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The membrane-associated solute-binding protein (SBP) MlaD of the maintenance of lipid asymmetry (Mla) system has been reported to help the transport of phospholipids (PLs) between the outer and inner membranes of Gram-negative bacteria. Despite the availability of structural information, the molecular mechanism underlying the transport of PLs and the ancestry of the protein MlaD remain unclear. In this study, we report the crystal structures of the periplasmic region of MlaD from Escherichia coli (EcMlaD) at a resolution range of 2.3-3.2 Å. The EcMlaD protomer consists of two distinct regions, viz. N-terminal β-barrel fold consisting of seven strands (referred to as MlaD domain) and C-terminal α-helical domain (HD). The protein EcMlaD oligomerizes to give rise to a homo-hexameric ring with a central channel that is hydrophobic and continuous with a variable diameter. Interestingly, the structural analysis revealed that the HD, instead of the MlaD domain, plays a critical role in determining the oligomeric state of the protein. Based on the analysis of available structural information, we propose a working mechanism of PL transport, viz. "asymmetric protomer movement (APM)". Wherein half of the EcMlaD hexamer would rise in the periplasmic side along with an outward movement of pore loops, resulting in the change of the central channel geometry. Furthermore, this study highlights that, unlike typical SBPs, EcMlaD possesses a fold similar to EF/AMT-type beta(6)-barrel and a unique ancestry. Altogether, the findings firmly establish EcMlaD to be a non-canonical SBP with a unique ligand-transport mechanism., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Providencia rustigianii is potentially enteropathogenic in humans. Recently, we identified a P. rustigianii strain carrying a part of the cdtB gene homologous to that of Providencia alcalifacines that produces an exotoxin called cytolethal distending toxin (CDT), encoded by three subunit genes ( cdtA , cdtB , and cdtC ). In this study, we analyzed the P. rustigianii strain for possible presence of the entire cdt gene cluster and its organization, location, and mobility, as well as expression of the toxin as a putative virulence factor of P. rustigianii . Nucleotide sequence analysis revealed the presence of the three cdt subunit genes in tandem, and over 94% homology to the corresponding genes carried by P. alcalifaciens both at nucleotide and amino acid sequence levels. The P. rustigianii strain produced biologically active CDT, which caused distension of eukaryotic cell lines with characteristic tropism of CHO and Caco-2 cells but not of Vero cells. S1-nuclease digested pulsed-field gel electrophoresis followed by Southern hybridization analysis demonstrated that the cdt genes in both P. rustigianii and P. alcalifaciens strains are located on large plasmids (140 to 170 kb). Subsequently, conjugation assays using a genetically marked derivative of the P. rustigianii strain showed that the plasmid carrying cdt genes in the P. rustigianii was transferable to cdt gene-negative recipient strains of P. rustigianii , Providencia rettgeri, and Escherichia coli. Our results demonstrated the presence of cdt genes in P. rustigianii for the first time, and further showed that the genes are located on a transferable plasmid, which can potentially spread to other bacterial species., Competing Interests: The authors declare no conflict of interest.

Polyurethane (PUR) is a soil and aquatic contaminant throughout the world. Towards bioremediation, in a previous study, a soil bacterium, Pseudomonas sp. AKS31, capable of efficiently degrading PUR was isolated. Polyurethanase (PURase) enzyme is capable of cleaving the ester bond of PUR and is considered as a key regulator of PUR biodegradation. Hence, for a high yield, easy purification, and further characterization, the aim of this study was to clone and overexpress the PURase gene of this isolate. The current study also investigated structural aspects of this enzyme through predictive bioinformatics analyses. In this context, the PURase gene of the isolate was cloned and expressed in E. coli using pET28(a) + vector. The obtained recombinant protein was found insoluble. Therefore, first, the protein was made soluble with urea and purified using nickel-NTA beads. The purified enzyme exhibited substantial activities when tested on the LA-PUR plate. Bioinformatics-based analysis of the protein revealed the presence of a lipase serine active site and indicated that this PURase belongs to the Family 1.3 lipase. Hence, the present study shows that active PURase can be produced in large quantities using a prokaryotic expression system and thus, provides an effective strategy for in-vitro PUR-degradation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Escherichia albertii is an emerging zoonotic foodborne pathogen. Several outbreaks of E. albertii have occurred, particularly in Japan. Although birds have been considered as one of the most important reservoirs of this bacterium, information regarding its prevalence in birds is still scarce. We performed a survey of E. albertii in wild birds in Japan and examined the characteristics of these isolates. E. albertii-specific genes were detected in five cloacal swabs from 156 birds by PCR. Four E. albertii strains were isolated from a swallow with two different E. albertii strains and two pigeons in a flock using XRM-MacConkey agar. These isolates were assigned to biogroup 3, showed no resistance to any tested antimicrobials, and were classified into two EAO-genotypes (EAOg2 and EAOg33) and were untypable. Similar to clinical E. albertii strains, these isolates carried virulence genes, including eae (n = 4), paa (n = 4), Eccdt-I (n = 2), and stx2f (n = 1), as well as Eacdt. Furthermore, stx2f genes in a strain were located on an inducible bacteriophage, which can confer the ability to produce Stx2f in E. coli. In conclusion, Japanese wild birds carried E. albertii at levels similar to the reported prevalence in birds. These isolates may have the potential to cause gastroenteritis in humans.

In Gram-negative bacteria, the maintenance of lipid asymmetry (Mla) system is involved in the transport of phospholipids between the inner (IM) and outer membrane. The Mla system utilizes a unique IM-associated periplasmic solute-binding protein, MlaD, which possesses a conserved domain, MlaD domain. While proteins carrying the MlaD domain are known to be primarily involved in the trafficking of hydrophobic molecules, not much is known about this domain itself. Thus, in this study, the characterization of the MlaD domain employing bioinformatics analysis is reported. The profiling of the MlaD domain of different architectures reveals the abundance of glycine and hydrophobic residues and the lack of cysteine residues. The domain possesses a conserved N-terminal region and a well-preserved glycine residue that constitutes a consensus motif across different architectures. Phylogenetic analysis shows that the MlaD domain archetypes are evolutionarily closer and marked by the conservation of a functionally crucial pore loop located at the C-terminal region. The study also establishes the critical role of the domain-associated permeases and the driving forces governing the transport of hydrophobic molecules. This sheds sufficient light on the structure-function-evolutionary relationship of MlaD domain. The hexameric interface analysis reveals that the MlaD domain itself is not a sole player in the oligomerization of the proteins. Further, an operonic and interactome map analysis reveals that the Mla and the Mce systems are dependent on the structural homologs of the nuclear transport factor 2 superfamily., (© 2021 Wiley Periodicals LLC.)

The aim of the study was to assess the presence of genes in ESBL-producing E. coli (ESBL-Ec) isolated from retail raw food in Nha Trang, Vietnam. A total of 452 food samples comprising chicken (n = 116), pork (n = 112), fish (n = 112) and shrimp (n = 112) collected between 2015 and 2017 were examined for the prevalence of ESBL-Ec. ESBL-Ec were detected in 46.0% (208/452) of retail food samples, particularly in 66.4% (77/116), 55.4% (62/112), 42.0% (47/112) 19.6% (22/112) of chicken, pork, fish and shrimp, respectively. Sixty-five out of the 208 (31.3%) ESBL-Ec isolates were positive for mcr genes including mcr-1, mcr-3 and both mcr-1 and mcr-3 genes in 56/208 (26.9%), 1/208 (0.5%) and 8/208 (3.9%) isolates, respectively. Particularly, there was higher prevalence of mcr-1 in ESBL-Ec isolates from chicken (53.2%, 41/77) in comparison to shrimp (22.7%, 5/22), pork (11.3%, 7/62) and fish (6.4%, 3/47). mcr-3 gene was detected in co-existence with mcr-1 in ESBL-Ec isolates from shrimp (9.1%, 2/22), pork (8.1%, 5/62) and fish (2.1%, 1/47) but not chicken. The 65 mcr-positive ESBL-Ec (mcr-ESBL-Ec) were colistin-resistant with the MICs of 4-8 μg/mL. All mcr-3 gene-positive isolates belonged to group A, whereas phylogenetic group distribution of isolates harboring only mcr-1 was B1 (44.6%), A (28.6%) and D (26.8%). PFGE analysis showed diverse genotypes, although some isolates demonstrated nearly clonal relationships. S1-PFGE and Southern hybridization illustrated that the mcr-1 and mcr-3 genes were located either on chromosomes or on plasmids. However, the types of mcr genes were harbored on different plasmids with varied sizes of 30-390 kb. Besides, the ESBL genes of CTX-M-1 or CTX-M-9 were also detected to be located on plasmids. Noteworthy, co-location of CTX-M-1 with mcr-1 or mcr-3 genes on the same plasmid was identified. The conjugation experiment indicated that the mcr-1 or mcr-3 was horizontally transferable. All mcr-ESBL-Ec isolates were multidrug resistance (resistance to ≥3 antimicrobial classes). Moreover, β-Lactamase-encoding genes of the CTX-M-1 (78.5%), CTX-M-9 (21.5%), TEM (61.5%) groups were found in mcr-ESBL-Ec. The astA gene was detected in 27 (41.5%) mcr-ESBL-Ec isolates demonstrating their potential virulence. In conclusion, mcr-1 and mcr-3 genes existed individually or concurrently in ESBL-Ec isolates recovered from retail raw food in Nha Trang city, which might further complicate the antimicrobial-resistant situation in Vietnam, and is a possible health risk for human., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gram-negative isolates producing carbapenemase enzymes is a great public health problem in developing countries and their control is challenging task due to the involvement of multiple factors including the practice of self-medication, use of antibiotics on animal farms, poor hospital hygiene, etc. During this study, we searched various databases for relevant publication on carbapenemase-producing isolates in Nepal. Various classes of carbapenemases had been reported in Nepal. Most frequent was the New Delhi Metallo beta lactamase with many variants where NDM-1 was most prevalent. Similarly, Oxacillinase and Klebsiella pneumoniae carbapenemase producers were also prevalent in Nepal. While other carbapenemases like VIM, IPM, and DIM also detected. The isolates producing carbapenemases were extremely drug-resistant as they also co-produced various other carbapenemases, beta-lactamases, 16S rRNA methylase. Most isolates were resistant to many members of carbapenem, cephalosporin, quinolone, penicillin, aminoglycoside group of antibiotics. Such isolates had very few treatment options as only last line drugs like colistin, fosfomycin, and tigecycline was effective against most of these isolates. Carbapenemase production by almost all major human pathogens including E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter. Citrobacter, Proteus, Providencia is a matter of concern because some of these enzymes are located on plasmids and pose rapid dissemination among various gram-negative pathogens. Timely surveillance for carbapenemase producers throughout the nation, their proper treatment, and proper hospital hygiene to prevent nosocomial infections by carbapenemase producers, controlled use of carbapenems, educating health care workers, students and the general public about the adverse effects of antimicrobial resistance is imminent.

Subclinical mastitis (SCM) is regarded as both a problem for dairy producers and a threat to human health worldwide owing to the potential bacterial contamination of milk and dairy products, particularly those made from raw milk. In the present study, we isolated Escherichia coli from 14 (9.3%) SCM milk samples. We serotyped each E. coli isolate (n = 14), and investigated its potential pathogenicity and antimicrobial resistance (AMR). The serotyping results showed that the E. coli isolates belonged to serotypes O55:H7 (n = 2), O111:H4 (n = 2), O127:H6 (n = 2), O128:HUT (n = 2), O26:HUT (n = 1), O44:H18 (n = 1), O114:H21 (n = 1), O86:HUT (n = 1), O124:HUT (n = 1), and O127:H7 (n = 1). Potential pathogenicity was detected in 93% (13/14) of the isolates. In particular, 13 isolates possessed at least one of the examined virulence genes. Ten isolates (71%) exhibited AMR to at least one of the tested antimicrobials, four (40%) were multidrug-resistant, and one isolate produced extended-spectrum β-lactamases. The obtained results indicate that SCM acts as a source for the spread of potentially pathogenic E. coli strains that are resistant to many groups of antimicrobials, and may constitute a hazard to both public and animal health.

This study was aimed to develop a multiplex PCR (m-PCR) for the detection of Escherichia coli attaching and effacing (eae), Shiga toxin (stx) and cytolethal distending toxin (cdt) genes encoding important virulence factors of diarrheagenic E. coli such as EPEC, STEC, and Escherichia albertii. For this purpose, the m-PCR was designed to detect eae, all the subtypes of stx (stx1, stx2a-g except stx2f) and cdt (I-V) genes. The m-PCR was validated with 58 and 55 target gene-positive and negative strains of different sources, respectively. Sensitivity and specificity of the m-PCR were 100%. The m-PCR could also detect the eae, stx and cdt genes in bacteria spiked into stool specimens with or without enrichment culture. Clinical specimens collected from children with diarrhea were tested by the m-PCR, and 27 eae and 32 cdt genes were detected. Among them, three cdt-II and one untypable cdt gene-positive bacteria were isolated and identified as E. albertii and Providencia rustigianii, respectively. This is the first report demonstrating the presence of cdtB gene in P. rustigianii. These results indicate that the m-PCR is useful for surveillance of eae, stx and cdt gene-positive bacteria, not only EPEC, STEC and E. albertii but also P. rustigianii.

The purpose of this study was to investigate the prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-Ec) in retail chicken meats in Japan. Fifty-six domestic and 50 imported (Brazil, n=36; United States, n=8; Thailand, n=6) chicken meat samples were analyzed. The 162 ESBL-Ec included 111 from 43 (77%) domestic samples and 51 from 26 (52%) Brazilian samples. Fifty-three and 30 of 111 and 51 ESBL-Ec from domestic and Brazilian chickens, respectively, were selected for ESBL genotyping. The bla CTX-M (91%), bla TEM (36%) and bla SHV (15%) genes were detected in ESBL-Ec isolated from domestic chickens, whereas bla CTX-M (100%) and bla TEM (20%) were detected in ESBL-Ec isolated from imported chickens. Among the bla CTX-M group, bla CTX-M-2 (45%) and bla CTX-M-1 (34%) were prevalent in domestic chicken isolates, whereas bla CTX-M-2 (53%) and bla CTX-M-8 (43%) were prevalent in imported chicken isolates. Domestic chicken isolates were mostly resistant to tetracycline (83%), followed by streptomycin (70%) and nalidixic acid (62%). Imported chicken isolates were resistant to streptomycin (77%), followed by nalidixic acid (63%) and tetracycline (57%). Notably, extensive multidrug resistance was detected in 60% (32/53) and 70% (21/30) ESBL-Ec from domestic and imported chickens, respectively. Virulence genes associated with diarrheagenic and extra-intestinal pathogenic E. coli were detected in ESBL-Ec isolated from domestic and imported chickens. These data suggest that ESBL-Ec in retail chicken meats could be a potential reservoir for antimicrobial resistance determinants and that some are potentially harmful to humans.

Cytolethal distending toxin (CDT)-producing Escherichia coli have been isolated from patients with diarrhea, sepsis and urinary tract infection. CDT of E. coli is divided into five types (CDT-I through CDT-V) based on differences in amino acid sequences and its genomic location. However, in our recent studies, a few strains of cdt-II gene-positive bacteria, initially identified as atypical E. coli, were re-identified as Escherichia albertii, an emerging enteropathogen, by extensive characterization including multilocus sequence (MLS) analysis and sugar utilization tests. This finding prompted us to investigate if bacteria previously identified as cdt-II gene-positive E. coli might be E. albertii. In the present study, we therefore re-examined the identity of 20 cdt-II gene-positive bacteria isolated from children with diarrhea, which were initially identified as atypical E. coli. By extensive sugar utilization tests, these bacteria showed a closer relatedness to E. albertii than E. coli, because they did not ferment any of the tested sugars including dulcitol, lactose, d-melibiose, l-rhamnose and d-xylose. Further, both phylogenetic analyses based on nucleotide sequences of 7 housekeeping genes (MLS analysis) and rpoB gene showed that all the cdt-II gene-positive bacteria belonged to a distinct lineage of E. albertii from those of E. coli and Shigella boydii. They were also positive by an E. albertii-specific PCR. Taken together, these data suggest that cdt-II gene-positive bacteria previously identified as E. coli are actually E. albertii. Therefore, we suggest a new definition for cdt-II gene-positive E. coli as E. albertii with the inclusion of CDT-II in E. albertii CDT., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

In this study, we attempted to isolate Escherichia coli from healthy adults in Ho Chi Minh City, Vietnam, and characterized its antimicrobial resistance profile, extended-spectrum β-lactamase (ESBL) genotype, phylogenetic grouping and virulence gene profile. A total of 103 E. coli isolates were obtained, and most of them were antimicrobial resistant such to streptomycin (80.6%), tetracycline (67.0%), ampicillin (65.0%), sulfamethoxsazole/trimethoprim (48.5%), nalidixic acid (43.7%), chloramphenicol (34.0%), cefotaxime (15.5%), ciprofloxacin (15.5%), kanamycin (12.6%), ceftazidime (10.7%), fosfomycin (4.9%) and gentamicin (2.9%). However, all these E. coli strains were susceptible to imipenem. Surprisingly, of 103 strains, 74 (71.8%) and 43 (41.7%) strains showed resistance to more than 3 and 5 classes of antimicrobials, respectively. Furthermore, 10 E. coli strains were ESBL-producers and positive for bla CTX-M genes (7 for bla CTX-M-9 and 3 for bla CTX-M-1 ), while five were additionally positive for bla TEM genes. S1-nuclease pulsed-field gel electrophoresis analysis revealed that 7 and 3 strains of E. coli carry bla CTX-M genes on their large plasmid and chromosome, respectively. Phylogenetic analysis exhibited that majority of the E. coli strains was grouped into A (44.7%), followed by B1 (23.3%), B2 (18.4%) and D (13.6%). Virulence genes associated with diarrheagenic E. coli, such as astA, EAF, eaeA, elt and eagg were also detected in ESBL-producing E. coli as well as antimicrobial resistant strains. These data suggest that commensal E. coli of healthy human could be a reservoir for antimicrobial resistance determinants and some of them might be harmful to human.

The objectives of this study were to investigate prevalence and pathogenic potential of Escherichia coli contaminating raw milk and its products in Egypt. Out of 187 dairy products including 72 raw milk samples, 55 Karish cheese and 60 Ras cheese, 222 E. coli isolates including 111, 89 and 22 were obtained from 55 raw milk samples (76.4%), 41 Karish cheese (74.5%), and 13 Ras cheese (21.7%), respectively. Isolated E. coli strains were examined for 24 representative virulence genes present in diarrheagenic E. coli (DEC) and extraintestinal pathogenic E. coli (ExPEC). Among DEC and ExPEC virulence factors, genes for enteropathogenic E. coli (eaeA, bfpA, EAF), enterohemorrhagic E. coli (stx1, stx2, eaeA), enterotoxigenic E. coli (elt, est), enteroinvasive E. coli (invE), enteroaggregative E. coli (Eagg, astA), diffusely adherent E. coli (daaD), ExPEC (cdt-I to cdt-V, cnf1, cnf2, hlyA) and putative adhesins (efa1, iha, ehaA, saa, and lpfAO113) were screened by colony hybridization assay. Out of 222 E. coli strains, 104 (46.8%) isolated from 69 (36.9%) samples carried one or more virulence genes. The most prevalent gene detected was lpfAO113 (40.5%), followed by ehaA (32.4%,), astA (3.15%,), iha (1.80%), hlyA (1.35%), stx1 (0.90%), stx2 (0.90%), eaeA (0.45%), cdt-III (0.45%) and cnf2 (0.45%). Two strains isolated from Karish cheese harbored 5 virulence genes (stx1, stx2, iha, ehaA, lpfAO113). Stx subtype was determined to be stx1 (not stx1c or stx1d) and stx2d. Indeed, expression of hemolysin A, CDT-III, CNF-II, Stx1 and Stx2d was confirmed by blood agar plate, cytotoxicity assay and Western blotting, respectively. Among the 222 E. coli strains, 54 (48.6%), 38 (42.6%) and 12 (54.7%) isolated from raw milk, Karish cheese and Ras cheese were potentially virulent, respectively. O-genotyping indicated that most of the potentially virulent E. coli isolates did not belong to clinically important O serogroups except O75, O91 and O166, which have been associated with human diseases. Phylogenetic grouping revealed that 150 (67.6%), 67 (30.2%) and 5 (2.30%) strains were clustered into A, B1 and D groups, respectively, which are considered to be associated with intestinal infection, indicating that these E. coli strains might have a potential to cause gastroenteritis. To the best of our knowledge, this is the first comprehensive study regarding prevalence and pathogenic potential of E. coli in dairy products in Egypt. Raw milk, Karish cheese and Ras cheese in Egypt are highly contaminated with E. coli including potentially pathogenic strains, which may impose a public health threat., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The phytase gene appAS was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppAS) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg(-1), respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.

Background: The aim of the present study was to observe the plasmid mediated intraspecies and interspecies transfer of vancomycin resistance, and possible antibacterial activity of nanoconjugated vancomycin against such resistant., Methods: Plasmids were isolated from a chosen vancomycin resistant Staphylococcus aureus strain (MMC-17). The obtained 890 bp plasmid was then transferred to vancomycin sensitive S. aureus (MMC-6) and Escherichia coli (RGK 26) strains., Results: The vancomycin sensitive S. aureus and E. coli developed vancomycin resistance. Plasmid analysis of the transformed MMC-6 and RGK 26 revealed that it contains 890 bp plasmid corresponding to the donor S. aureus, which may harbor the vanA gene. Nanoconjugated vancomycin shows its efficient drug action through transport of vancomycin into transformed MMC-6 and RGK 26., Conclusions: Plasmid mediated intraspecies and interspecies transfer of vancomycin resistance is accomplished by the vanA gene. Nanoconjugated vancomycin shows effective drug delivery in plasmid mediated vancomycin resistance in S. aureus and E. coli.

Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89th position was replaced by aspartic acid and in the other both glycine and valine at the 88th and 89th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88-120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E.