for 24 h. (i) IF detection of ICP4, (ii and iii) immuno-FISH for detection of ICP4 or ICP8 and HSV-1 genome, and (iv–vii) immuno-FISH for the detection of PML, Daxx, ATRX or SUMO-2/3 and the HSV-1 genome. (B) and (C) Neurons were infected at 38.5°C for 48 h with <i>in</i>1374 and <i>in</i>1330, respectively. (i–iv) immuno-FISH for the detection of PML, Daxx, ATRX or SUMO-1 and the HSV-1 genome. (D) RT-qPCR for the detection of <i>LacZ</i> transcripts in <i>in</i>1374 infected neurons treated or not with trichostatin A (TSA). Results show means (± SD) of two independent experiments. (E) RNA-DNA FISH combined with IF for detection of <i>LacZ</i> transcripts (blue), HSV-1 genomes (red), and PML (green) in <i>in</i>1374 infected neurons treated or not with TSA. Three different patterns are shown. (F) RNA-DNA FISH combined with IF for detection of LAT transcripts (blue), HSV-1 genomes (red), and PML (green) in mouse TG neurons at 6 to 8 dpi. Scale bars represent 10 μm.</p

HSV-1 MA pattern corresponds to vDCP-NBs and contains SUMO proteins.

<p>(A) Immuno-DNA-FISH showing HSV-1 genomes (red), promyelocytic leukemia nuclear bodies (PML-NB)–associated proteins (green), and cellular chromatin (DAPI, blue). (B) WB of ATRX and Daxx in TG of uninfected or infected mice during acute infection. Infected (left TG) and not-infected (right TG) TGs of the same mouse (two mice) were harvested 6 dpi, and treated to perform WB. Actin is shown as a loading control. NIH3T3 is shown as a cellular control. (C) and (D) Immuno-DNA-FISH showing HSV-1 genomes (red), small ubiquitin modifier (SUMO) proteins (green), and cellular chromatin (DAPI, blue/grey). (C) SUMO-1 and (D) SUMO-2/3 detection in (i) non-infected neurons, (ii) RC-containing neurons and (iii, iv) MA/vDCP-NBs or S/vDCP-NB-containing neurons. (E) IF for detection of PML (green) in uninfected TG neurons and satellite cells. Arrows point out PML-NBs in neurons or satellite cells, arrowheads point out neurons without PML-NBs. (F) Immuno-DNA-FISH showing HSV-1 genomes (red, RC) and (i) PML or (ii) CENP-A (green), in neurons. DAPI staining (grey) shows nuclei. Scale bars represent 10 μm.</p