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Objective: To evaluate the typing and clinical application effect based on clustered regularly interspaced short palindromic repeats (CRISPRs), serotype, and Multilocus Sequence Typing (MLST). Methods: The spacers, serotype and sequence type (ST) were obtained with CRISPRsFinder, SeroTypeFinder and MLST. PCR was used to amplify the CRISPRs, and the spacers were used to predict serotype and ST, then comparing with the serotype and ST. Results: We defined the I-E CRISPR/Cas as CT-Ⅰ, I-F CRISPR/Cas as CT-Ⅱ, and only CRISPR3-4 as CT-Ⅲ. We designated each unique arrangement spacer profile as a unique CRISPRs type. A total of 79 CT types, 76 serotypes, and 66 STs were identified. The CRISPRs typing was the most discriminating, with the Simpson index of 0.936, having the highest correlation with serology with the adjusted Rand index of 0.908. The CRISPRs type could divide the same serotype (ST) into two subtypes [O157∶H7(ST11), O104∶H4(ST678), and O26∶H11(ST21)]. The detection rates of CRISPR1, CRISPR2, CRISPR3, CRISPR4, and CRISPR3-4 were 81.1%, 94.5%, 1.4%, 1.4%, and 4.6%, with the accuracy rate of 95.0% and 100.0% according to the spacers to forecast O157∶H7 (ST11) and ST131. Conclusion: Based on the CRISPRs spacer, this method can be used as an essential molecular typing for E.coli , as it presents a good typing and clinical application effect.

Objective: To analyze the effect of the identification and evaluation of Escherichia ( E .) coli and Shigella , based on the upstream flanking sequences of CRISPR1. Methods: Both CRISPR and cas sequences were obtained through the BLAST with repeating sequences against the publicly complete genome in GenBank that related to E. coli and Shigella . Clustal X was used to perform multi-sequences alignment of the flanking sequences. PCR method was used to amplify the upstream flanking sequences of CRISPR1 in order to appraise the effect of identification and evaluation of upstream flanking sequences on E. coli and Shigella , which were based on the upstream flanking sequences of CRISPR1. Results: The results showed that 73.4% of the strains containing the I-E CRISPR/Cas that belonged to the phylogroups A, B1, D while 8.4% strains carried the I-F CRISPR/Cas. Another 17.2% of the strains owned CRISPR3-4 (non-CRISPR/Cas) only belonged to the phylogroups B2. All the Shigella strains carried I-E CRISPR/Cas. More than 99% of similarity the CRISPR1 upstream-flanking sequences was seen in E. coli (except B2) and Shigella and E. coli (B2). Both sensitivity and specificity were greater than 91% after PCR amplification in the region to identify the E.coli and Shigella . Conclusion: The upstream of CRISPR1 could achieve a preliminary identification effect on E.coli and Shigella .

The emergence and dissemination of carbapenem-resistant Escherichia coli (E. coli) strains is a main risk for global public health, but little is known of carbapenemase producing E. coli in Henan, China. The study was undertaken to investigate the prevalence and mechanism of carbapenem-resistant E. coli strains in a hospital in Xinxiang, Henan, China, 2014. A total of 5 carbapenemase-producing E. coli strains were screened from 1014 isolates. We found that they were all resistant to meropenem and imipenem. Amikacin showed the best sensitivity, with gentamicin coming up next. The positive rate of bla NDM was 80% (4/5). The sequencing results showed that two isolates belonged to bla NDM-1 whereas other 2 isolates carried the bla NDM-5. Other carbapenemase genes including bla IMP , bla VIM , bla KPC and bla OXA-48 were not detected. The bla CTX-M-15, bla TEM-1, sul2, aad, and aac(6")-Ib-cr were also detected. MLST analysis showed that NDM-producing E. coli were sporadic. Conjugation test indicated bla NDM could be transferred. In conclusion, the bla NDM was the principal resistance mechanism of carbapenem-resistant E. coli in the hospital, Henan, China., (Copyright © 2017. Published by Elsevier Ltd.)

Objective: A new method related to molecular biomarker with CRISPR/Cas (clustered regularly interspaced short palindromic repeats-cas) in Escherichia (E.) coli was developed and used for surveillance programs., Methods: CRISPR/Cas sequence that containing 135 strains with complete sequence and 203 strains with whole genome shotgun sequence of E. coli in GenBank by BLAST and 361 strains of E. coli (including 38 strains of E. coli O157∶H7) in laboratory were identified by PCR and analyzed with the CRISPR Finder. Spacers were compared with DANMAN and the phylogenetic trees of cas gene were constructed under Clustal Ⅹ and Mega 5.1., Results: With new perspective, a descriptive method was developed targeting on the position of CRISPR/cas in E. coli. The CRISPR1 was detected in 77.04%, 100.00% and 75.62% and the CRISPR2 was detected in 74.81%, 100.00% and 92.24% and the CRISPR3 and CRISPR4 were detected in 11.85%, 0 and 1.39% for 135 strains with complete sequence, 203 strains with whole genome shotgun sequence and 361 strains in the laboratory, respectively. One strain downloaded in GenBank with whole genome sequencing and 2 strains in the our laboratory were identified that containing four CRISPR locus. The other E. coli strain was with insertion sequence in downstream of the non-cas CRISPR1. The unique CRISPR was found in 8 strains of O55∶H7, in 180 strains of O157∶H7, in 8 strains of O157∶HNM, in 40 strains of O104∶H4, in 4 strains of O145∶H28, in all the 699 E. coli strains. The phylogenetic tree could be divided into two groups-cas with type I-E or type I-F., Conclusions: CRISPR/Cas might be used as a valuable molecular biomarker in epidemiological surveillance studies to identify the high virulent strains or new strains of E. coli. Phage night be related to the missing or obtaining of spacers.

Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [(14)C]phenyl-1-thio-beta-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of beta-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.