A high-throughput sequencing determination method for upstream genetic structure (UGS) of ISEcp1-blaCTX-M transposition unit and application of the UGS to classification of bacterial isolates possessing blaCTX-M.

INTRODUCTION: Because blaCTX-M is responsible for resistance of bacteria to the third generation cephalosporins, location of blaCTX-M could be a good indicator for classifying bacterial isolates harboring blaCTX-M in molecular epidemiology. However, determination of blaCTX-M location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of blaCTX-M location. METHODS: Extracted DNA samples obtained from 168 Escherichia coli isolates possessing blaCTX-M were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer. RESULTS: Nucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures. CONCLUSIONS: Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-blaCTX-M transposition unit was useful for classification of bacterial isolates harboring blaCTX-M.

Characterization of blaCTX-M-14 transposition from plasmid to chromosome in Escherichia coli experimental strain.

Mostly, blaCTX-M is found on transferable plasmids as a component of the blaCTX-M transposition unit containing an insertion sequence, ISEcp1, which exists on the upstream region of blaCTX-Ms. Several recent studies conducted in clinical and community settings have reported the presence of chromosomally located blaCTX-M in extended spectrum ß-lactamase (ESBL)-producing bacterial isolates. In this study, we observed the frequency and molecular nature of the ISEcp1-mediated transposition of blaCTX-M-14 from a plasmid to a chromosome by using an experimental strain of Escherichia coli. We determined 102 different chromosomal transposition sites of blaCTX-M-14 in 126 E. coli isolates following five independent screening procedures. The characterization of the 102 different chromosomal transposition sites of blaCTX-M-14 observed in this study revealed the presence of 5-bp direct repeat (DR) sequences and identical left terminal inverted sequences in 80 E. coli isolates. However, 5'-flanking sequences of the right terminal DR sequences in the 80 E. coli isolates were highly diverse, and consensus sequences of the right terminal inverted repeat sequences were not observed. In case of our E. coli experimental strain, the frequency of the ISEcp1-mediated transposition of blaCTX-M-14 from a plasmid to a chromosome was determined to be 0.51% (SD = 0.37). Collectively, the molecular nature of ISEcp1 could plausibly be a factor contributing to the high detection rates of E. coli possessing chromosomally located blaCTX-M-14 in both clinical and community settings.

Characterization of CTX-M type ESBL-producing Enterobacteriaceae isolated from asymptomatic healthy individuals who live in a community of the Okinawa prefecture, Japan.

This study was performed to characterize CTX-M type extended spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae carriage in asymptomatic health individuals, which has not been well investigated, in a community of the Okinawa prefecture, Japan. Fecal samples were voluntary collected from asymptomatic healthy individuals who were going to take a routine medical checkup. The collected fecal samples were inoculated on MacConkey agar supplemented with 2 µg/ml of cefotaxime and incubated at 37 °C. Randomly selected three lactose-fermented colonies per each sample were analyzed. Genetic relatedness among the CTX-M type ESBL-producing Enterobacteriaceae isolates were performed by pulsed-field gel electrophoresis (PFGE) after confirmation of ESBL phenotype and determination of bacterial species. Location of blaCTX-M was confirmed by S1-PFGE, I-CeuI-PFGE and the Southern blotting hybridization. ESBL-producing Enterobacteriaceae was isolated from 32 (12.2%) of the collected 263 fecal samples, and 96 ESBL-producing Enterobacteriaceae isolates were obtained. CTX-M type ESBL-producing Escherichia coli B2 were major (67 isolates, 72.0%) and 40 (59.7%) of the 67 CTX-M type ESBL -producing E. coli B2 were E. coli B2-ST131. Three CTX-M type ESBL-producing E. coli B2-ST131 isolates from asymptomatic healthy individuals showed similar PFGE band patterns as five CTX-M type ESBL -producing E. coli B2-ST131 isolates from a hospital locates in the same area of the target community. Chromosomally-transferred blaCTX-M was observed in 10.0% of the examined CTX-M type ESBL-producing Enterobacteriaceae isolates. We report current situation CTX-M type ESBL-producing Enterobacteriaceae carriage in asymptomatic healthy individuals of the Okinawa prefecture, Japan. In addition, our results indicated that worldwide distributed CTX-M type ESBL-producing E. coli B2-ST131 has been spread in a community. Therefore monitoring of ESBL-producing Enterobacteriaceae in healthy individuals is important.

Current status of extended spectrum ß-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan.

Enterobacteriaceae producing extended spectrum ß-lactamase (ESBL) are distributed worldwide. In this study, 114 ESBL-producing Enterobacteriaceae were isolated by analyzing 1672 clinical isolates of Enterobacteriaceae collected from an Okinawa prefectural hospital in Japan between June 2013 and July 2014. The overall prevalence of ESBL-producing Enterobacteriaceae was 6.8%; the prevalence of different bacterial species among the ESBL-producing isolates was as follows: 11.5% Escherichia coli (90 of 783 isolates), 6.2% Klebsiella pneumoniae (19 of 307 isolates), and 11.1% Proteus mirabilis (5 of 45 isolates). The ESBL types blaCTX-M-1, -3, -15, -2, -14, -27, and mutants of blaSHV-1 were detected. Among them, blaCTX-M-15 (33.3%), blaCTX-M-14 (27.8%) and blaCTX-M-27 (33.3%) were dominant in the E. coli isolates, whereas a blaSHV mutant which possessed four mutations (Tyr7Phe, Leu35Gln, Gly238Ser and Glu240Lys) in the amino acid sequence of SHV-1 dominated in the K. pneumoniae isolates (11 of 19, 57.9%). The pandemic E. coli ST131 clone was found to constitute 3.3% of the overall examined isolates and 62.2% of the ESBL-producing E. coli isolates. Our results suggest that the genetic combination of blaCTX-M, and blaSHV and antibiotics-resistant profile were different from that in other regions such as other areas of Japan, Asia, Europe, and North America, especially in the ESBL-producing K. pneumoniae isolates and in the E. coli B2-O25b-ST131 isolates possessing blaCTX-M-15 (40.7% of the E. coli B2-O25b-ST131 isolates). Taken together, our results indicate that the ESBL-producing Enterobacteriaceae in Okinawa, Japan, might be of a unique nature.

Carriage of Escherichia coli Producing CTX-M-Type Extended-Spectrum ß-Lactamase in Healthy Vietnamese Individuals.

Healthy carriage of CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli was examined by thrice collecting fecal samples from the same 199 healthy Vietnamese subjects every 6 months. Using pulsed-field gel electrophoresis (PFGE), identical PFGE patterns throughout the three samplings were not observed, although prevalence of E. coli in the subjects was around 50% in the three samplings. Our results suggested a short carriage period of the CTX-M-type ESBL-producing E. coli in healthy Vietnamese subjects.

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds.

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates.

ER stress elicits non-canonical CASP8 (caspase 8) activation on autophagosomal membranes to induce apoptosis.

The VPS37A gene encodes a subunit of the endosomal sorting complex required for transport (ESCRT)-I complex that is frequently lost in a wide variety of human solid cancers. We have previously demonstrated the role of VPS37A in directing the ESCRT membrane scission machinery to seal the phagophore for autophagosome completion. Here, we report that VPS37A-deficient cells exhibit an accumulation of the apoptotic initiator CASP8 (caspase 8) on the phagophore and are primed to undergo rapid apoptosis through the intracellular death-inducing signaling complex (iDISC)-mediated CASP8 activation upon exposure to endoplasmic reticulum (ER) stress. Using CRISPR-Cas9 gene editing and comparative transcriptome analysis, we identified the ATF4-mediated stress response pathway as a crucial mediator to elicit iDISC-mediated apoptosis following the inhibition of autophagosome closure. Notably, ATF4-mediated iDISC activation occurred independently of the death receptor TNFRSF10B/DR5 upregulation but required the pro-apoptotic transcriptional factor DDIT3/CHOP to enhance the mitochondrial amplification pathway for full-activation of CASP8 in VPS37A-deficient cells stimulated with ER stress inducers. Our analysis also revealed the upregulation of NFKB/NF-kB signaling as a potential mechanism responsible for restraining iDISC activation and promoting cell survival upon VPS37A depletion. These findings have important implications for the future development of new strategies to treat human cancers, especially those with VPS37A loss.Abbreviations: ATG: autophagy related; BMS: BMS-345541; CASP: caspase; CHMP: charged multivesicular body protein; DKO: double knockout; Dox: doxycycline; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; gRNA: guide RNA; GSEA: gene set enrichment analysis; GSK157: GSK2656157; iDISC: intracellular death-inducing signaling complex; IKK: inhibitor of NFKB kinase; IPA: ingenuity pathway analysis; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NFKB/NF-kB: nuclear factor kappa B; OZ: 5Z-7-oxozeaenol; RNA-seq: RNA sequencing; UPR: unfolded protein response; TFT: transcription factor target; THG: thapsigargin; TUN: tunicamycin; VPS: vacuolar protein sorting.

Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure.

VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.

High Prevalence of Colistin-Resistant Escherichia coli with Chromosomally Carried mcr-1 in Healthy Residents in Vietnam.

The wide distribution of colistin-resistant bacteria in developing countries has become a common phenomenon. To understand the mechanisms underlying their distribution, we studied the mcr genetic background of colistin-resistant Escherichia coli isolates from the fecal microbiota of healthy human residents from a community in Vietnam with a high prevalence of colistin-resistant E. coli with mcr Fifty-seven colistin-resistant isolates were obtained from 98 residents; one isolate was collected from each individual and analyzed for mcr We found that 36.8% of the isolates carried chromosomal mcr-1 Further, 63.2% and 1.8% of the isolates carried mcr-1 on the plasmid and the plasmid/chromosome, respectively. Whole-genome sequencing of genetically unrelated isolates showed that the majority (6 of 7) of the isolates had the chromosomal mcr-1 in a complete ancestral mcr-1 transposon Tn6330, ISApl1-mcr-1-PAP2-ISApl1, which was inserted at various positions on the chromosomes. In addition, the majority (87.5%) of Tn6330 of mcr-1-carrying plasmids (n = 8) lacked both upstream and downstream ISApl1 transposons. The results obtained in this study indicate that plasmid-to-chromosomal transfer of mcr-1 may have occurred recently in the fecal microbiota of the residents. Additionally, Tn6330 on the chromosome may lose ISApl1 from the transposon during multiplication to gain a more stable mcr-1 state on the chromosome. Stabilization of resistance by the chromosomal incorporation of mcr-1 would be an additional challenge in combating the dissemination of resistant bacteria.IMPORTANCE Elucidation of the mechanism of the wide dissemination of colistin-resistant bacteria in communities of developing countries is an urgent public health issue. In this study, we investigated the genetic background of the colistin resistance gene mcr in E. coli isolates from the fecal microbiota of healthy human residents living in a community in Vietnam with a high prevalence of colistin-resistant E. coli Our study revealed for the first time, a surprisingly high percentage (36.8%) of colistin-resistant E. coli carrying chromosomal mcr-1, the emergence of which may have occurred recently, in the fecal microbiota of the community residents. The mcr-1 transposon on the chromosome may develop into a more stable genotype by the loss of insertion sequences (ISs). Our results are valuable in understanding the mechanism underlying the increasing prevalence of colistin-resistant bacteria within a community.

Characterisation of chromosomally-located blaCTX-M and its surrounding sequence in CTX-M-type extended-spectrum ß-lactamase-producing Escherichia coli isolates.

OBJECTIVES: Although it has been regarded that the CTX-M-type extended-spectrum ß-lactamase (ESBL) gene blaCTX-M is mainly carried by antimicrobial resistance plasmids, Escherichia coli possessing chromosomally-located blaCTX-M has been reported in previous studies. This study aimed to characterise the genetic structure of the chromosomally-located blaCTX-M transposition unit and its surrounding sequence in ESBL-producing E. coli isolated in a Japanese hospital. METHODS: A total of 81 ESBL-producing E. coli isolates were studied. The existence of chromosomally-located blaCTX-M was confirmed by S1 nuclease-digested pulsed-field gel electrophoresis (PFGE) and Southern blot hybridisation and by sequencing analysis of the PCR-amplified DNA fragments. RESULTS: Chromosomally-located blaCTX-M was confirmed in 22 (27.2%) of the 81 E. coli isolates examined; five and four location types of chromosomally-located blaCTX-M-14 and blaCTX-M-15 were determined, respectively. Among the 22 E. coli isolates, 15 (68.2%) possessed single chromosomally-located blaCTX-M gene, probably due to single transposition of a plasmidic blaCTX-M to the chromosome. In isolate N0057, the blaCTX-M-15 transposition unit was transferred from a plasmid into two different chromosomal regions. In addition, 'recurrent' transposition of already existing chromosomally-located blaCTX-M-14 to another chromosomal region was observed in isolates N0211, N0214, N01127, N1682 and N1753; consequently, these isolates possessed two copies of chromosomally-located blaCTX-M-14. CONCLUSION: Considering that isolates N0211, N0214, N01127, N1682 and N1753 in which the 'recurrent' transposition event occurred were genetically related according to PFGE, these data suggest the possibility of accumulation of blaCTX-M on the chromosome in CTX-M-type ESBL-producing E. coli.