Escherichia coli is an important pathogen that causes diarrhea in both humans and animals. To determine the relationships between putative virulence factors and pathotypes or host taxa, many molecular studies on diarrhea-associated E. coli have been reported. However, little is known regarding genome-wide variation of E. coli from animal hosts. In this study, we performed whole genome sequencing of 127 E. coli isolates from sheep and swine with diarrhea in China. We compared isolates to explore the phylogenomic relatedness based on host origin. We explored the relationships of putative virulence factors across host taxa and pathotypes. Antimicrobial resistance was also tested.E. coli genomes in this study were diverse with clear differences in the SNP, MLST, and O serotypes. Seven putative virulence factors (VFs) were prevalent (> 95%) across the isolates, including Hcp, csgC, dsdA, feoB, fepA, guaA, and malX. Sixteen putative VFs showed significantly different distributions (P < 0.05) in strains from sheep and swine and were primarily adhesion- and toxin-related genes. Some putative VFs were co-occurrent in some specific pathotypes and O serotypes. The distribution of 4525 accessory genes of the 127 strains significantly differed (P < 0.05) between isolates obtained from the two animal species. The 127 animal isolates sequenced in this study were each classified into one of five pathotypes: EAEC, ETEC, STEC, DAEC, and EPEC, with 66.9% of isolates belonging to EAEC. Analysis of stx subtypes and a minimum spanning tree based on MLST revealed that STEC isolates from sheep and EAEC isolates from sheep and swine have low potential to infect humans. Antibiotic resistance analysis showed that the E. coli isolates were highly resistant to ampicillin and doxycycline. Isolates from southeast China were more resistant to antibiotics than isolates from northwest China. Additionally, the plasmid-mediated colist in resistance gene mcr-1 was detected in 15 isolates, including 4 from sheep in Qinghai and 11 from swine in Jiangsu.The E. coli isolated from animal sources. Distinguishable differences between swine and sheep isolates at the genomic level provides a baseline for future investigations of animal E. coli pathogens.Our study provides insight into the genomes of The online version of this article (10.1186/s12864-019-5588-2) contains supplementary material, which is available to authorized users. Escherichia coli is a part of the normal microflora of the human body, and also inhabits the intestinal tracts of other mammals. However, some E. coli pathotypes have acquired various putative virulence factors (VFs) from their environment. Pathogenic E. coli strains are classified as extraintestinal E. coli (ExPEC) or intestinal E. coli based on their ecological niche. Intestinal E. coli, also called diarrheagenic E. coli (DEC), can cause diarrhea in mammals; their classification is further broken down into six well-described categories: enteropathogenic E. coli (EPEC), shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffuselyadherent E. coli (DAEC) [i (DAEC) .E. coli stands out as an important agent associated with acutediarrhea [E. coli have been reported in different countries [E. coli can occur in humans from domestic animals, especially from pigs, cattle and sheep [E. coli isolated from pigs, sheep and goats were found to be similar to strains from humans, suggesting that pigs and ruminants could be a potential source of infection in humans [Diarrhea is a majorcause of mortality and morbidity inhumans and young domestic animals all over the world, especially in developing countries. diarrhea . A largeountries . Postweaountries , 4. Foodnd sheep . Addition humans –8.E. coli isolates from animals with diarrhea in China. The goal of this study was to provide large-scale genomic data on E. coli from host animal species, and examine putative VFs across species, as well as antimicrobial resistance, in order to contribute to the understanding of E. coli from different hosts.With the development of next-generation sequencing technologies, analysis of whole genome data from large numbers of clinically relevant bacterial isolates is now possible. However, most comparative genome analyses have focused on human isolates, and there is very little information regarding genome comparisons of large numbers of animal isolates. Here, we performed whole genome sequencing and comparative analysis of 127 intestinal E. coli isolates from the two animal taxa (i.e. pigs and sheep) were extensively distributed across the phylogenetic tree, while there is no major clustering of host sources on the tree. The results of multilocus sequence typing (MLST) assay were largely concordant with the phylogenomic results  in both swine and sheep isolates. H2, H8, H11and H31 were prevalence (≥ 5 isolates) in sheep isolates, while H5, H10 and H45 were prevalence (≥ 5 isolates) in swine isolates  belonging to iron acquisition/transport systems and biosynthesis, were most prevalent (> 95%) across the isolates  and one toxin gene (cadA) were more prevalent (P < 0.05) in swine isolates than in sheep isolates  and one iron acquisition/transport gene (sepA) were found in 12.9 to 22.9% of the swine isolates but were absent from sheep isolates .Swine and sheep tes Fig. A. Five tLT, eatA, and CS6 fimbrial subunits A and B were significantly more common (P < 0.05) in sheep EAEC isolates than in swine EAEC isolates, while csgA,CS1 and fimA were significantly more common (P < 0.05) in swine EAEC isolates than in sheep EAEC isolates  between swine and sheep isolates. The significantly different accessory genes between swine and sheep isolates covered 23 COGs, and were most enriched in carbohydrate transport and metabolism, followed by the mobilome (prophages and transposons), cell wall/membrane/envelope biogenesis, and transcription , ETEC , STEC , DAEC  and EPEC . EAEC has been reported as the most frequent or second most frequent DEC in humans , 15. EAEE. coli strains isolated from human clinical cases. Five ST  from swine were also observed in sheep. The remaining STs were only observed in one host  were resistant to ampicillin, and 50% were resistant to doxycycline  between isolates from southeast and northwest China, most of which were aminoglycoside- and sulfonamide-resistance genes  and seven stx2 subtypes  [stx types and subtypes may be associated with differences in STEC pathogenicity. STEC carrying stx1a, stx2a, stx2c, and stx2d are associated with severe clinical symptoms, while STEC carrying stx1c and stx2b are mainly associated with diarrheal disease [Stx2e has been reported in association with ED in pigs and is probably not pathogenic to humans [Stx2a, stx2c and stx2d have been recognized in relation to severe STEC infections in humans [stx1c and stx2b subtypes, while the 12 STEC isolates from swine were positive only for stx2e. These results corresponded to previous reports [VFs play an important role in the infection process of  genomes . Putativith ETEC , 23, andomal RNA . Stx can disease . Stx2e ho humans . Stx2a, n humans , 28. She reports  and sugget al. [ompA, fimH, vat, traT, and iutA were highly prevalent (> 60%) in swine ExPEC strains isolated in China, and fimH was the most prevalent (81.2%) adhesion factor. Similarly in our study, fimH and traT had a high prevalence (> 60%) in swine isolates, and fimH was present in 87.1% of swine isolates, suggesting that fimH and traT were prevalent both in ExPEC and intestinal E. coli. In this study, ompA was excluded as it was found in 199 E. coli genomes in the NCBI GenBank database  and chicken (14.0%) farms [mcr-1 was present in 15.7% and 7.0% of swine and sheep isolates, respectively. All the mcr-1-positive strains were tested for antibiotic resistance to polymyxin. Ten strains were resistant to polymyxin and five  showed intermediate resistance to polymyxin (data not shown). The results of antibiotic resistance tests showed that isolates from southeast China were more resistant to antibiotics; this might due to the antibiotic is greater used in southeast China. As most of the sheep came from one province (Qinghai) whereas the swine came from other regions, the antibiotic resistance differences may also be because swine are more likely to be treated with antibiotics than sheep.In recent years, drug resistance has become a serious problem, which has attracted increasing attention from researchers. As the incidence of carbapenem-resistant Enterobacteriaceae increased worldwide, polymyxins have been adopted as the last line of defense against Gram-negative bacterial infections . Resistad by Liu , is a pl%) farms  in ChinaE. coli isolates from swine, and sheep with diarrhea. To differentiate between E. coli strains obtained from different hosts, various aspects of these E. coli isolates, including putative VFs, accessory genes, antibiotic resistance, MLST, O serotypes, pathotypes, and phylogenomic trees were analyzed. No specific putative VFs were found to be completely present or absent in isolates from any one animal group. However, some putative VFs co-occurred in some specific pathotypes and O serotypes. The frequency of some VFs and accessory genes present in swine and sheep isolates differed significantly. We have described the genomic profiles of intestinal E. coli isolates from different animals with diarrhea, which will provide a baseline for future research into DEC.In this study, comparative genomic analysis was performed for 127 E. coli strains were isolated from sheep (n = 57) and swine (n = 70) with diarrhea from 1972 to 2013 in China. Additional file A total of 127 Genomic DNA was extracted using a Bacterial DNA Kit . The genomic DNA was fragmented by ultrasonication, and library preparation was performed using Illumina TruSeq DNA Sample Prep Kits . Paired-end sequencing was performed on an Illumina HiSeq 2000 system.http://tree.bio.ed.ac.uk/ so/ software/figtree/). Roary [adk, fumC, gyrB, icd, mdh, purA and recA) were blast according to the protocol of the E. coli MLST database (https://pubmlst.org/data/) [Low quality reads were filtered using the DynamicTrim Perl script within SolexaQA . Raw rea). Roary  is a hig). Roary . Seven cg/data/) .E. coli strains were identified by PCR or sequence alignment. Multiplex PCR was performed as previously described to detect the pathotype of the strains [stx gene; ETEC if positive for genes coding for heat-stable enterotoxin or heat-labile enterotoxin; EPEC if positive for the gene coding for the outer membrane protein intimin; EAEC if positive for the gene coding for transporter protein Aat; EIEC if positive for the gene coding for the invasion protein IpaH; and DAEC if positive for the gene coding for an accessory protein with a function in F1845 fimbriae production.Pathotypes of  strains . An isolE. coli ATCC 25922 was used as the positive control. The antibiotic-resistance database  [Antibiotic resistance was determined using the agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The following antibiotics were tested: ciprofloxacin, ampicillin, cefotaxime, gentamycin, kanamycin, and doxycycline. The reference strain ter.ca/)  was usedE. coli putative VF reference sequences . Strains lacking matched sequences were detected by classical serum agglutination tests.O serotypes were determined by blasting genome sequences from this study against sequences of 184 O antigen biosynthesis gene clusters as previously reported . H serotE. coli isolates were entered in NCBI and accession numbers for each sample are listed in Additional file All sequences from the 127 Additional file 1:Table S1. Strains information. Host, collection year, geographical location, sample type, MLST type, O antigen, pathotype, number of scaffolds, and average scaffold size of the 127 isolates used in this study. Table S2. Percentage of strain type. Isolates from this study were compared to putative virulence factor (VF) reference sequences using BLAST, and each putative VF was considered matched with a query sequence if the sequence identity was > 60% and the aligned length coverage was > 50%. The proportion of putative VF in distinct groups was included. Fisher’s exact test was used to determine significant differences of each putative VF between two different groups. P-values were adjusted using Bonferroni correction. Table S3. Putative VF in MG1655. Putative VF detected in the E. coli MG1655 genome. Table S4. Average putative VF numbers of each strain type. This table contains the numbers of putativeVF in each strain and the average numbers of putative VF in the two strain types. Table S5. Percentage of VFs in EAEC strains. Isolates from this study were compared to putative virulence factor (VF) reference sequences using BLAST, and each putative VF was considered matched with a query sequence if the sequence identity was > 60% and the aligned length coverage was > 50%. The proportion of putative VFs in distinct groups is included. Fisher’s exact test was used to determine significant differences in each putative VF between two different groups. P-values were adjusted using Bonferroni correction. Table S6. Percentage of VFs in STEC strains. Isolates from this study were compared to putative virulence factor (VF) reference sequences using BLAST, and each putative VF was considered matched with a query sequence if the sequence identity was > 60% and the aligned length coverage was > 50%. The proportion of putative VFs in distinct groups is included. Fisher’s exact test was used to determine significant differences in each putative VF between two different groups. P-values were adjusted using Bonferroni correction. Table S7. Distribution of VFs in O20 and O8 isolates. Isolates from this study were compared to putative virulence factor (VF) reference sequences using BLAST, and each putative VF was considered matched with a query sequence if the sequence identity was > 60% and the aligned length coverage was > 50%. Table S8. Distribution of putative virulence factors in different O serotypes. Isolates from this study were compared to putative virulence factor (VF) reference sequences using BLAST, and each putative VF was considered matched with a query sequence if the sequence identity was > 60% and the aligned length coverage was > 50%. Table S9.Stx subtype information. This table lists the stx subtypes as well as host, collection year, geographical location, sample type, MLST type, and O antigen of 20 STEC strains in this study. Table S10. Antibiotic resistance of strains. The antibiotic resistance to six drugs  of the 127 isolates in this study. Table S11. Reference E. coli genomes. The putative VF reference sequences in this study were collected from the Virulence Factor Database and from previous studies, and the core genes of 199 E.coli genomes in the NCBI GenBank database were then removed. This table contains the name and accession numbers of the 199E.coli genomes. Table S12. Accession numbers of the strains. All genome data from this study were submitted to the NCBI GenBank database. This table contains the accession numbers of the genomes of all 127 E. coli isolates. (PDF 1165 kb)Additional file 2:Figure S1. Presence of antibiotic-resistance genes in E. coli isolates. Red and green bars on the x-axis represent a geographical location in southeast and northwest China, respectively. The bright red and dark blue regions represent the presence or absence of genes in a particular isolate, respectively. This figure shows that significantly more antibiotic resistance genes were found in isolates from southeast than northwest China. (PDF 46 kb)Additional file 3:Dataset S1. putative VF reference sequences. This table contains putative VF reference sequences used in this study. All putative VFs were classified according to their function. (XLSX 163 kb)


Asinibacterium sp. strains OR43 and OR53 belong to the phylum Bacteroidetes and were isolated from subsurface sediments in Oak Ridge, TN. Both strains grow at elevated levels of heavy metals. Here, we present the closed genome sequence of Asinibacterium sp. strain OR53 and the draft genome sequence of Asinibacterium sp. strain OR43. Asinibacterium sp. strains OR43 and OR53 belong to the phylum Bacteroidetes and were isolated from subsurface sediments in Oak Ridge, TN. Both strains grow at elevated levels of heavy metals. Here, we present the closed genome sequence of Asinibacterium sp. strain OR53 and the draft genome sequence of Asinibacterium sp. strain OR43. Asinibacterium sp. strains OR43 and OR53 (formerly Sediminibacterium sp. strains OR43 and OR53) are Gram-negative, nonmotile, aerobic bacteria. The type strain Asinibacterium lactis was isolated from donkey milk powder  in Oak Ridge, TN, with the diffusion chamber approach  between the genomes was calculated with the Microbial Species Identifier (MiSI) method (Asinibacterium spp. to withstand and grow in the presence of uranium.The genome size for ) method  at 96.4%ATYE00000000 (Asinibacterium sp. strain OR43) and AZXP00000000 (Asinibacterium sp. strain OR53). The raw reads were deposited in the SRA under SRP078705 (Asinibacterium sp. strain OR53) and SRP078706 (Asinibacterium sp. strain OR43).This whole-genome project has been deposited at DDBJ/EMBL/GenBank under the accession numbers


Monolluma quadrangula is a succulent bush traditionally used to treat diabetes and peptic ulcer. The present study aimed to investigate the effect of M. quadrangula hydroethanolic extract on glucose tolerance, insulin sensitivity, glucose metabolizing enzymes, lipid profile, and adiponectin expression in type 2 diabetic rats. In addition, the study evaluated the antioxidant and anti-inflammatory activities of the M. quadrangula extract. Type 2 diabetes was induced by feeding rats a high-fat diet (HFD) for 8 weeks followed by 30 mg/kg streptozotocin (STZ). Diabetic rats received 300 or 600 mg/kg M. quadrangula extract for 4 weeks. HFD/STZ diabetic rats showed impaired glucose tolerance, reduced insulin secretion, and insulin resistance. HFD and STZ induced a significant increase in serum cholesterol, triglycerides and proinflammatory cytokines, and liver lipid peroxidation. Treatment with M. quadrangula extract ameliorated these metabolic disturbances and increased liver glycogen, hexokinase activity, and antioxidants. M. quadrangula declined the activity of liver glucose-6-phosphatase and fructose-1,6-biphosphatase. In addition, M. quadrangula extract increased serum adiponectin levels and hepatic adiponectin expression in HFD/STZ diabetic rats. In conclusion, M. quadrangula exerts antidiabetic effect mediated via ameliorating glucose tolerance, insulin sensitivity, glucose metabolizing enzymes, and antioxidant defenses. Increased adiponectin levels and expression seems to mediate, at least in part, the antidiabetic effect of M. quadrangula. Diabetes mellitus (DM) is a metabolic syndrome characterized by hyperglycemia that occurs as a result of deficient insulin secretion and/or action. Uncontrolled hyperglycemia can lead to micro- and macrovascular complications, nephropathy, cardiomyopathy, and retinopathy. Type 2 DM is the common form of DM and represents a public health concern worldwide . OxidatiAdiponectin, a 30 kDa multimeric protein, is secreted mainly by adipose tissue; however, it is expressed in the liver, myocytes, placenta, epithelial cells, and osteoblasts –12. SeveMonolluma quadrangula (Forssk.) extract, focusing on its role in modulating glucose metabolizing enzymes, oxidative stress, and adiponectin expression. M. quadrangula is a succulent bush with a yellow flower and irregularly branched and a compressed stem [Caralluma quadrangula and has been used in folk medicine for the treatment of DM and peptic ulcer. M. quadrangula showed an antioxidant effect in ethanol-induced peptic ulcer [M. quadrangula ameliorated serum lipids, hepatic and cardiac oxidative stress, and the expression of fatty acid synthase (FAS) and low density lipoprotein- (LDL-) receptor in HCD-fed rats [M. quadrangula could be a promising candidate for the treatment of diabetes. The antihyperglycemic effect of methanol, chloroform, and n-butanol extracts of M. quadrangula has been recently tested by Abdel-Sattar et al. [M. quadrangula on glucose tolerance, insulin sensitivity, lipid profile, oxidative stress, and adiponectin expression in type 2 DM has not been investigated.The present study aimed to investigate the antihyperglycemic and insulin sensitizing effects of sed stem . It is kic ulcer  and highic ulcer . Recentlfed rats . Therefor et al.  which shM. quadrangula samples and preparation of hydroethanolic extract were conducted as we recently described [M. quadrangula collected from Abha-Al-Taif road (Saudi Arabia) were air-dried, grounded into fine powder in an electric grinder, and soaked for 24 h in water/ethanol (1 : 1 vol/vol). After filtration of the mixture and evaporation of the solvent in a rotary evaporator, the dried residue was collected and used for animal treatments.The collection of escribed . Brieflyad libitum for 8 weeks and then received a single intraperitoneal (ip) injection of STZ (30 mg/kg) dissolved in freshly prepared cold citrate buffer (pH 4.5). Seventy-two h after STZ injection, blood glucose was measured and rats having fasting blood glucose of more than 200 mg/dL were considered diabetic. A corresponding group of rats fed with a normal diet and received a single ip injection of citrate buffer served as a control group.Male Wistar rats were fed a HFD All animals included in this study were obtained from the animal house of King Saud University (Saudi Arabia), and all procedures were approved by the ethical committee at Princess Nourah bint Abdulrahman University . The animals were housed under standard laboratory conditions as we previously reported .M. quadrangula extract dissolved in distilled water via oral gavage for 4 weeks [The total of six normal control rats was used as group I (control) and received a daily dose of distilled water via oral gavage for 4 weeks. The diabetic rats were allocated randomly into 3 groups, each group has six, as follows: group II (diabetic) included diabetic rats which received distilled water orally and daily for 4 weeks, group III and group IV included diabetic rats which received daily doses of 300 and 600 mg/kg  4 weeks .At the end of the experiment, all groups were fasted overnight and sacrificed under anesthesia. Blood samples were collected to separate the serum and the rats were dissected to collect the liver. Samples from the liver were homogenized in a cold 0.1 M phosphate buffer (pH 7.4), centrifuged at 8000 rpm, and used for biochemical assays. The other samples were kept frozen at -80°C for RNA isolation.Oral glucose tolerance test (OGTT) was performed on the day before the sacrifice. Overnight-fasted rats received 3 g/kg glucose solution orally, and blood samples were collected from the tail vein at 30, 60, 90 and 120 min . GlucoseTotal serum cholesterol , HDL-choα), and adiponectin were assayed using ELISA kits (Merck Millipore), USA.Serum insulin, interleukin-6 (IL-6), tumor necrosis factor alpha  × fasting glucose (mmol/L)/22.5.HOMA − IR = fasting insulin , and catalase (CAT) were assayed in the liver homogenate using OxiSelect kits (USA).ΔΔCt- method [To analyze the gene expression levels of adiponectin in the liver, we used qPCR as previously described , 34. In t method .P value less than 0.05 was considered significant.The results were analyzed using GraphPad Prism . The results were presented as mean ± standard error (SEM), and one-way ANOVA test followed by Tukey's test was used for statistical comparisons. A M. quadrangula for four weeks improved the glucose tolerance as shown in the OGTT results  as compared to the control group. On the other hand, diabetic rats which received 300 and 600 mg/kg body weight hydroethanolic extract of M. quadrangula for four weeks showed improved serum insulin levels (P < 0.001) as compared to the diabetic control animals  as compared to nondiabetic rats. M. quadrangula extract at both 300 and 600 mg/kg doses significantly improved insulin sensitivity (P < 0.01) as shown by decreased HOMA-IR values  and in the activity of hexokinase (P < 0.01) as compared to the control rats as shown in Figures M. quadrangula extract significantly improved (P < 0.01) hepatic glycogen content and hexokinase activity.The data showing the effect of P < 0.001). The treatment of the diabetic rats with both doses of M. quadrangula extract significantly improved the activity of hepatic glucose-6-phosphatase (P < 0.001) and fructose-1,6-biphosphatase (P < 0.01).In contrast, HFD/STZ rats showed a significant increase in the activity of hepatic glucose-6-phosphatase and fructose-1,6-biphosphatase  as compaP < 0.001) elevation of serum triglycerides (M. quadrangula extract showed a significant improvement of serum triglycerides and cholesterol and its fractions (P < 0.001). On the other hand, HFD/STZ diabetic rats showed a significant (P < 0.05) decrease in serum levels of HDL and treatment with 300 and 600 mg/kg M. quadrangula extract did not induce significant changes as represented in HFD/STZ diabetic rats showed a significant . HFD/STZ diabetic rats treated with the two doses of M. quadrangula extract significantly increased the lowered liver GSH content and SOD and CAT activities.The data presented in Figures α and IL-6, were significantly elevated in the serum of HFD/STZ diabetic rats (P < 0.001) as compared to the control rats. The treatment of the HFD/STZ diabetic rats with 300 and 600 mg/kg M. quadrangula extract ameliorated the levels of TNF-α and IL-6 as compared to the nontreated diabetic group (P < 0.001).As presented in M. quadrangula extract on serum adiponectin and hepatic adiponectin gene expression are presented in P < 0.01) as compared to the control rats (M. quadrangula extract showed a significant increase (P < 0.05) in serum adiponectin. The 600 mg/kg M. quadrangula extract induced a significant increase (P < 0.01) in serum adiponectin levels.The data showing the effect of rol rats . The diaP < 0.01) in the liver of HFD/STZ diabetic rats as compared to the control rats as presented in M. quadrangula extract increased the gene expression of adiponectin in the liver of the diabetic rats (P < 0.01).Adiponectin gene expression significantly decreased (M. quadrangula has been used in tradition as a medicine to diabetes and peptic ulcer. However, its antidiabetic mechanisms are not known. Recently, we reported the antihypercholesterolemic effect of M. quadrangula in HCD-fed rats [M. quadrangula ameliorated serum lipids, hepatic and cardiac oxidative stress, and the expression of FAS and LDL-receptor in HCD-fed rats. Given its potent lipid-lowering effect, M. quadrangula could be a promising candidate for the treatment of diabetes.fed rats . M. quadβ-cells, diminished insulin secretion and sensitivity, reduced peripheral glucose uptake, and increased hepatic glucose production [M. quadrangula extract for 4 weeks improved blood glucose, insulin secretion, and insulin sensitivity, demonstrating a potent antihyperglycemic effect.HFD/STZ diabetic rats in the present study showed an impaired glucose tolerance and insulin sensitivity as shown by the increased blood glucose, decreased insulin, and increased HOMA-IR value. The combination of HFD and STZ has been previously reported to induce type 2 DM characterized by an impaired glucose tolerance and insulin resistance –38. Theroduction , 41. TheM. quadrangula extract in HFD/STZ diabetic rats. Our results showed an improved hepatic glycogen content in HFD/STZ diabetic rats following the 4-week treatment with M. quadrangula extract. Liver glycogen is a valuable marker to evaluate the hypoglycemic effect of drugs or plant extracts [M. quadrangula extract increased the activity of hexokinase and decreased glucose-6-phosohatase and fructose-1,6-biphosphatase in the liver of the diabetic rats. In diabetes, the rate of glycogenolysis and gluconeogenesis increases, leading to an increased hepatic glucose output [In addition to increased insulin sensitivity, we assumed that an improved peripheral glucose uptake and a decreased hepatic glucose production participate in the antihyperglycemic effect of extracts . In addie output . Previoue output , 42. Dece output , 45.M. quadrangula extract for 4 weeks showed an improvement in their lipid profile marked by a decreased serum triglycerides, and total-, LDL- and vLDL-cholesterol. There are no previous reports showing the hypolipidemic effect of M. quadrangula extract in diabetic rats; however, we recently reported that M. quadrangula decreased serum lipids in rats which received HCD for 8 weeks [M. quadrangula on hepatic FAS and LDLR expressions mediates its hypolipidemic effect [The diabetic rats in the present study showed a significant increase in triglycerides, and total-, LDL- and vLDL-cholesterol in addition to a decreased HDL-cholesterol. This altered lipid profile can lead to the development of cardiovascular disease and is known as the atherogenic lipid profile . In acco 8 weeks . This imc effect .M. quadrangula extract for 4 weeks showed a decreased lipid peroxidation and an increased GSH, SOD and CAT. These findings agreed to the study of Ibrahim et al. [M. quadrangula extract exerts antioxidant effect in ethanol-induced gastric ulcer in rats. In this study, pretreatment with M. quadrangula prevented lipid peroxidation and ameliorated the gastric SOD and CAT [M. quadrangula extract for 8 weeks prevented oxidative stress and improved antioxidants in the liver of HCD-fed rats [M. quadrangula might be attributed to its rich content of glycosides and phenolics [M. quadrangula might have a role in suppressing oxidative stress in the liver of the diabetic rats. This hypothesis is supported by studies showed that altered lipidemic status is a risk factor for oxidative stress and cell injury [Oxidative stress driven by hyperglycemia can impair insulin signaling and induce insulin resistance. In our study, HFD/STZ-induced rats showed oxidative stress as increased liver lipid peroxidation and decreased antioxidants, GSH, SOD, and CAT. In diabetes, ROS can react and induce the peroxidation of the cell membrane polyunsaturated fatty acids, leading to cell damage. The resultant oxidative stress is an important factor in the development of diabetic complications such as nephropathy and retinopathy. Hyperglycemia can also lead to declined cellular antioxidants. In agreement with our data, Mahmoud et al.  reportedm et al.  which sh and CAT . Recentlfed rats . The anthenolics , 51. In l injury , 8.M. quadrangula on oxidative stress has been associated with anti-inflammatory effect as shown by the decreased serum TNF-α and IL-6. HFD/STZ diabetic rats had increased serum TNF-α and IL-6 levels as previously reported [α and IL-6 are correlated with insulin resistance, impaired glucose tolerance, and type 2 DM [M. quadrangula are mediated, at least in part, via its antioxidant and anti-inflammatory activities. The ability of M. quadrangula extract to reduce inflammation has been recently reported in our work where it decreased the serum levels of proinflammatory cytokines in HCD-fed rats [The suppressive effect of reported . These cype 2 DM , 53. Botype 2 DM –56. Therfed rats .M. quadrangula extract in HFD/STZ diabetic rats. Thus, we measured the effect of M. quadrangula extract in serum adiponectin and the adiponectin expression in the liver of HFD/STZ diabetic rats. In the present study, diabetic rats showed a significant decrease in serum adiponectin and hepatic adiponectin gene expression as previously reported [M. quadrangula extract for 4 weeks showed increased levels of serum adiponectin and its hepatic expression. These results were correlated with an improved glucose tolerance, insulin sensitivity, hepatic glucose output, and peripheral glucose uptake. Adiponectin has been reported to stimulate AMP-activated protein kinase resulting in an increased insulin sensitivity and regulation of glucose metabolism [α, decrease the hepatic and skeletal muscle triglyceride content [We assumed that the upregulation of adiponectin mediates the antidiabetic effect of reported , 39. Lowreported . Diabetitabolism . In additabolism . Further content , and enh content .M. quadrangula extract improves insulin sensitivity and glucose tolerance in HFD/STZ type 2 diabetic rats. M. quadrangula increased peripheral glucose uptake, improved lipid profile, suppressed hepatic glucose output, and prevented oxidative stress and inflammation in diabetic rats. In addition, M. quadrangula extract increased the serum adiponectin levels and adiponectin gene expression in the liver of the diabetic rats. These findings point to the role of adiponectin in mediating the antidiabetic effect of M. quadrangula; however, further studies to determine its exact mechanism of action are recommended.In conclusion, our results showed for the first time that


Kosakonia sp. strain CCTCC M2018092 is a fucose-rich exopolysaccharide producer that was isolated from spring water in Chongqing, Southwest China. In this study, the whole-genome sequence and genetic characteristics of this strain were elucidated. This genome contained 4,789,478 bp, with a G+C content of 56.08% (excluding the plasmid). The genome information in this study will facilitate understanding of the mechanism of high yield of fucose-rich exopolysaccharide produced by Kosakonia sp. Kosakonia sp. strain CCTCC M2018092 is a fucose-rich exopolysaccharide producer that was isolated from spring water in Chongqing, Southwest China. In this study, the whole-genome sequence and genetic characteristics of this strain were elucidated. This genome contained 4,789,478 bp, with a G+C content of 56.08% (excluding the plasmid). The genome information in this study will facilitate understanding of the mechanism of high yield of fucose-rich exopolysaccharide produced by Kosakonia sp. CCTCC M2018092. Kosakonia sp. strain CCTCC M2018092 was determined to be a Gram-negative, facultatively anaerobic bacterium with a rod cell shape. Most Kosakonia species are isolated from soil or trees, and several of them can promote plant growth. However, some other Kosakonia species, such as Kosakonia cowanii, are associated with human beings . Whole-genome sequencing of strain CCTCC M2018092 was carried out on PacBio RS and Illumina sequencing platforms . A TruSeq DNA sample prep kit and a TruSeq sequencing by synthesis (SBS) kit v3-HS (200 cycles) were used for library construction and DNA sequencing (Illumina HiSeq platform), respectively. The genomic DNA was disrupted into 8- to 10-kbp fragments by the g-TUBE method, followed by PacBio RS platform sequencing on two single-molecule real-time (SMRT) cells. After adapter trimming, 40,025 total reads from PacBio data and 18,637,752 total reads from Illumina data, with 548-fold average coverage, were obtained. The FastQC tool was used to assess the read quality (http://www.bcgsc.ca/platform/bioinfo/software/abyss) was used to perform genome assembly with multiple kmer parameters in order to obtain optimal assembly results (https://github.com/marbl/canu) was used to assemble the corrected PacBio long reads. The GapCloser software (https://sourceforge.net/projects/soapdenovo2/files/GapCloser/) was subsequently applied to fill the remaining local inner gaps and correct the single-base polymorphisms for the final assembly results (https://www.ncbi.nlm.nih.gov/), Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/), and Clusters of Orthologous Groups (COG) of proteins database (https://www.ncbi.nlm.nih.gov/COG/). All software programs were used with default parameters, unless otherwise stated.Kosakonia sp. CCTCC M2018092 is closest to Kosakonia cowanii JCM10956 (GenBank accession number CP019445), and the two of them are classified in the same branch. Furthermore, the two strains are far from the other strains of the genus Kosakonia in classification, which indicates that they may not belong to the genus Kosakonia.The 16S rRNA genes were used to construct a phylogenetic tree with the MEGA 7.0 software . The neiCP034225. The raw data were submitted to the SRA under accession number PRJNA506809.The whole-genome sequence has been deposited in the NCBI GenBank database under accession number


Mycoplasma genitalium is rising globally with resultant clinical treatment failure. We investigated the prevalence of mutations in the macrolide and fluoroquinolone resistance-determining regions of M. genitalium in Johannesburg, South Africa, and ascertained their association with HIV serostatus.Antimicrobial resistance in M. genitalium positive specimens, collected from STI and HIV patients enrolled in the Gauteng STI National Microbiological Surveillance programme (2007–2014) and a large HIV outpatient clinic-based study (2007) in Johannesburg, were tested for antimicrobial resistance.Stored gyrA and parC genes in 266 M. genitalium positive DNA extracts. No macrolide resistance-associated mutations were detected in any of the specimens analysed. QRDR mutations with known M. genitalium-associated fluoroquinolone resistance were not detected in gyrA, however, one specimen (0.4%) contained a D87Y amino acid alteration in parC, which has been linked to fluoroquinolone treatment failure. The most common parC amino acid change detected, of unknown clinical significance, was P62S (18.8%). We found no significant association between QRDR mutations in M. genitalium and HIV-infection.We determined the prevalence of 23S rRNA gene mutations conferring macrolide resistance and mutations in the quinolone resistance-determining regions (QRDR) of the M. genitalium is essential, as macrolide resistance may emerge given the recent incorporation of azithromycin into the 2015 South African national STI syndromic management guidelines.Ongoing antimicrobial resistance surveillance in  Mycoplasma genitalium is a known cause of non-gonococcal urethritis (NGU) in men and cervicitis in women [M. genitalium infection in women is also associated with an increased risk of pelvic inflammatory disease (PID), infertility, preterm delivery and spontaneous abortion [M. genitalium infection is a possible cofactor for the acquisition and transmission of human immunodeficiency virus (HIV) [M. genitalium infection ranges between 1.1 and 3.3% in the general population and can be as high as 35% among men with symptomatic non-chlamydial NGU [M. genitalium infections are common among women and often remain undiagnosed [in women . A recenabortion . Furtherus (HIV) –5. The pdial NGU . Asymptoiagnosed .M. genitalium and Chlamydia trachomatis infections [Escherichia coli numbering) in the V region of the 23S rRNA gene of M. genitalium [gyrA) and the topoisomerase IV (parC) genes [M. genitalium in cell culture remains problematic and therefore in vitro antimicrobial susceptibility testing of M. genitalium strains is rarely performed [M. genitalium strains from South Africa is important for studies of antimicrobial resistance, which in turn enables the development of effective treatment algorithms for syndromic management of patients with genital discharge.The current STI Management Guidelines 2015) for South Africa include the use of 1 g azithromycin (a macrolide), orally, as a single dose for the treatment of male urethritis syndrome (MUS) and vaginal discharge syndrome (VDS) . The Eurfections . The widfections –12. A rafections , 13, 14. for Soutnitalium . MoxifloC) genes , 17–21. C) genes –25. The erformed . The genM. genitalium positive specimens at the National Institute for Communicable Diseases (NICD), South Africa. We sequenced the region V of the 23S rRNA gene and QRDRs of the gyrA and parC genes in M. genitalium positive specimens obtained from Gauteng STI National Microbiological Surveillance (NMS) (2007–2014), as well as in M. genitalium positive specimens obtained from HIV positive patients previously recruited to an urban HIV outpatient clinic in Johannesburg, South Africa (2007). Additionally, we sought to determine the association between HIV serostatus and the presence of mutations in the antimicrobial resistance-determining regions of M. genitalium.We evaluated the prevalence of macrolide and fluoroquinolone resistance in stored Neisseria gonorrhoeae, C. trachomatis, Trichomonas vaginalis and M. genitalium using a validated in-house multiplex real-time PCR assay on the Rotor-Gene platform  [M. genitalium DNA, of which 222/2509 (8.8%) were from males and 236/2222 (10.6%) from females. These 458 M. genitalium positive DNA specimens were stored at -70 °C at the STI Section, CHIVSTI and were used for the study. Additional stored M. genitalium DNA specimens, obtained from a previous study that determined the burden of asymptomatic STIs among people living with HIV/AIDS (PLWHA) at an urban HIV outpatient clinic in Johannesburg, were also included in this study [M. genitalium DNA in these specimens was determined using the same methodology described above. A total of 68/1108 (6.1%) of PLWHA specimens also tested positive for M. genitalium DNA; these positive DNA extracts were stored at -70 °C at the STI Section, CHIVSTI and used in this analysis. The total number of M. genitalium-positive specimens that were available for this study is depicted in Table M. genitalium DNA extracts were re-tested to confirm the presence of M. genitalium DNA using a validated, commercially available Sacace M. genitalium Real-TM PCR assay , according to manufacturer’s instructions. The HIV serostatus of each de-identified NMS participant was determined as part of the main NMS study protocol.The STI Section of the Centre for HIV & STI (CHIVSTI) at the NICD, Johannesburg, has conducted annual STI National Microbiological Surveillance (NMS) surveys among STI patients  attending the Alexandra Health Centre in Gauteng, South Africa, since 2007. This facility is a community-based primary healthcare centre offering HIV, AIDS, STI and TB-related treatment, care and support services and is situated in the most populous province in South Africa. Clinical and microbiological data were linked using survey numbers and delinked from patient identifiers. Genomic DNA was initially extracted from urine (men) and endocervical swabs (women) using an automated DNA extractor . Extracted DNA specimens were tested for Germany) . A totalis study . The preM. genitalium positive specimens. Published primers targeting a unique 147 bp region of the M. genitalium 23S rRNA gene flanking the mutations found in region V of the 23S rRNA gene were used [E. coli DNA gyrase genes (gyrA) and the topoisomerase IV (parC) genes in M. genitalium were analysed. PCR primers for the amplification of the gyrA and parC genes have been published previously [Macrolide resistance mediating mutations in region V of the 23S rRNA-gene were detected by DNA sequencing of amplicons obtained from the ere used . Fluoroqere used . Mutatioeviously . The PCReviously , 29. Cycp = 0.05.Contigs obtained from initial sequencing were assembled using Sequencher software version 5.4.6  and the FASTA sequences obtained were aligned with reference sequences using MEGA version 7.0.20 . A chi-sM. genitalium positive using the Sacace M. genitalium Real-TM PCR assay (Table p = 0.002) in the M. genitalium-infected cohort analysed.A total of 266/526 (50.6%) stored DNA specimens were confirmed as ay Table . These DgyrA and parC genes were detected in 9/266 (3.4%) and 54/266 (20.3%) specimens, respectively . HIV serostatus was not associated with the presence or absence of mutations in either gyrA (p = 0.99) or parC (p = 0.51).Macrolide resistance-associated mutations (A2058G/A2059G) were not detected in any of the specimens analysed. Mutations in the QRDR of the gyrA mutations comprised two single nucleotide polymorphisms (SNPs) resulting in altered amino acids at positions 69 (M69I) and 118 (H118R). The clinical relevance of these SNPs is unknown. The M69I alteration was detected in two specimens  sampled in 2013 while the H118R alteration occurred only once  in a sample collected in 2007. An additional six specimens contained single synonymous substitutions at nucleotide positions 231 (G231A), 288 (A288G), 318 (A318G), 321 (C321T) and 348 (T348C), resulting in no amino acid changes.QRDR parC were more frequently observed and included amino acid alterations at positions 62 (P62S), 87 (D87Y) and 113 (D113G) . No macrolide resistance-associated mutations were found in the 23S rRNA genes of M. genitalium analysed in this study, and only one specimen (0.4%) contained a known fluoroquinolone resistance-associated mutation (G259 T) in the parC gene resulting in amino acid change D87Y.We report the baseline prevalence rates of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium is a known cause of non-gonococcal urethritis (NGU) worldwide but syndromic management of NGU is mainly focused on the successful treatment of C. trachomatis infections. Three antimicrobial classes, namely tetracyclines, macrolides and fluoroquinolones, have shown antimicrobial activity against mycoplasmas. In South Africa, prior to 2008, ciprofloxacin and doxycycline were the recommended syndromic treatment options for patients presenting with genital discharge syndromes but due to the rapid emergence of fluoroquinolone-resistant N. gonorrhoeae in the country, the treatment regimen was changed in 2008 to single-dose cefixime and doxycycline [C. trachomatis infections but has a low eradication rate for M. genitalium infection. Given concerns over the subsequent emergence of cefixime-resistant N. gonorrhoeae isolates in South Africa, and in the face of already high-level tetracycline resistance among gonococci, the treatment guidelines were once again changed in 2015. A key change was to replace doxycycline with azithromycin, a potent macrolide with a long half-life and good tissue penetration [C. trachomatis and, although not ideal, may also treat co-existing M. genitalium infections. Azithromycin has now been adopted in many countries as the first line treatment option for NGU [ycycline . Doxycycetration , 31. Sin for NGU . The add for NGU .M. genitalium strains has been reported in many parts of the world, signifying a strong association between the presence of specific mutations in the 23S rRNA gene of M. genitalium and macrolide treatment failure [M. genitalium infections are unlikely to respond to either regimen [M. genitalium analysed [M. genitalium infections has heralded calls to use an extended azithromycin regimen or revert to doxycycline as first-line treatment of NGU. Moxifloxacin is recommended in settings where macrolide resistance is already well established [An increase in the prevalence of macrolide resistant  failure , 34–37.  regimen , 39. Theanalysed , 40–43. ablished .M. genitalium [M. genitalium-positive specimens collected between 2011 and 2012 in the Mopani District, Limpopo Province [M. genitalium detected in females attending a pregnancy termination clinic at the George Mukhari Academic Hospital in Pretoria in 2012 and 2016 [M. genitalium-positive 2016 specimens contained the A2059G 23S rRNA mutation [M. genitalium-positive specimens analysed in our study but could emerge due to the recent incorporation of azithromycin into the 2015 South African national STI treatment guidelines.Limited information is available on the prevalence of macrolide resistance-associated mutations in South Africa with only two studies reporting prevalence rates of mutations in the 23S rRNA gene of nitalium , 45. Theand 2016 . No macrmutation . MacroliM. genitalium infection but vary in terms of efficacy and in vitro activity against the organism [M. genitalium include moxifloxacin, gatifloxacin, sitafloxacin and some of the newer fluoroquinolones, while ciprofloxacin, ofloxacin and levofloxacin are less efficacious for the treatment of M. genitalium infections [M. genitalium infections. However, recent reports of moxifloxacin treatment failure with reduced cure rates of macrolide-resistant M. genitalium infections in the Asia-Pacific region have emerged; these are all linked to the presence of fluoroquinolone resistance-associated mutations in gyrA and parC [gyrA and parC have been described [parC was higher than in gyrA (19.5% v 1.1%) [Fluoroquinolones are often recommended as a second-line treatment option for organism . Fluoroqfections , 47, 48.and parC . A numbeescribed , 49–51.  v 1.1%) , 49, 50.E. coli) [M. genitalium strain (M6282) that originated from a male with non-chlamydial NGU in Miyazaki, Japan [parC, however this strain has demonstrated in vitro phenotypic susceptibility to fluoroquinolones [M. genitalium-positive individuals in Melbourne, Australia and in 14.8% of M. genitalium-positive men-who-have-sex-with-men (MSM) in Alabama, USA [parC indicates that it may play a role in the evolution of fluoroquinolone resistance but current evidence suggests it does not confer phenotypic resistance [M. genitalium strains in the community, and this could explain the presence of QRDR mutations observed in M. genitalium from this study. They may also have been exposed to quinolones used as treatment for other indications such as urinary tract infections. Moxifloxacin is currently one of five second-line drugs used to treat multi-drug resistant Mycobacterium turberculosis (TB) in South Africa, but due to concerns of widespread unrestricted use in primary healthcare centres and consequently the development of moxifloxacin resistance in TB, it is unlikely to be included in future national STI treatment guidelines.The most commonly reported amino acid alterations associated with fluoroquinolone resistance are found at positions 83 and 87 of ParC (corresponding to positions 80 and 84 in E. coli) , 49–51. E. coli) , 49–52. i, Japan . The M62inolones . The P62ama, USA , 54. Thesistance . STI patgyrA and parC detected in this study and patient treatment outcomes. Patients presenting with genital discharge at primary health care clinics in South Africa are treated syndromically and they might not return to the same clinic for follow-up assessment and treatment if initial treatment failed to cure their infection. Antimicrobial susceptibility assays for M. genitalium are also rarely performed due to the technical difficulties in culturing M. genitalium from clinical specimens, and therefore attempts to associate phenotypic resistance with genotypic findings are lacking [M. genitalium-positive specimens that were stored at -70 °C. Unsuccessful amplifications were mostly observed in older specimens and may have been due to DNA degradation during storage or due to low bacterial loads of M. genitalium in these specimens. Carlson and Jensen (2010) demonstrated that freezing of M. genitalium-positive clinical specimens, as well as extracted DNA, leads to a significantly lower M. genitalium DNA load and a decreased sensitivity to that obtained by testing fresh specimens, especially in those with an initial low DNA load [Limitations of the study included the inability to determine the association between the presence of amino acid alterations in the QRDR of  lacking . AnotherDNA load . FinallyM. genitalium-positive patients presenting with urogenital discharge to the Alexandra Health Centre between 2007 and 2014 and among asymptomatic M. genitalium-positive, HIV-positive individuals attending an urban HIV outpatient clinic in Johannesburg in 2007. The low number of known fluoroquinolone resistance-associated mutations observed in the QRDR of M. genitalium in this study could strengthen the case for reverting to this class of antibiotic if macrolide resistance become widespread in South Africa. However, resistance to macrolides could emerge due to the incorporation of azithromycin in the 2015 South African national STI treatment guidelines. Countries with high-level macrolide resistance in M. genitalium could implement rapid M. genitalium and macrolide resistance testing at point-of-care, using commercial assays such as the SpeeDX Resistance Plus™ MG test to facilitate appropriate targeted treatment [M. genitalium positive cases is warranted and will be performed in our setting to support the continued use of azithromycin, which is included in the current national STI syndromic management guidelines.In conclusion, we report the absence of macrolide resistance-associated mutations in reatment . Ongoing


Lactobacillus paracasei, GCRL 46, we observed temporal up-regulation of proteins in the 40–45 kDa molecular weight range for whole-cell extracts when culturing in fermenters at a controlled pH of 4.0 versus optimum growth pH of 6.3. Up-regulation of proteins in this size range was not detected in SDS-PAGE gels of the cytosolic fraction, but was routinely detected following growth at low pH in whole cells and cell debris obtained after bead beating and centrifugation, indicating a cell surface location. N-terminal sequencing and in silico analyses showed sequence similarity with proteins in the L. casei group  which were variously annotated as uncharacterized proteins, surface antigens, possible TrsG proteins, CHAP -domain proteins or putative peptidoglycan d,l-endopeptidase due to the presence of a CwlO domain. This protein is a homologue of the p40 (Msp2) secreted protein of L. rhamnosus LGG, which is linked to probiotic functionality in this species, and is phylogenetically related to structurally-similar proteins found in Enterococcus, Streptococcus and Bifidobacterium species, including the glucan-binding (GbpB), surface antigen (SagA) proteins detected in pathogenic group A streptococci species as secreted, immunoglobulin-binding (SibA) proteins . Three-dimensional (3D) modelling predicted structural similarities in the CHAP proteins from the L. casei group and streptococcal species, indicating retention of overall architecture despite sequence divergence, and an implied retention of function during evolution. A phylogenetically-related hydrolase also contained the CwlO domain with a NLPC_P60 domain, and showed similar overall but distinct architecture to the CHAP proteins. We concluded that the surface-located, CHAP protein in L. casei is up-regulated during long-term exposure to acidic conditions during growth but not during acid shock.Bacterial cell wall hydrolases, including amidases and peptidases, play a critical role in peptidoglycan turnover during growth, impacting daughter cell separation, and cell death, through autolysis. When exploring the regulation of protein expression across the growth cycle of an acid-resistant strain of  Firmicutes, including Lactococcus, Enterococcus, Oenococcus, Pediococcus, Streptococcus and Lactobacillus. LAB have in common the ability to convert sugars into lactic acid either homo- or hetero-fermentatively . They occupy a variety of ecological niches in nature, notably associated with human and animal gastrointestinal tracts (GIT) and mucosal surfaces, and have been used traditionally in the production of fermented meat, plant and dairy products [Lactobacillus comprise one of the largest genera of the LAB, with 237 species currently reported in the list of prokaryotic names with standing in nomenclature (http://www.bacterio.net/lactobacillus.html). Several Lactobacillus species are used in food fermentations as starter cultures, which have a primary function of rapid acidification of the food matrix, while others occur as non-starter LAB (NSLAB) which develop as part of the food microbiota in maturing cheeses [Lactobacillus confer human health benefits [Lactobacillus species are associated with cell surface structures and surface plus secreted proteins [The gram-positive lactic acid bacteria (LAB) belong to several genera in the phylum products ,2. Fermeproducts ,4. Lacto cheeses . While c cheeses ,7, probibenefits ,9,10, wibenefits ,12. Manyproteins ,14,15. Tproteins  and deteLactobacillus consists of the peptidoglycan (PG) sacculus, which is made up of glycan chains containing N-acetylglucosamine and N-acetylmuramic acid that are attached to short peptides which cross-link to form a mesh-like structure. In addition to potential modification to the PG by O-acetylation or N-deacetylation, the surface can be decorated by teichoic acids and other surface polysaccharides [Lactobacillus also synthesize an S-layer, which is an outer microcrystalline protein sheath which has been implicated in probiotic function, including infection control and adhesion [The cell wall of charides . Some spadhesion ,17,18. Tadhesion . PG , in contrast to acid shock conditions where there is a considerable and established literature for LAB [Firmicutes and their bacteriophages, prophages or plasmids, with the p40/Msp2 homologue showing modelled structural similarities to Streptococcus homologues . The CHAP-CwlO protein was only detected in a limited number of Lactobacillus species. The PcsB protein has been proposed as a candidate for new pneumococcal vaccine development, given that it is essential for normal cell growth and is highly conserved in this genus [Lactobacillus species, will inform the discussion on this matter.While characterizing the acid tolerance of a dairy strain of  for LAB . One up-is genus . UnderstLactobacillus species using PCR primers described by Dubernet et al. [L. paracasei [L. rhamnosus and other L. casei group members [L. casei and L. paracasei using similar approaches is problematic [L. casei, ATCC 393, plus a small group of genetically-related strains, constitute a clade which the authors recommend remain designated as L. casei [L. casei and L. paracasei, form a distinct, separate clade (A) and it was recommended that all of these strains be renamed L. paracasei. Despite the majority of strains in this clade demonstrating >98% average nucleotide identity (ANI) similarity to each other, and to the L. paracasei type strain ATCC 25302 and ATCC 334 , the issue of nomenclature remains unresolved. In the present study, we use the names L. casei and paracasei synonymously and use the species names that have occurred in prior literature for consistency; broader in silico analysis against published genomes of the L. casei group members classified GCRL 46 in clade A and we refer to this strain as L. paracasei in the text.All strains were confirmed as t et al. ,32. GCRL members  differenblematic . Recent L. casei . Other sL. paracasei GCRL 46 in shake-flask culture using buffered media indicated the optimal pH for growth was 6.5 [L. acidophilus ATCC 4356 was selected to benchmark acid resistance of L. paracasei GCRL 46 as this strain has been reported as acid tolerant and showed greater resistance than L. casei strains tested in parallel [L. plantarum [Previous characterization of  was 6.5 : the maxparallel . Figure lantarum .L. paracasei GCRL 46 was harvested hourly from fermenters for cultures with set pH of 6.3 and 4.0 and proteins in whole cell preparations separated on SDS-PAGE . The corresponding whole cells, cell debris and the cytosolic fractions were compared on SDS-PAGE c. AlthouGiven that analysis of the cytosolic fraction would not have been useful in exploring the nature of the surface proteins upregulated by growth at low pH, proteins in whole cell lysates were separated by preparative SDS-PAGE and N-terminal protein sequencing performed. Although it was likely that the broad protein band contained several proteins, a clear N-terminal sequence was obtained, with only the amino acid in the eleventh position reported as equivocal: DTSDSIASNK(S/Q/D)ETNALLKQI.L. casei group species where the proteins were variously annotated as hypothetical (secreted) proteins, peptidoglycan hydrolases, surface antigens, possible TrsG proteins and analogues of the p40 surface antigen or secreted protein of L. rhamnosus (L. rhamnosus and L. casei/L. paracasei strains.The 20 amino acid (AA) N-terminal sequence was subjected to BLASTp in KEGG, using all three possible amino acids in the eleventh position: matched protein sequences were only detected when serine was in this position, with 17 hits in hamnosus a. The coL. casei strains GCRL 163 and MJA 12: a unique protein was detected in both genomes and this sequence was used in BLASTp to explore alignment with other Lactobacillus proteins. The phylogram for Lactobacillus species , where this was 36 AA. Other single amino acid substitutions were noted in the signal peptide, N-terminal and catalytic domain sequences, in addition to deletions and substitutions which accounted for MW differences.The N-terminal sequence was used to search the genome of  species b again s species b. WithinL. rhamnosus strains showed >88% sequence identity with the GCRL 163 protein. Phylogenetically the nearest neighbors to these CHAP-domain proteins were L. sakei and L. curvatus species (384–412 AA), although the alignment scores were 46–50%, indicating sequence divergence. The CHAP domain (pfam identifier PF05257) is commonly found in amidases, including peptidoglycan hydrolases, and is a member of the NLPC_P60 superfamily [d,l-endopeptidases, based on the presence of the CwlO motif (K21471). The CwlO motif is the only member of the cl25603 superfamily described as the uncharacterized N-terminal domain of peptidoglycan hydrolases: peptidoglycan lytic activity associated with the presence of this motif in proteins was originally described in Bacillus subtilis [The CHAP-domain proteins (416 AA) in the second, closely-related clade of erfamily . KEGG gesubtilis .L. rhamnosus proteins were distinct from the L. casei/L. paracasei proteins  so two clades were observed again. The L. casei NLPC_P60 proteins showed an alignment score of 32.6% with the CHAP hydrolases (L. casei strains), indicating some sequence similarity in the CwlO motif but divergence in the endopeptidase domain region, despite the CHAP domain being in the NLPC_P60 superfamily. Plasmid and bacteriophage proteins (818–825 AA) were distinguishable from the smaller CHAP- and NLPC_P60-domain endopeptidases, although they contained domains annotated as surface antigen or CHAP  [L. casei group, including GCRL 163, but not in all strains [L. casei group strains. Interestingly, strain JCM 8130 contained three genes for CHAP-domain proteins, LBPC_0020 , LBPC_0475 , LBPC_2403  as well as the gene specifying the CwlO-NLPC_P60 (LBPC_1969) protein. or CHAP , whether (46.6%) , also su strains , and exeL. casei group endopeptidases was similar  [Firmicutes (currently 2753 entries).Although the degree of sequence identity was low, the overall domain architecture of the  similar b, consisroteins) , which hL. acidipiscus, however, showed a different architecture: a region containing multiple LysM motifs (approximately 40 AA globular domain) followed the SP, with a conserved C-terminal CHAP domain, with lower hydrophobicity in the coding region beyond the signal sequence. In contrast, the architecture of the 825 AA bacteriophage protein lacked a SP and showed hydrophobic regions around AA 175–225 (noted as a periplasmic-specific motif in CDD searches) and across the three catalytic domains detected, indicating a transmembrane location but unlikely secretion.A CHAP-domain, glucan-binding protein in L. casei GCRL 163 as queries are shown in Streptococcus pyogenes and other Streptococcus species proteins, whereas for the NLPC_P60 protein was more closely related to similar proteins in Clostridium and Listeria species. When phylograms extended to the top 500 hits  and S. aureus domain structure was LysM-CHAP, similar to the L. acidipiscus domain architecture in Lactobacillus species, including L. acidophilus, L. plantarum, L. helveticus, L. reuterii, L. delbrueckii and L. johnsonii. In contrast, the NLPC_P60 domain, which is more widely spread in the Bacteria and occurs in proteins with a variety of functions and accompanying domains [Lactobacillus species in UniProt, including the L. acidophilus group. In the top 500 hits for this protein in a KEGG search using the full sequence of the protein, other genera detected included Streptomyces, Bacillus, Enterococcus but not Streptococcus species: while this observation may be explained by low sequence similarity and annotation failure, it suggests that the spread of genes, or their composite domains, through genera and species was different for the two cysteine peptidases.BLASTp searches using the sequences for the CHAP and NLPC_P60 proteins from the genome of 500 hits , the CHA domains , was detS. pneumoniae CwlO-CHAP protein, called PcsB , has been reported [S. mutans (GbpB) and S. agalactiae and homologous proteins occur in all Streptococcus and Lactococcus species , all with >99% confidence (probability that the sequence and template are homologous) for regions aligned with templates. Models for the L. casei CwlO-CHAP proteins were highly similar to the S. mutans and S. pyogenes proteins. However, the L. rhamnosus homologues showed variability in predicted architecture, despite multiple modelling over an extended period–often producing models with the coiled-coil regions bunched. L. rhamnosus (sequence from NCBI), where the latter shows the four features seen in streptococci structures but was distinct from the p40 model: the AA sequence differed by three AAs between the p40 protein in LGG and the multi-species CHAP-domain protein sequence, one in the signal sequence and two in the catalytic domain, so differences in the models cannot necessarily be explained by minor sequence differences. N-terminal differences in the models are in line with the observed size differences seen for the signal peptide , the key difference was the relative position of the catalytic region. Attempted modelling of another protein with an NLPC_P60 domain detected in the genome of L. casei  was not successful, as 54% of the sequence was predicted as disordered. The LysM-CHAP protein detected in L. acidipiscus, which lacked the CwlO motif, showed a catalytic region (green structure in model 10) but no parallel coiled regions, consistent with the domains detected.Although the general architecture of the modelled NLPC_P60 proteins showed features similar to the All of the proteins had high-confidence predicted binding sites for Zn and some for Mg, usually in the coiled regions; the NLPC_60 proteins had predicted metal binding also in the catalytic regions.Firmicutes, particularly in Streptococcus species [Lactobacillus species [Lactobacillus or other LAB in terms of protein synthesis during exposure to stress [L. casei has one of the highest numbers of predicted surface and secreted proteins in the genus Lactobacillus [Streptococcus species. In the present study, several proteins were observed as up-regulated when L. paracasei GCRL 46 was cultured in fermenters at low pH, or when the pH of the medium was decreased to 4.0 and sustained at this pH during culture, but similar changes were not detected when cells were temporarily shifted to low pH then returned to optimum (acid shock). An up-regulated, surface-located protein detected by N-terminal sequence analysis was variously annotated as a surface antigen, uncharacterized, possible hydrolase and TrsG protein in L. casei group species, L. casei, L. paracasei and L. rhamnosus, although the sequence identity between the L. casei/paracasei and L. rhamnosus clades was 88%. TrsG was originally detected in S. aureus plasmids as part of the transfer machinery [L. casei W56), which had previously been reported as one of the major secreted proteins in probiotic L. casei and L. rhamnosus strains [L. casei group, p75/Msp1, was characterized as a PG hydrolase by degradation of muropeptides [L. casei group species: knock-out mutants either failed to cause phenotypic changes in L. casei, due to redundancy, or were not generated in L. rhamnosus [L. casei BL23 for Msp2 (which were positive for Msp1) [L. rhamnosus LGG for gels loaded with 20 µg of purified protein [L. rhamnosus LGG, implicating this protein in PG turnover and daughter cell separation. Only one other PG hydrolase in the L. casei group has been functionally characterized as an autolysin from its mureinolytic activity [L. casei group species.How LAB respond to stressors has been the subject of increasing research at the proteomic and transcriptomic level over the last 20 years, due to the importance of understanding the underlying mechanisms of coping with potentially-lethal or growth-limiting assaults that are encountered during passage through the GIT or during food fermentations ,29. The  species , and the species ,27,49,50o stress ,29,51. Oo stress ,52. In sbacillus ,52. Howebacillus  noted thbacillus ,43. Furtbacillus  and multachinery  and cont strains ,26,27. Apeptides , the rolhamnosus ,26,27. F protein , indicatactivity , which iBacillus, Clostridium, Streptococcus and Listeria, and a signal peptide. Furthermore, these authors demonstrated that both p40 and p75 presumptive hydrolases had a surface and extracellular location by extraction of surface proteins with 1.5 M LiCl and detection by Western blotting using immune serum prepared against p40 and p75  with 34% similarity to the CHAP protein. Although the sequence similarity was <50%, p40 homologues were detected in a limited number of Lactobacillus species, including the most closely related sequence in strains of L. sakei and L. curvatus for proteins with the same overall domain layout and structural architecture as p40, indicating that p40 is not unique to the L. casei group. However, p40 homologues were not detected in the L. acidophilus group species although NLPC_P60-domain proteins were, as reported previously following in silico analysis of published genomes of L. acidophilus for detecting presumptive autolysins [L. casei/paracasei p40 homologue was phylogenetically most closely related to Streptococcus PcsB  proteins, whereas the CwlO-NLPC_P60 protein was more similar to Clostridium, Listeria and (with less similarity) Bacillus homologues. The difference in analysis by Bäuerl et al. [Lactobacillus and other genomes now sequenced and the differentiation between detecting the common CwlO domain in structures that lack the CHAP domain but may contain the NLPC-P60 domain. There are no published studies on the role of the CwlO-NLPC_P60 protein in L. casei group strains, including its possible role in PG turnover. The modelled structure of this protein showed similarities to the p40 protein which suggests putative PG hydrolase activity: the question of whether this enzyme, or other uncharacterized PG hydrolases, complement p40 activity so that inactivation of p40 would result in no phenotypic changes remains a matter for further exploration through transcriptomics, proteomics and physiological studies.Many of the features of the p40/Msp2 protein in the current report were originally described by Bäuerl et al. , who ideeviously ). These eviously  concludetolysins . The L. l et al.  and the Lactobacillus, Streptococcus, Staphylococcus, Listeria and other genera [L. casei group species, given the distinct phylogeny of the two classes of CwlO-containing proteins. This is supported by our observation for strain L. paracasei subsp. paracasei JCM 8130, which contained multiple genes encoding proteins with CHAP domains, including one with GC content indicative of recent acquisition but with high sequence similarity to the p40 CHAP domain detected in this study . It is likely that genes are exchanged regularly between phage and plasmids, as predicted in Bacillus species [L. casei is indicated from GC content similar to the average for the genome. The genomes of L. casei/paracasei consist of a core and variable regions which make up the pan-genome, indicative of a species adapted to specialized environments [L. rhamnosus LGG can modify during product storage indicating genetic rearrangements in genome islands containing insertion elements [Lactobacillus species and how probiotic bacteria exert their beneficial effects, including pathogen exclusion in species with a similar genetic makeup and PG hydrolase complement.The CHAP domain was also detected in proteins lacking the CwlO domain, but containing other domains including LysM in modular structures, particularly in prophage-related proteins with presumptive activities related to modification of the cell wall but lacking signal peptides. Bacteriophage endolysins are often large proteins with multiple modular domains, including CHAP, which occur in phage that infect or reside in r genera . It is l species , but ancronments ,58. Furtelements . UnderstB. subtilis, S. aureus and S. pneumoniae: the WalKR regulon includes PG hydrolases involved in cell separation, such as PcsB in S. pneumoniae (see ref 30). Although the PcsB protein is well characterized and contains the putative catalytic domains linked with PG hydrolase activity (CwlO-CHAP), demonstrating hydrolytic activity has not been successful [L. casei group strains showed high similarity with the Streptococcus PcsB/SibA/CHAP proteins PcsB, it is likely that members of this family of proteins will behave similarly in L. casei group and other low GC Gram positive species where the architecture of the proteins is conserved. The L. casei group p40 protein has not been crystallized nor has the mechanism of its regulation been explored, but it is likely that its location and regulation would be similar to Streptococcus. Accumulation of this protein at the cell surface during prolonged acid stress in GCRL 46 may suggest altered transcriptional regulation of expression and that p40 activity is insufficient to achieve cell separation alone. The CwlO-NLPC_P60 showed a similar, but distinct, architecture where the hydrolase ‘head’ was not in the same position relative to the coiled regions of the PcsB and p40 homologue. This may be an artefact of modelling which would be clarified by protein crystallization and analysis of the atomic distances to determine whether the FtsEX model would apply to this enzyme also. The SagA/PcsB proteins are considered targets for vaccine or new antimicrobial development, given the essential nature of this protein in pathogenic streptococci [The regulation of PG hydrolases in cells involves a crucial balance between transcription and post-translational control of enzyme activity, to provide sufficient expression of protein synthesis for PG turnover and cell separation without exerting the potentially lethal process of autolysis ,57. Regu see ref . Althoug see ref . Althoug see ref . AlthougL. acidophilus [Lactobacillus species, including L. casei group, lack an S-layer and the concentration of proteins in the extracellular medium and at the surface appears to be low [L. casei [L. casei in prior reports [Our prior work with GCRL 46 showed that LiCl-extractable proteins were altered following growth at low pH . The p40dophilus ,50,62: to be low ,63, hencL. casei . Failure reports ,48 is reLactobacillus were originally obtained as a gift from the Commonwealth Scientific and Industrial Research Organization (CSIRO), Food Science Australia laboratories  or purchased from the American Type Culture Collection . Type strains used were: L. acidophilus ATCC 4356, L. casei ATCC 393 and L. rhamnosus ATCC 7469. L. paracasei VUP 12006 was initially stored in the Victoria University, Werribee, VIC, Australia, culture collection [L. casei GCRL 163 was originally isolated from maturing Cheddar cheese [Strains of llection  and was llection . L. caser cheese  and its r cheese . StrainsLactobacillus genus-specific primers for ISRs (R16-1/LbLMA1-rev) were described by Dubernet et al. [L. acidophilus [L. casei [L. casei (IDL11F/IDL03R) and L. rhamnosus (IDL04F/IDL73R). PCR amplification conditions and amplicon separation techniques are reported in Pepper [https://blast.ncbi.nlm.nih.gov/Blast.cgi) or the BLASTn tool in Kyoto Encyclopedia of Genes and Genomes  to determine the closest matches with known 16S rRNA gene sequences. ClustalW (KEGG BLAST tools) was used for sequence alignment.Genomic DNA was extracted from overnight MRS broth cultures of all strains as described previously ,65 usingt et al. ; universt et al. , as modit et al. , were pudophilus  and PR1/L. casei ,70. PrimL. casei  were alsn Pepper  and Shahn Pepper . Amplicog, 10 min), concentrated 10-fold in MRS and vessels seeded aseptically, in duplicate for each test condition, to achieve an initial optical density (600 nm) (OD600) of 0.13–0.23 . The vessels were stirred at 150 rpm, sparged with nitrogen gas (0.1 L/min), with temperature and pH maintained at set points. In some experiments, the set pH was changed during growth or the pH control not initiated so the pH in the vessels decreased over time. Samples were normally removed hourly for the determination of dry weight, OD600, glucose concentration and collection of cells for later protein analysis. If not used immediately, all samples were stored at −21 °C. Growth rates were determined using the Monod equation [max), calculated automatically using the BASIC program first developed by Viega and Gutierrez [Culture was performed in MRS broth using fully-instrumented, water-jacketed one L Applikon fermenters , allowing continuous measurement and control of temperature at 37 °C (± 0.5) and pH values (± 0.2). Set pH values in the range of 2 to 8 were achieved using sterile 2 M HCl or NaOH as required prior to inoculation and by automatic addition during culture. Media were autoclaved  in the vessels and sparged with oxygen-free nitrogen to maintain anaerobic conditions. Oxygen levels were monitored throughout using an oxygen probe and the volume of acid or base dosed during growth recorded. Starter cultures were prepared from frozen stocks by seeding 20 mL MRS broth with two cryobeads or 50 µL glycerol stock culture and incubating 6–8 h at 37 °C before sub-culturing 10 mL of starter culture into 200 mL of fresh medium then incubating overnight at 37 °C without mixing. Cells were collected by centrifugation  with 10% or 12% running gels and 4% stacking gel, as described previously [600, 50 mL culture samples from fermenters were centrifuged, the supernatant removed and cells resuspended in a volume of Tris buffer  to concentrate to a standard OD600 equivalent to 15. A sample of cell suspension was added to an equal volume of double-strength sample buffer, mixed by vortexing and heated at 100 °C for 5 min, followed by centrifugation  to remove unbroken cells and debris. Equal volumes of each whole cell extract were loaded onto SDS-PAGE gels when comparing time-course samples. Alternatively, cells were disrupted by bead beating using a tissue homogenizer : an equal volume of concentrated cell suspension and glass beads  were placed in a stainless-steel homogenizer bottle and treated in 30 s bursts for a total 2 min, with cooling on ice. The homogenate was centrifuged to remove debris  and the supernatant fluid collected (cytosolic fraction). The cell debris was retained for SDS-PAGE analysis, resuspending in the original sample volume with single-strength sample buffer before heating at 100 °C for 5 min and brief centrifugation  to pellet beads. Protein concentration in the cytosolic fraction was determined by a modified Lowry assay [http://www.protocol-online.org/, accessed from August 1999 to 10 February 2019) and gels scanned using a Gel-Pro analyzer  to determine the molecular weight and relative density of the proteins. Gel images were also scanned using UN-SCAN-IT Gel Analysis software 7.1 for relative density (% pixels) and molecular weight estimation .One dimensional SDS-PAGE was performed according to the method of Laemmli  using veeviously . After rry assay  against v/v, methanol, pH 11.0) for 5 min and electroblotted to polyvinylidene difluoride (PVDF) membranes [® Cell  according to the supplier’s instructions. Bands were visualized using Amido Black 10B , small samples of membrane excised and sent to the Australian Proteome Analysis Facility (APAF)  for N-terminal sequencing.Proteins in whole cell extracts were applied to SDS-PAGE gels in the PROTEAN II XL system and electrophoresed until the 31.5 kDa size standard had reached the bottom of the gel, allowing greater separation of proteins in the size range of 40–50 kDa. Gels were soaked in transfer buffer  [https://www.ebi.ac.uk/Tools/msa/clustalo/) for pair-wise and multiple sequence alignment. Domain analysis employed the NCBI conserved domain database (CDD) [https://img.jgi.doe.gov/, most recently accessed on 10 February 2019). Guanine-cytosine (GC) content was calculated using the Endmemo DNA/RNA GC content calculator .BLASTp (KEGG) of the N-terminal protein sequence, using all three combinations of the possible identity of the eleventh amino acid, was used for initial sequence similarity searches. This sequence was also used to search the annotated genomes of 1000000)  to obtaise (CDD) , the KEGse (CDD) . Phylogese (CDD) ,81 with se (CDD) . The layhttps://web.expasy.org/protscale/, most recently accessed on 10 February 2019) with window size of 9. The Phyre2 web portal for protein modelling, prediction and analysis [The UniProt platform  was usedanalysis  was emplanalysis . Models analysis  with def


The objective of this study was to investigate pretreatment systemic inflammatory response (SIR) markers in patients who underwent initial intravesical treatment for high-risk non-muscle invasive bladder cancer (NMIBC). A total of 385 patients who underwent initial intravesical Bacillus Calmette-Guerin treatment after transurethral resection of bladder tumor (TURB) were included. We analyzed the relationship between oncological outcomes and ratios of SIR markers, including neutrophil-to-lymphocyte ratio (NLR), derived neutrophil-to-lymphocyte ratio (dNLR), and platelet-to-lymphocyte ratio (PLR). Each SIR marker was used for analysis. Their cut-off values were determined through receiver operation characteristics curves analysis. Patients were divided into two groups according to pretreatment NLR (<1.5 vs. ≥1.5), dNLR (<1.2 vs. ≥1.2), and PLR values (171< vs. ≥171). Patients with NLR ≥ 1.5 and dNLR ≥ 1.2 were associated with poor prognosis in terms of overall survival and cause-specific survival. However, no serum SIR marker was associated with prognosis in recurrence-free survival or progression-free survival. Cox multivariate analysis revealed that age, NLR, dNLR, hemoglobin, and pathologic T stage were significant factors predicting overall survival. Age, NLR, and pathologic T stage were significant factors predicting cancer-specific survival, NLR and tumor number were the most important predictors of bladder preserving survival. NLR before treatment was correlated with both oncological outcomes and survival outcome in NMIBC patients undergoing initial intravesical BCG treatment after TURB. Increased NLR reflects a poor prognosis of these outcomes. Bladder cancer is the 9th most commonly diagnosed cancer worldwide and the 13th most frequent cause of cancer death worldwide .in situ (CIS), and Ta low grade tumor with multiple, recurrent, and large (>3 cm) tumor (in situ (CIS) are at high risk of recurrence and progression . Ninety percent of UC cases originate in the bladder. Of these, 75% are diagnosed as non-muscle invasive bladder cancer (NMIBC) with mucosal or submucosal confined disease , 4. NMIBd large > cm tumorm) tumor . Patientgression . The stagression , 3. In agression . These sgression . Pretreagression . In bladTo prevent recurrence and rapid progression, we performed intravesical Bacillus Calmette-Guerin (BCG) therapy. In 1976, Morales et al. confirmed the efficacy of BCG therapy for bladder cancer patients and then for NMIBC patients , 6. IntrThe Institutional Review Board of Seoul National University Hospital Clinical Research Institute approved this study . This was a retrospective study. It was exempt from the requirement of patient written consent. The study protocol and all related items followed the Declaration of Helsinki guidelines. We retrospectively reviewed medical records of 3,127 patients who received TURB at Seoul National University Hospital from 1991 to 2015. Patients who underwent initial intravesical BCG treatment after TURB were included. There was a difference in treatment duration from 1991 to 2015. Indication and recommended method of intravesical BCG treatment also differed for different ages. Therefore, we retrospectively included patients who underwent intravesical BCG treatment among patients who met our defined high-risk group criteria. All patients were high-risk NMIBC patients with T1, high-grade tumor, or CIS. Patients without UC, with short follow-up (<2 years), and those who were suspected of infections accompanied by fever or leukocytosis were excluded. Finally, 385 patients were included.Pretreatment systemic inflammatory response markers were investigated. We collected clinicopathological information from patient's medical records at the time of initial intravesical treatment after TURB. Clinicopathological information included age, sex, body mass index (BMI), hypertension, diabetes, lung disease, absolute neutrophil, lymphocyte, and platelet counts, hemoglobin, pre-intravesical treatment NLR, dNLR, and PLR values, creatinine, estimated glomerular filtration ratio (eGFR), pathological T stage, tumor grade, CIS, lymphovascular invasion (LVI), tumor number, and tumor size. We also collected various oncologic prognoses and outcomes, including upper urinary tract recurrence, recurrence in bladder, history and timing of radical cystectomy, progression, cancer-specific mortality, and overall mortality. SIR markers calculated included neutrophil-to-lymphocyte ratio , derived neutrophil-to-lymphocyte ratio [dNLR (absolute neutrophil count/white blood cell count) – neutrophil count], and platelet-to-lymphocyte ratio .We performed TURB and intravesical treatment according to established principles of our hospital. Specimens resected by TURB were histologically examined and reviewed by two experienced urological pathologists. They determined the TNM stage and tumor grade according to the 2010 American Joint Committee on Cancer classification and the 2004 World Health Organization/International Society of Urologic Pathology consensus classifications principles. Almost all patients were followed-up every 3 months for 3 years first. They were then followed up every 6 months for 2 more years (year 5) and annually thereafter. At each follow-up, basic laboratory tests, urine analysis, urine cytology, and cystoscopy were performed. Computed tomography (CT) was performed annually to check progression and upper tract recurrence. Intravesical BCG treatment was performed. The BCG schedule was as follows: BCG induction therapy (weekly BCG instillation for the first 6 weeks) and maintenance therapy for up to 3 years .P < 0.1 in univariate analysis. By identifying explanatory variables that were significantly related to response variable, dozens of variables were considered one at a time. A less restrictive level (P < 0.1) is often used to identify many explanatory variables that can be associated with response variables in univariate analysis. Thus, we used a limited number of variables with P < 0.1 in the univariate analysis for the multiple regression model. We used receiver-operating characteristic (ROC) curve to find appropriate cut off values for SIR markers. The optimal cut-off value was selected as the point at which the sensitivity and specificity were maximized for the prediction of oncologic outcomes. In the ROC curve analysis , dNLR , and PLR  showed predictive power to distinguish OS. Continuous variables are presented as mean values with standard deviation (SD) while categorical variables are presented as ratio of events (%). Statistical significance was defined at p ≤ 0.05. IBM SPSS Statistics version 22.0  was used for all statistical analysis.The primary end point was overall survival (OS). Secondary end points were recurrence-free survival (RFS), progression-free survival (PFS), bladder preserving survival (BPS), and cancer-specific survival (CSS). Kaplan-Meier survival analysis and log rank test were used for all survival analyses. Cox proportional hazard regression analysis was used to analyze various independent predictors of oncological outcomes. Univariate analysis was used for various factors related to patient characteristics and tumor characteristics before initial intravesical BCG treatment. Multivariate analysis was performed only for variables with A total of 385 high risk NMIBC cases were diagnosed after TURB and initial intravesical BCG treatment was performed. The mean follow-up time was 80 months [Standard deviation ± 50.7 months]. Most (84.9%) patients were males. Nearly half (48.3%) of these patients experienced recurrence and 11.4% of patients had disease progression. Radical cystectomy was performed for 13.2% of patients. The overall mortality rate was 18.2%. Cancer-specific mortality rate was 4.7%. The mean time to recurrence was 43 months while the mean time to progression was 31 months. Median values of SIR markers were: NLR, 1.86 (IQR: 0.26–3.50); dNLR, 1.36 (IQR: 0.22–3.09); and PLR, 113.25: (IQR 35.3–200.8) , CSS , and BPS . However, no serum SIR marker was associated with prognosis in terms of RFS or PFS. Unexpectedly, PLR did not help predict any oncologic or survival outcome in patients with intravesical BCG treatment , dNLR (<1.2 vs. ≥1.2), and PLR values (171< vs. ≥171). Patients with NLR ≥ 1.5 and dNLR ≥ 1.2 were associated with poor prognosis in terms of OS .Important predictors of OS in multivariate analysis were age, NLR, dNLR, Hb, and pathologic T stage Table . Importap = 0.04 and p = 0.034, respectively) and CSS . However, there was no statistically significant relationship of NLR or dNLR with BPS, PFS, or RFS. PLR was not significantly associated with oncologic survival outcomes  and radical cystectomy. These studies suggest that NLR before radical cystectomy may help predict tumor prognosis –21. The Unlike previous studies, our results showed that pretreatment NLR or dNLR did not increase the presence of tumor grade, number, size, concomitant CIS, or LVI. This may be due to insufficient patient pool. In addition, our study was performed on patients after TURB. In mechanistic view, advanced stage and aggressiveness may be associated with increased host immune response. However, in this study, all patients were NMIBC patients with low tumor burden after TURB. Such patient status seems to be difficult to reflect characteristics of the tumor.This study has several limitations. First, it was a retrospective study of a single institution. Thus, it cannot be free from selection bias. Second, the timing of pretreatment NLR measurements was not exactly the same. Third, a single test does not represent the entire systemic immune response state. Fourth, values and confidence intervals of the AUC of the ROC curves cannot show solid results to establish these values as cutoff points with sufficient prediction.In conclusion, NLR before treatment was correlated with both oncological outcomes and survival outcome in NMIBC patients undergoing initial intravesical BCG treatment after TURB. Increased NLR reflects poor prognosis of these outcomes. Although large-scale prospective studies are needed to apply NLR to clinical practice, NLR will be a useful tool to help improve risk assessment and treatment guidelines for intravesical treatment in high-risk NMIBC patients.HY, CJ, HK, and JK contributed to the concept and project planning. HY, CJ, and CK contributed significantly to the writing of this paper. JK contributed significantly to the editing of the final draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salmonella enterica serovar Dublin strains, 03EB8736SAL and 03EB8994SAL, isolated from raw-milk cheese and milk filtrate, respectively. Analysis of the draft genomes of the two isolates reveals the presence of the viaA, viaB, and ompB loci of the Vi capsular polysaccharide antigen (Vi antigen).Here, we report the genome sequences of two  Salmonella enterica serovar Dublin strains, 03EB8736SAL and 03EB8994SAL, isolated from raw-milk cheese and milk filtrate, respectively. Analysis of the draft genomes of the two isolates reveals the presence of the viaA, viaB, and ompB loci of the Vi capsular polysaccharide antigen (Vi antigen).Here, we report the genome sequences of two  Salmonella enterica serovar Dublin is adapted to cattle and can be transmitted to humans via the consumption of contaminated raw milk and raw-milk cheeses  , 5 genes for Vi antigen export , and open reading frame 11 (ORF11). The ompB locus contains two regulatory systems (rscB-rscC and ompR-envz), and it controls the regulation of Vi polysaccharide synthesis  is commonly found in strains of human-adapted typhoidal aratyphi . It contaratyphi , 8. The . Dublin , 9, it i. Dublin , 10. Exp (SPI-7) . The via species . The viaSalmonella Dublin according to the White-Kauffmann-Le Minor scheme (https://www.pasteur.fr/sites/default/files/veng_0.pdf). Genomic DNA was extracted using the QIAamp DNA minikit . The quality of DNA was checked using gel electrophoresis, and DNA quantity was determined using the Qubit quantification platform . Genomic DNA libraries were prepared using the Nextera XT library preparation kit , following the manufacturer’s protocol. Whole-genome sequencing (WGS) of multiplexed libraries was carried out on the Illumina HiSeq platform using a 250-bp paired-end protocol. The total number of reads for each S. Dublin isolate is provided in The two strains, 03EB8736SAL and 03EB8994SAL, were identified as belonging to http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter sequences were removed using ea-utils package version 1.04.807 . Sequencing data were de novo assembled using SPAdes version 3.11  (http://quast.bioinf.spbau.ru/). The assembly data  for each S. Dublin isolate are provided in viaB locus of the Vi antigen were determined using BLASTn version 2.2.25  was used as a reference. Moreover, analysis of harbored plasmid/s within the draft genome of the two S. Dublin isolates, 03EB8736SAL and 03EB8994SAL, was performed using PLSDB with search strategy Mash screen, and the default values were a maximum P value of 0.1 and minimum identity of 0.99 (https://ccb-microbe.cs.uni-saarland.de/plsdb/) http://wwn 2.2.25  with abon 2.2.25  was used/plsdb/) . Interes/plsdb/) .S. Dublin strains might play an important role in the ability of bacteria to cause invasive illness in humans. Further studies will be carried out to evaluate the expression of Vi antigen and determine the exact role of Vi antigen in S. Dublin virulence and pathogenesis.The presence of Vi chromosomal loci within nontyphoidal S. Dublin isolates, 03EB8736SAL and 03EB8994SAL, have been deposited in the European Nucleotide Archive (ENA) under project number PRJEB30372. The raw sequence reads are available under accession numbers ERS3015385 and ERS3015387, and the draft genome sequences are available under accession numbers ERS3015643 and ERS3015644, respectively.The raw sequence reads and draft assemblies of the two