Escherichia coli (ESBL-E. coli) may occur as outbreaks due to common-source exposures. Yet, it is currently unknown if they cluster geographically as would be expected as part of an outbreak.Urinary tract infections caused by extended-spectrum beta-lactamase (ESBL)-producing E. coli bacteriuria in a safety-net public healthcare system from January 2014 to March 2020 . We assessed the presence of spatial clusters of (1) ESBL-E. coli bacteriuria episodes, and (2) individuals with any ESBL-E. coli bacteriuria episode, with Global and Local Moran’s I. We evaluated differences in prevalence of bacteriuria recurrence by ESBL-production by Poisson regression.We collected electronic health record data on all patients living in San Francisco with culture-documented community-onset E. coli bacteriuria episodes (n = 461) compared to non-ESBL-E. coli bacteriuria episodes . Spatial clusters of individuals with any bacteriuria caused by ESBL-E. coli were not identified (p = 0.43). Bacteriuria recurrence was more likely to occur with ESBL-E. coli , particularly after an initial ESBL-E. coli bacteriuria episode .Out of 4,304 unique individuals, we identified spatial clusters of ESBL-E. coli bacteriuria episodes. However, this was partly explained by clustering within individuals more than between individuals, as having an ESBL-E. coli bacteriuria was associated with recurrence with ESBL-E. coli. These findings may help better tailor clinical treatment of patients with recurrent urinary tract infections after an initial episode caused by ESBL-E. coli.We found spatial clusters of ESBL-The online version contains supplementary material available at 10.1186/s13756-023-01320-1. Escherichia coli (ESBL-E. coli) bacteriuria episodes, but not at the individual level. ESBL-E. coli was more likely to cause recurrent bacteriuria.• In a large public healthcare system, from 2014 to 2020, we found spatial clusters of all extended-spectrum beta-lactamase-producing The online version contains supplementary material available at 10.1186/s13756-023-01320-1. Escherichia coli (ESBL-E. coli) infections has been well established with increasing prevalence in community settings [E. coli [E. coli in close geographic vicinity of one another which would suggest possible outbreaks.In 2019, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae were identified as “serious threat” pathogens in a Centers for Disease Control and Prevention (CDC) report on antimicrobial resistance [settings . Novel r[E. coli –15. Thus[E. coli . Indeed,[E. coli –20. HoweE. coli detected within a large urban public healthcare system from 2014 to 2020. We compared the frequency of recurrent bacteriuria by ESBL-production as potential clusters could be explained by bacteriuria recurrence in the same individual. Our goal was to provide new insight into spatial clustering of these infections in community settings that could inform public health surveillance efforts and help reduce drug-resistant pathogen transmission in the community.Here, we sought to identify spatial clusters of community-onset bacteriuria episodes caused by ESBL-E. coli bacteriuria receiving care from the San Francisco Health Network and San Francisco General Hospital, a safety-net public healthcare system, from January 2014 to March 2020. This healthcare system includes 15 primary care clinics and an acute care hospital and serves a multiethnic, low-income, and under-studied population residing in various San Francisco neighborhoods. The hospital microbiology laboratory conducts all laboratory testing for the entire system. Bacteriuria episodes, representing either urinary tract infection or asymptomatic bacteriuria, were defined as a single urine culture growing E. coli on a unique date. If multiple cultures were sent on the same day, only one culture was considered, to avoid artificially inflating the number of bacteriuria episodes. Urine cultures from separate days were considered to be separate episodes, as we could not differentiate between an untreated and a repeat infection. Recurrent bacteriuria episodes were defined as > = 2 E. coli bacteriuria episodes in the last 6 months or > = 3 in the last year [E. coli bacteriuria episodes only, defined as cases in which a urine culture, obtained in (a) an outpatient clinic or emergency department setting, or (b) within 48 h of inpatient admission, grew E. coli. We excluded patients who were hospitalized within 90 days of the urine culture or whose urine was sent for culture 48 h after inpatient admission. We excluded patients with a documented residence outside of San Francisco County or those living at the public skilled nursing facility. This study was approved by institutional review boards from UCSF and SFGH (IRB number 19-27233).This is an observational study focused on patients with culture-proven ast year . For thiE. coli bacteriuria cases identified from January 2014 to March 2020 by the UCSF CTSI data abstraction services. We extracted data on the urine culture organism as well as antimicrobial susceptibility testing (AST). The microbiology laboratory performs AST with Microscan and disk diffusion tests, reporting resistance based on CLSI breakpoint standards [E. coli as an E. coli strain resistant to ceftazidime or cefotaxime and inhibited by clavulanic acid using broth microdilution, per 2016 CLSI guidelines [E. coli was the main outcome of interest. Other variables included age at time of culture ; gender (women or men); self-identified race and ethnicity ; preferred language ; and insurance type . Patients were considered as Latine if they identified having Latine ethnicity regardless of specified race. While analyses did not focus on differences by race and ethnicity, we conceptualize race and ethnicity as a marker of differential environmental exposures driven by cultural mores such as diet and travel.Data were extracted from electronic health records (EHR) for all tandards . The micidelines  BacteriuE. coli bacteriuria episodes versus non-ESBL-E. coli bacteriuria episodes at multiple levels: (1) episodes and (2) individuals. Here, we define spatial clusters of ESBL-E. coli bacteriuria episodes as clusters of all infections, including repeat and recurrent infections, in the same individual or in individuals living near one another as a result of repeat common-source exposures. Spatial clusters of individuals with any ESBL-E. coli bacteriuria episodes during the study period would represent clusters of any such infection in different individuals living near one another or at the same residential address due to common-source exposures within a community.As part of system-wide efforts, addresses listed in the EHR in 2019 of all patients receiving care in the public healthcare system were geocoded to latitude and longitude coordinates using ArcGIS Business Analyst 2016 (ESRI) . We condE. coli bacteriuria episodes in the same individual within the same month were treated as the same episode. Analyses at the individual level were also stratified by race and ethnicity  and preferred language  to identify potential differences in exposure due to patient sociodemographic characteristics. The parameters for Global Moran’s I included Euclidean distance, inverse distance for conceptualization of spatial relationships, and no standardization of spatial weights. When global clusters were identified, local clusters were assessed with Local Moran’s I, with inverse distance bands, to understand their contribution to the global clustering statistic. We applied a false discovery rate (FDR) correction which adjusts the p-value to control for multiple testing and spatial dependence [Sensitivity analyses were conducted separately for study periods 2014–2016 and 2017–2020 to identify possible differences in spatial clustering given changes in guidelines for ESBL identification in 2016 . In addipendence , 26. WheE. coli vs. non-ESBL-E. coli, for individuals with an initial ESBL-E. coli bacteriuria episode vs. non-ESBL-E. coli bacteriuria episode was assessed by Poisson regression models, adjusting for age category, gender, race and ethnicity, and use of antibiotics in the last 6 months. Analyses were conducted by RStudio4 version 4.0.4. We report 95% confidence intervals to characterize uncertainty in our effect estimates.Descriptive statistics, including frequencies and percentages for categorical data, were used to summarize variables. The likelihood of recurrent bacteriuria, (1) overall and (2) with ESBL-E. coli was identified in 9,028 (67%) isolates . There were 6,291 unique patients with an E. coli bacteriuria episode. Of the 5,576 patients who met the definition of a community-onset E. coli bacteriuria, 4,304 had a valid San Francisco address. Our analyses include 5,938 community-onset E. coli bacteriuria episodes from those 4,304 unique patients with a San Francisco address. Most patients were between the ages of 35 and 64 (46%) , compared to non-ESBL-E. coli episodes. In Local Moran’s I models, with inverse distance band, we found evidence of local spatial autocorrelation of ESBL-E. coli bacteriuria episodes (high-high clusters) in the neighborhoods of SOMA/Tenderloin, the Mission, and Excelsior. Sensitivity analyses excluding bacteriuria episodes within the same month also identified global and local spatial clusters (p < 0.001), even when correcting with false discovery rate.All 5,938 E. coli bacteriuria episode vs. unique individuals with only non-ESBL-E. coli bacteriuria episodes (p = 0.43), meaning that there was no clustering between different individuals with any ESBL-E. coli bacteriuria episodes. We did not identify such spatial clusters of individuals with any ESBL-E. coli bacteriuria vs. only non-ESBL-E. coli in analyses stratified by racial and ethnic groups  or preferred language . Sensitivity analyses for years 2014–2016 and 2017–2020 showed no spatial clusters. We restricted data to densely populated neighborhoods, where many patients receiving care within this healthcare system live and where evidence of spatial clustering had been found when mapping all episodes. No evidence of spatial clustering of individuals with any ESBL-E. coli bacteriuria vs. individuals with any non-ESBL-E. coli bacteriuria for each neighborhood separately was identified .Using the same parameters, we did not identify spatial clusters of unique individuals with any ESBL-E. coli bacteriuria episodes were identified (p < 0.0001) vs. those with recurrent non-ESBL bacteriuria episodes and single bacteriuria episodes.Spatial clusters of individuals with recurrent ESBL-E. coli bacteriuria episodes, 220 (4%) represented recurrent episodes  non-recurrent episodes caused by ESBL-E. coli bacteriuria. Twenty-three (58%) recurrent episodes caused by ESBL-E. coli occurred after an initial ESBL-E. coli bacteriuria episode, compared to 8 (4%) recurrent episodes caused by ESBL-E. coli that occurred after an initial non-ESBL-E. coli bacteriuria episode. In Poisson regression models, recurrence was more likely to occur with ESBL-E. coli than non-ESBL E. coli  and initial ESBL-E. coli bacteriuria episodes as part of recurrent episodes were more likely to be followed by ESBL-E. coli . Moreover, with Asian American patients as the reference group, Black or African American patients  and White patients  had increased odds of recurrent bacteriuria episodes. Latine patients , White patients , and patients identifying as other  had greater odds of having a recurrent bacteriuria episode caused by ESBL-E. coli.Of all 5,938 E. coli causing bacteriuria. We detected evidence of spatial clusters of ESBL-E. coli bacteriuria episodes within a large urban safety net healthcare system, with data spanning 6 years. Such clusters were not replicated when mapping unique individuals with any ESBL-E. coli bacteriuria episode during the study period. In further analyses, we found that patients with an initial ESBL-E. coli bacteriuria episode were more likely to have recurrent bacteriuria episodes, particularly caused by ESBL-E. coli. Thus, the spatial clusters of ESBL-E. coli bacteriuria episodes may be explained by bacteriuria recurrence in the same patients. This suggests the lack of evidence of local geographic E. coli sources affecting multiple individuals. However, it may be that certain individuals may be exposed to the same E. coli sources, leading to recurrent bacteriuria episodes.To our knowledge, this is the first report identifying spatial clusters of community-onset ESBL-E. coli causing urinary tract infections in urban areas in the West of Ireland [Few reports to date have assessed the presence of spatial clusters of infections caused by ESBL-producing Enterobacteriaceae and all have used different methods to report such clusters. In a 2-year study based on community-onset infections, Sarda et al. found spatial clusters of ceftriaxone-resistant Enterobacteriaceae isolates by census tracts from a single healthcare center in Cook County, Illinois . Patient Ireland  Yet, mosE. coli, as suggested by studies finding infection or colonization with fluoroquinolone-resistant E. coli to be associated with neighborhood-level fluoroquinolone consumption [Enterobacteriaceae were located close to rivers and hospitals [E. coli bacteriuria episode were more likely to have recurrent episodes, particularly with ESBL-E. coli. While others have reported such recurrence patterns, they have not focused on community-onset infections [There are multiple factors that could explain spatial clustering of drug-resistant organisms. First, geographical patterns in antibiotic prescribing or consumption, at the neighborhood-level, may drive selection of drug-resistant sumption , 29, 30.ospitals  Third, afections , 32.E. coli bacteriuria episode [E. coli bacteriuria episodes we found may be entirely explained by recurrent ESBL-E. coli bacteriuria. Yet, it is also feasible that ESBL-E. coli bacteriuria episodes are caused by a variety of E. coli genotypes representative of multiple common source exposures throughout San Francisco. Mapping specific genotypes, as opposed to drug-resistant phenotypes, would help elucidate this hypothesis. Relatedly, Nobrega et al. reported spatial clusters of bloodstream infections caused by ST131-C2 subclade in a North East Calgary sector without long-term care facilities [E. coli. [E. coli did not occur in spatially distinct patterns given the widespread food distribution in metropolitan areas through large supermarket chains.When we conducted sub-analyses by race and ethnicity and preferred language, given historical segregation and the presence of ethnic enclaves in San Francisco, we found no evidence of spatial clusters of individuals with any ESBL- episode . Sub-anacilities . ExposurE. coli. , 35 ThusE. coli bacteriuria from a large urban healthcare system caring for diverse populations, our study has several limitations. First, we report data from one healthcare system, which limited our sample size and may have resulted in selection bias. Future studies may include other healthcare systems in San Francisco to better identify spatial clusters involving the entire prevalence of uropathogenic ESBL-E. coli in San Francisco. Second, we did not have access to pathogen genotype data, as it is not routinely collected for clinical management purposes, and thus we could not assess spatial patterns of specific genotypes producing ESBL. We did not conduct spatial clusters analyses of antibiotic susceptibility phenotypes as a proxy for genotype for the following reasons: (1) genotypes may harbor or lose mobile genetic elements which may change antibiotic susceptibility phenotype and (2) our study was not powered to assess spatial clustering of the various antibiotic susceptibility phenotypes. Third, we used patients’ last known geocoded address as of 2019, which may have resulted in some geographic misclassification. This may have led to a small margin of error, as about 10% of the population within the most represented neighborhoods in our study moved within the same county, and about 5% moved from a different county throughout the study period [E. coli spatial prevalence due to sample size. Indeed, sensitivity analyses conducted for years 2014–2016 and 2017–2020 did not identify any ESBL-E. coli bacteriuria episode spatial clusters. Finally, our data, as any other healthcare system-based data, were limited to individuals who had a processed urine culture. However, current IDSA guidelines do not recommend routine urine culture testing in all patients with symptoms suggesting urinary tract infection [Although this is the first multi-year study evaluating the presence of spatial clusters of community-onset ESBL-y period . Fourth,nfection . As suchE. coli bacteriuria episodes among patients in community settings. However, analyses among unique individual did not show such clusters, suggesting that the clusters we initially found may be explained by the recurrence of ESBL-E. coli bacteriuria in the same individuals. These findings have important implications in understanding the epidemiology of ESBL-E. coli and in the clinical treatment of patients with past ESBL-E. coli bacteriuria, such as the greater odds of recurrent bacteriuria after an initial episode caused by ESBL-E. coli. Future work will include identification of spatial clusters of genotypes as such level of information would be more adequate markers of common-source exposures.In analyses spanning 6 years at a large urban safety-net healthcare system, we found spatial clustering of ESBL-Below is the link to the electronic supplementary material.Supplementary Material 1


Objectives: Ancillary staff members perform operational support functions and play an active role in enhancing the patient care experience. Infection prevention practices among ancillary staff play a critical role in preventing transmission of microorganisms, which ensures the safety of patients. Low hand hygiene compliance was found among porters in a cross-institutional hand hygiene audit in 2021. A quality improvement team was formed to improve hand hygiene compliance, especially during the COVID-19 pandemic. Methods: A focus-group discussion and survey were conducted to understand hand hygiene knowledge and challenges among porters. Using the findings, the team initiated Glo–germ education tools, pocket alcohol hand-rub agents, pocket moisturizer, poster display, and a toolbox messaging system via conversion of group roll call to satellite-area roll call. Respective satellite teams were sent hand hygiene reminders, and prompt corrective action was taken following noncompliance events. Analytic comparisons of pre- and postsurvey data were performed using the χ2 test, and P < .05 was regarded as statistically significant. Results: In total, 572 ancillary staff participated in the survey. Knowledge of hand hygiene practices improved significantly following the interventions, as shown in the comparison of pre- and postintervention results: knowledge of the hand hygiene steps (P < .001), knowledge of the duration of hand rub (P < .001), and knowledge of duration of handwashing (P < .001). Also, 295 staff members (97.68%) stated that implementation measures increased their awareness of the importance of hand hygiene. Moreover, the hand hygiene compliance rate improved from 77.8% to 100%. There were no significant differences related to sex (P = .089), age group (P = .355), years of working (P = .359), education level (P = .268), or difficulty in reading English (P = .906). Conclusions: Evaluating staff hand hygiene knowledge and understanding the challenges faced among porters helped toward the development of appropriate interventions and assurance of success in project.


Staphylococcus aureus is a gram-positive spherical bacteria and the most common cause of nosocomial infections in the world. Given its clinical significance, the genome sequence of S. aureus has been elucidated to enhance our comprehension of its lifestyle and pathogenicity. The research aimed to summarize a potential hypothetical protein that may play an important role in S. aureus virulence and pathogenicity, covering its anticipated structure, probable biological functions, and importance in this context.S. aureus, was chosen for analysis and modeling by several bioinformatics tools and databases in this work. According to primary and secondary structure analyses, YP_498675.1 is a stable hydrophilic protein with a significant proportion of α–helices. Subcellular localization predictions by CELLO, PSORTb, and SOSUI server indicate that it is a cytoplasmic protein. NCBI-CDD, Pfam, and InterProScan functional genomics research revealed that the hypothetical protein may include the pyridoxal phosphate (PLP)-dependent 2, 3-diaminopropionate biosynthesis protein SbnA domain. In the homology modeling method, the HHpred server was employed to create its 3D structure using the template structure of a Staphyloferrin B precursor biosynthetic enzyme SbnA bound to PLP (PDB ID: 5D84_A), an X-ray diffraction model having 100% sequence identity with the hypothetical protein. After energy minimization, several quality assessments and validation factors determined that the generated protein model was reliable and of reasonable quality.A hypothetical protein, YP_498675.1 with 281 amino acid residues of S. aureus. Further experimental validation would aid in determining the actual function of YP_498675.1 as well as confirm the protein’s value as a therapeutic target.The present study has characterized and functionally annotated the hypothetical protein YP_498675.1 of  S. aureus have been there since the beginning of time, they were first discovered as a disease causing agent in the nineteenth century. In 1880, Alexander Ogston first observed grape-like clusters of bacteria in pus from a surgical abscess in a knee joint and named them Staphylococcus [taphylococcus aureus is a gram-positive, spherical bacterium with a diameter of around 1 μm, which are responsible for a wide range of clinical illnesses [S. aureus carriers [.S. aureus is one of the main causes of hospital and community-acquired infections, which can result in serious consequences [S. aureus diseases. S. aureus can also lead to bone, joint, and endovascular diseases [S. aureus can result in ventilator assisted pneumonia as well as central venous catheter-associated bacteremia. Moreover, it causes serious deep-seated infections, such as endocarditis and osteomyelitis [S. aureus frequently causes toxin-mediated diseases such as toxic shock syndrome, scalded skin syndrome, and staphylococcal foodborne illnesses (SFD) [S. aureus (MRSA) that may lead to septicemia, pneumonia, and dermatitis [S. aureus demands new strategies for treating infections caused by this pathogen. One potential avenue is the development of a vaccine or drug targeting a HP unique to S. aureus, which could help to overcome the limitation posed by antibiotic resistance. By thoroughly characterizing the properties and function of a HP, researchers can gain crucial knowledge about its potential as a vaccine candidate and explore its effectiveness in eliciting an immune response capable of combating S. aureus infection.Scientists may gather massive volumes of data in a relatively short period of time using next-generation sequencing (NGS). As more organisms are being sequenced, the challenge of assigning functions to genes is increasing , 2. In mlococcus . In 1884lococcus . Staphylllnesses . It has carriers . The mosequences . Circuladiseases . Infectimyelitis . Along wrmatitis . The emeS. aureus measures about 2.82 Mp in size, with a mean GC content of 32.90%. It has so far been discovered with 2872 genes, and 2767 proteins. To date, a total number of 1511 proteins of S. aureus have been identified with no known function. About half of the genomic proteins in reference strain S. aureus NCTC 8325 are hypothetical [S. aureus was used in this work since its structural characteristics are unknown, but its core amino acid sequence is known. The goal of this study was to investigate the physiochemical and secondary structural characteristics of the putative S. aureus protein (YP_498675.1), construct its first three-dimensional (3D) model through homology modeling, and conduct functional and comparative genomics research using Basic Local Alignment Search Tool for proteins (BLASTp) and multiple sequence alignment (MSA) analysis. The current study aims to enhance our understanding of the functional roles performed by members of the staphylococci community, thereby offering valuable insights into potential therapeutic targets.The genome of S. aureus was sourced from National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) database. Initially, approximately 1500 HP from S. aureus were chosen for subsequent in silico analysis. Exclusion criteria were applied to eliminate HP with amino acid sequences shorter than 50 residues, as proteins below this length are known to exhibit compromised folding characteristics. Out of the initial pool of 746 HP with amino acid sequences longer than 50 residues, their protein physicochemical properties were analyzed using ProtParam tool. Approximately 405 HP were excluded from further analysis due to exhibiting unstable characteristics according to ProtParam results. The remaining stable HP (341) underwent subcellular localization analysis using CELLO v2.5, PSoRTb, SOSUI, and PSLpred. HP that consistently displayed the same result across all the four tools were then selected for protein domain and motif prediction. After undergoing subcellular localization analysis and subsequent protein domain and motif screening steps, a total of 94 HP were rejected from further analysis. In the subsequent step, we employed homology modeling to predict the three-dimensional (3D) structure of HP. HP displaying less than 80% sequence similarity to any published protein structure are rejected for downstream analysis. At this stage of screening, we were able to delimit the HP number to 39. These predicted models were then subjected to protein quality assessment to evaluate their reliability and accuracy. As a result of this screening process, the number of HP was successfully reduced to below ten, as several models did not meet the quality assessment criteria and were therefore excluded from further consideration. From this narrowed-down selection, we carefully considered the clinical significance and function of all predicted HP, and the best-performing HP, YP_498675.1, consisting of 281 amino acids, was chosen as the representative for our manuscript. The overall workflow of this screening process is depicted in Fig. The proteomic data of https://web.expasy.org/protparam/) tool was used to characterize HP in terms of their physicochemical features. Parameters such as molecular weight, aliphatic index (AI), extinction coefficients, amino acid composition, grand average of hydropathy (GRAVY), isoelectric point (pI), and estimated half-life were analyzed.The ExPASy ProtParam  (https:/http://cello.life.nctu.edu.tw/) [https://www.psort.org/psortb/) [https://harrier.nagahama-i-bio.ac.jp/sosui/mobile/) [https://webs.iiitd.edu.in/raghava/pslpred/submit.html) [The putative subcellular localization of the HP (YP_498675.1) was determined by CELLO v.2.5 (edu.tw/) , an analpsortb/) , SOSUI (mobile/) , and PSLit.html) . SOSUI dhttps://structure.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [http://pfam.xfam.org/) [http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=iprscan5&sequence=uniprot:KPYM_HUMAN) [https://www.genome.jp/tools/motif/) InterProscan tool. Pfam is a protein family database that uses hidden Markov models (HMMs) in order to generate annotations and multiple sequence alignments. To determine the protein sequence motif, MOTIF Search (https://www.genome.jp/tools/motif/) tool was used [NCBI Conserved Domain Search (NCBI CD-Search) (psb.cgi) , Proteinam.org/) , and IntM_HUMAN)  were usewas used .https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) [https://clc-sequence-viewer.software.informer.com/8.0/) was employed to perform multiple sequence alignment and generate a phylogenetic tree for a specific subset of sequences.In order to identify the homologs of the HP (YP_498675.1), a protein-BLAST (BLASTp) (roteins)  from NCBhttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) [http://bioinf.cs.ucl.ac.uk/psipred/) [Two-dimensional structure of the YP_498675.1 protein was determined using SOPMA  (ma.html)  and PSI-sipred/) . Result https://toolkit.tuebingen.mpg.de/tools/hhpred) [HHpred server (/hhpred)  was used/hhpred) .https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/) [https://servicesn.mbi.ucla.edu/Verify3D/) [https://swissmodel.expasy.org/qmean/) [Structural evaluations of the protein model were performed by using several programs called PROCHECK (OCHECK/) , Verify rify3D/) , ERRAT [rify3D/) , and Qua/qmean/)  programs/qmean/) . The acc/qmean/) . A score/qmean/) . ERRAT s/qmean/) . These aThe 3D structure of the hypothetical protein YP_498675.1 was refined by performing YASARA energy minimization server . In ordehttp://sts.bioe.uic.edu/castp/) server was used to find out the ligand binding sites of the hypothetical protein YP_498675.1. CASTp obtains the topographical features of a protein in a detailed, comprehensive, and quantitative manner. CASTp predicts active pockets located on protein surfaces and in the interior site of the 3D structure, the regions and key residues of protein which interact with ligands. As a result, it has become an essential tool for predicting regions and key residues of protein which interact with ligands [Computed atlas of surface topography of proteins (CASTp) -dependent enzyme at the position of 1 to 254 amino acid residues with an E-value of 1.5e-46.NCBI-CD Search, Pfam, and InterProScan annotation tools were used to identify conserved domains and potential function of the YP_498675.1. The specific hit explored by conserved domain (CD) search tool predicted the query protein belongs to tryptophan synthase beta superfamily (fold type II) (Try-synth-beta_ II). Protein of this family is pyridoxal phosphate (PLP)-dependent enzyme covers 1 to 269 amino acid residues with an Staphylococcus species (Table Staphylococcus species (WP_000570813.1 and WP_0000808.1) and Escherichia coli (HAI9356092.1) appear to have common ancestor with the WP_047424351.1 and WP_047530432.1 proteins of S. schweitzeri. The scale bar estimates sequence divergence, and amount of genetic change is represented by the line segment with the number (0.015).The BLASTp search against the non-redundant database showed homology (up to 100% sequence similarity) with other known type of tryptophan synthase beta superfamily protein from different es Table . A totales Table . Using tes Table . The tarThe query sequence was submitted into the HHpred server for protein homology detection and structure prediction . The 3D PROCHECK, Verify 3D, QMEAN, and ERRAT were used to evaluate the quality of our modeled 3D structure. To validate protein models, the program PROCHECK employs several evaluation metrics, including the generation of a Ramachandran plot and the analysis of torsion angles, surface areas, bond angles, and atomic distances. These calculations play a crucial role in assessing the structural integrity and accuracy of protein models . AccordiThe identification and characterization of active site residues are key steps in the design of a drug or inhibitor. The CASTp server was used to assess the active site of the model structure, as well as to determine the active site amino acid residues. The top active sites of the model protein were determined in one of the largest pockets using the area of 1198.087 and the volume of 1046.218 amino acids. According to CASTp prediction, the model protein’s active residues are shown in . The spectrum of SAg-mediated diseases included from relatively benign food poisoning to life-threatening toxic shock syndrome (TSS). The major secreted SAgs of S. aureus include TSS toxin 1 (TSST-1) and enterotoxin (SE) serotypes A to Q, excluding F [S. aureus, but in clinical trials, no vaccine candidates have been found to be effective. Characterization of HPs YP_498675.1 of S. aureus can aid in understanding bacterial metabolic regulations, formulating disease control strategies, and developing effective therapeutics. Various computational resources were employed in this study to characterize the hypothetical protein YP_498675.1 of S. aureus from structural and functional aspects. The physiochemical properties’ analysis revealed that the protein consists of 281 amino acid sequence, have a molecular weight of 30,872.44, the grand average of hydropathicity (GRAVY) score of − 0.119, and a theoretical PI of 5.78  biosynthesis. SbnA and SbnB together appear to synthesize 2, 3-diaminopropionate, a precursor of certain siderophores and other secondary metabolites [S. aureus and other related organisms  and motif (PLP-dependent enzyme), providing clues about its putative biological roles. By thoroughly examining YP_498675.1, we have expanded our knowledge regarding its putative involvement in vital cellular processes and interactions. Our research advances knowledge of the genetic and proteomic profile of S. aureus, identifying putative targets for development of a drug or vaccine against this pathogenic bacterium. While this study represents a crucial initial steps towards the functional significance of YP_4986675.1, it is warranted to conduct further experimental validation and functional characterization to confirm the predicted structural and functional attributes. Nonetheless, our comprehensive in silico analysis lays a solid foundation for future research, offering valuable insights into the potential roles of YP_4986675.1 and its implications with the realm of S. aureus physiology and pathogenesis.Our study delved into the in silico structural and functional annotation of a novel hypothetical protein YP_498675.1, derived from


Interestingly, the natural quinone-based compounds even reduced the symptom severity of the necrotrophic fungi, i.e., the grey mold pathogen B. cinerea and the Southern Leaf Spot fungus C. heterostrophus, to some extent. The urease inhibitors p-BQ, HQ, and DCC interfered with appressorial penetration and confirmed the appropriateness of urease inhibitors as novel fungicidal agents.The development of new anti-ureolytic compounds is of great interest due to the newly discovered role of urease inhibitors in crop protection. Purine degradation and the generation of ammonium by urease are required for the full virulence of biotrophic and hemibiotrophic fungal plant pathogens. Accordingly, chemicals displaying urease inhibitor activity may be used as a novel class of fungicides. Several urease inhibitors belonging to different chemical classes are known, and some compounds have been developed as urea fertilizer additives. We tested whether the natural urease inhibitors  Coccioides posadasii and the basidiomycete Cryptococcus neoformans, and in bacteria like Helicobacter pylori, Proteus mirabilis, and others [H. pylori is associated with the occurrence of gastritis and gastric cancer. H. pylori grows well at pH values of 6 to 8 but does not survive at pH values below 4 [H. pylori infection. The role of the urease in pH adjustment and tissue damage was not only found in H. pylori. Studies employing the human pathogenic fungi C. posadasii and C. neoformans suggested a conserved role of urease in bacterial and fungal pathogenesis. In these pathogens, ammonia-mediated cell damage is required for lung invasion, inhibition of phagocyte function, and for accessing the bloodstream. After getting trapped in small capillaries, urease-generated ammonia again causes cell damage and allows the fungus to grow from the capillary, propagate, and cause severe damage in the host tissue [C. neoformans were significantly reduced in virulence, and higher concentrations of antigen-presenting cells were found in the brains of mice inoculated with the urease-positive strain compared with the control animals infected with urease-negative strains [Urease has been identified as an important virulence factor in several mammalian pathogens, including dimorphic fungi such as the ascomycete d others . Great i below 4 . The aci below 4 ,4. Moreot tissue ,6. Accor strains ,7,8. How strains . For fur strains .Agrobacterium tumefaciens-mediated random mutagenesis experiments have identified the allantoicase gene of the maize anthracnose fungus Colletotrichum graminicola as a virulence factor [ure1 deletion strains [C. graminicola WT strain and strengthened the idea that chemical inhibitors of urease activity may represent a novel class of agents protecting plants from fungal disease [While the role of urease in human pathogens can be considered well-understood, little is known about the virulence function of this enzyme in plant pathogens. e factor  and sugg strains . Moreove strains , reduced disease .The plant surface is extremely low in nitrogen ,13, and Uromyces viciae-fabae secretes the plant cell wall-degrading enzyme polygalacturonate lyase when haustorial mother cells are differentiated in the host, i.e., immediately at the site of host cell invasion. This enzyme has an extremely alkaline pH optimum of higher than 10 [The role of urease in pH increases in plant-infecting pathogens may also be considered. The broad bean rust fungus  than 10 , and incUrease inhibitors belonging to several chemical groups  are wideZea maize cv. Mikado; KWS SAAT SE, Einbeck, Germany), winter wheat , broad bean , and on strawberry (Fragaria x ananassa cv. Ostara) fruits. Experiments were performed in growth chambers  at 22 °C. Light intensities were ~140 μmol/m2/s, except for Z. mays, which was grown at ~300 μmol/m2/s at 25 °C). Strains and infection experiments for Blumeria graminis f. sp. Tritici, Uromyces viciae-fabae, and Colletotrichum graminicola were as described [B. graminis f. sp. tritici is a field single-spore-isolate (BgtEV01) collected at the Experimental Station of Martin Luther University, and maintained on susceptible T. aestivum cv. Winnetou plants were grown in a greenhouse at 21 ± 1 °C. The broad bean rust fungus, U. viciae-fabae, isolate I2, was provided by Kurt Mendgen, University of Konstanz, Germany. The wild-type strain CgM2 (syn. M.1.001) of C. graminicola was from the strain collection of Ralph L. Nicholson, Purdue University, IN, USA, and maintained as described [Cochliobolus heterostrophus (lab strain C12) was provided by Stefan Wirsel  and the grey mold fungus Botrytis cinerea was from Paul Tudzynski . C. heterostrophus and B. cinerea were kept on Potato Dextrose Agar .Infection experiments were performed on leaves of maize  [p-benzoquinone (p-BQ), hydroquinone (HQ), isopropoxy carbonyl phosphoric acid amide (iCPAA), benzyloxy carbonyl phosphoric acid amide (bCPAA), and dipropyl-hexamino-1,3-diphosphacenium chloride (DDC). To test the effect of urease inhibitors on powdery mildew infection, 1, 5, or 10 mM of the inhibitors were sprayed onto leaves until run-off and allowed to dry for 2 h prior to inoculation. To analyze the effect of the compounds on rust infection structure differentiation in vitro, urediniospores of U. viciae-fabae were suspended in 0.02% (v/v) Tween 20. The inoculum was adjusted to contain 1, 5, or 10 mM of the inhibitors and sprayed onto thigmo-inductive polyethylene sheets [C. graminicola, detached maize leaves were inoculated with 10 µL droplets of a suspension containing 106 conidia/mL 0.02% (v/v) Tween 20 and incubated at 23 °C in darkness. The development of disease symptoms was scored and photographed daily. To analyze appressorial penetration rates by microscopy, leaves were inoculated with 10 µL droplets of a suspension containing 105 conidia/mL 0.02% (v/v) Tween 20, and penetration rates were counted at 48 hpi [B. cinerea infections, strawberry fruits were surface sterilized by incubation in 70% ethanol for 3 min, followed by three washes in sterile water. Spray inoculation with B. cinerea to the point of run-off was performed with 104 spores/mL 0.02% (v/v) Tween 20. To analyze the effect of urease inhibitors, p-HQ, HQ, iCPAA, bCPAA, and DDC were added to the conidial suspensions at a concentration of 10 mM. Inoculated strawberries were kept in a climate chamber at 23 °C, 50% relative humidity, and a 12 h photoperiod. For C. heterostrophus infections, 10 µL of a spore suspension (104 spores per ml of 10 mM urease inhibitors in 0.02% (v/v) Tween 20) were droplet-inoculated onto detached leaf segments as described for C. graminicola. For phytotoxicity tests, 10 mM solutions of each compound were sprayed onto leaf segments or fruits. Plants were incubated as described above, and the formation of chloroses or necroses was checked daily until 5 days post-treatment (dpt).For urease inhibitor treatment, spore suspensions of Germany) , or 1, 5t 48 hpi . For B. Canavalia ensiformis) urease  dissolved in 0.1 M K-phosphate buffer, pH 7.3. To quantify urease activity, liberation of ammonium from urea was determined spectrophotometrically at λ620 nm, using the Berthelot method in a two-step reaction with hypochlorite and salicylate [v/v) free chlorine) in 0.5 M NaOH and 0.1 M salicylic acid in 1.0 M NaOH were added, and color development was measured after 15 min of incubation at room temperature. The urease inhibitors were used at concentrations of 1, 5, or 10 mM.To test the efficiency of the inhibitors, in vitro activity assays were performed using Jackbean  microscopy was performed with a Nikon Eclipse 600 microscope . Digital images were taken using a DS-5M camera . Image processing was done with the software package Lucia 4.61 . Image analysis was performed using ImageJ .p ≤ 0.01 for the plant infection assays or p ≤ 0.05 for the urease activity test. Significant differences were indicated by asterisks in the results.All plant infection assays were performed as three independent replicates. Urease activity assays also represent data from three independent measurements. Statistical significance was analyzed by applying the Tukey post hoc test with one-way analysis of variance at either Since the plant surface is essentially devoid of nitrogen, fungi have developed lifestyle-specific strategies to access the nitrogen of their host plants. The pre-penetration infection structures of biotrophic and hemibiotrophic fungi develop in the absence of external nitrogen . TherefoUrease inhibitors used in this study A exhibitAt 12 days post-inoculation (dpi), dense powdery mildew pustules had formed on non-treated wheat leaves A. MicrosImportantly, the phytotoxic effects of the inhibitors used in this study were not observed, as on leaves treated with 10 mM concentrations, neither chloroses nor necroses occurred. As an example, inhibitor-treated maize leaves are shown .To investigate the effect of urease inhibitors on another biotrophic fungus, we employed the broad bean rust fungus U. viciae-fabae. Like most rust fungi, U. viciae-fabae deposits an appressorium on a stomatal pore  to invadAs rust hyphae developing in the intercellular space are difficult to visualize, polyethylene sheets with thigmo-inductive ridges  were inoTo investigate whether the urease inhibitors tested here would protect maize leaves from anthracnose disease, we added 1, 5, or 10 mM of each substance to the infection droplet. In the absence of urease inhibitors, the WT strain of C. graminicola caused significant disease symptoms at 3 dpi A, arrowsTo test for the stability of the urease inhibitors on the leaf surface, we applied the compounds by spraying the entire leaf surface with 10 mM concentrations to the point of run-off, and droplet-inoculated the leaves five days after this treatment with conidial suspensions of C. graminicola. Disease symptoms were analyzed macroscopically and appressorial penetration rates were quantified microscopically at 3 dpi . Maize lThe effect of urease inhibition on fungi with a necrotrophic lifestyle, i.e., the Southern Leaf Spot fungus C. heterostrophus and the grey mold fungus B. cinerea , were tep-BQ also reduced the disease symptoms caused by C. heterostrophus on maize leaf segments, with mild water-soaked lesions visible at 48 hpi (Colletotrichum or Magnaporthe species are spatially separated from the nutrient resources of the host [B. cinerea and the maize pathogen C. heterostrophus. The putative urease inhibitors used in this study had previously been identified as compounds affecting urea transformation in soils. We took advantage of these in vitro inhibitor studies to test the disease-protecting activities of five different inhibitors belonging to different chemical classes. Using commercially available jackbean urease, the urease inhibitory activity of the chemistries used was demonstrated, three of which were described as urease inhibitors for the first time in the present study. AHA, which like DDC was a strong inhibitor of jackbean urease activity in vitro, protected plants from infection by all the biotrophs and the hemibiotrophs tested here. Interestingly, the phosphoric acid amides, iCPAA and bCPAA, effectively inhibited jackbean urease at 10 mM concentrations in vitro, but only iCPAA interfered with wheat powdery mildew and broad bean rust infection. Neither iCPAA nor bCPAA affected the hemibiotroph C. graminicola nor were these chemistries effective against the necrotrophs tested. Reversely, HQ was moderately inhibitory in the urease activity assay only at the highest concentration tested but like p-BQ, effectively inhibited mildew, rust, and C. graminicola infections. These apparent discrepancies between inhibitory effects on purified jack bean urease and activity against the ureolytic bacterium Klebsiella pneumoniae have also been observed in a large screen comparing 71 commercially available urease inhibitors [C. heterostrophus and B. cinerea, leading to the hypothesis that, in contrast to biotrophic and hemibiotrophic fungal pathogens, necrotrophs gain immediate access to the host nitrogenous compounds by killing host cells prior to invasion. Like AHA, neither DDC nor iCPAA or bCPAA were able to inhibit infection by these pathogens. Symptoms caused by B. cinerea and C. heterostrophus, although to different extents, appeared to be slightly reduced by HQ and p-BQ. Despite the reduced severity of disease symptoms at the early stages of pathogenesis, microscopy revealed that these necrotrophic fungi had invaded the host tissues, and severe symptoms occurred with some delay. The differences in the efficacies of quinone-based compounds and the other inhibitors tested may be due to the peculiar mode of action of p-BQ and HQ [Botrytis spp. generate ROS, which kill host cells and cause major tissue damage well in advance of the growing hyphae. The protective effect of radical scavengers, such as quinone-based chemistries against ROS, has been extensively discussed and reviewed elsewhere [p-BQ- and HQ-treated maize leaves and strawberry fruits by necrotrophs may indeed be due to the ROS-scavenging effect of these compounds.Plant pathogenic fungi exhibiting a biotrophic or hemibiotrophic lifestyle gain access to the nitrogen of their host plants only after establishing a compatible parasitic interaction ,31. Apprthe host . Therefothe host . Implicihibitors . It is vhibitors . The resQ and HQ . These cQ and HQ . In planlsewhere ,35,36, aC. graminicola, the powdery mildew fungus B. graminis f. sp. tritici, and the rust fungus U. viciae-fabae with four out of five different inhibitors belonging to different chemical classes has been shown to reduce disease symptoms. In comparison, the inhibitors only slightly affected infections by the necrotrophic pathogens B. cinerea and C. heterostrophus, demonstrating that the inhibitory effect strongly depends on the fungal lifestyle.Overall, the role of urease in the early stages of infection, as evidenced by previous gene deletion studies , was conThe discovery of new fungicide targets has significant relevance as more and more active ingredients are banned, resulting in reduced numbers of pesticides exhibiting distinct modes of action. It is widely accepted that reducing the range of registered fungicides may lead to the rapid development of resistance to the few remaining fungicides. It is also accepted that chemistries with three distinct modes of action are needed to avoid the development of fungicide resistance . Importa


Trichoglottis exhibits a range of rich variations in colors and shapes of flower and is a valuable ornamental orchid genus. The genus Trichoglottis has been expanded by the inclusion of Staurochilus, but this Trichoglottis sensu lato (s.l.) was recovered as a non-monophyletic genus based on molecular sequences from one or a few DNA regions. Here, we present phylogenomic data sets, incorporating complete plastome sequences from seven species (including five species sequenced in this study) of Trichoglottis s.l. (including two species formerly treated as Staurochilus), to compare plastome structure and to reconstruct the phylogenetic relationships of this genus. The seven plastomes possessed the typical quadripartite structure of angiosperms and ranged from 149,402 bp to 149,841 bp with a GC content of 36.6–36.7%. These plastomes contain 120 genes, which comprise 74 protein-coding genes, 38 tRNA genes, and 8 rRNA genes, all ndh genes were pseudogenized or lost. A total of 98 (T. philippinensis) to 134 (T. ionosma) SSRs and 33 (T. subviolacea) to 46 (T. ionosma) long repeats were detected. The consistent and robust phylogenetic relationships of Trichoglottis were established using a total of 25 plastid genomes from the Aeridinae subtribe. The genus Trichoglottis s.l. was strongly supported as a monophyletic group, and two species formerly treated as Staurochilus were revealed as successively basal lineages. In addition, five mutational hotspots  were identified based on the ranking of PI values. Our research indicates that plastome data is a valuable source for molecular identification and evolutionary studies of Trichoglottis and its related genera. Trichoglottis Blume is a genus of Aeridinae within the family Orchidaceae, which was established by Carl Blume in 1825 with the type species Trichoglottis retusa Blume [Trichoglottis are mainly distributed in tropical Asia and the northwest Pacific [Trichoglottis are mainly characterized by monopodial growth, an axillary inflorescence with one or several brightly colored flowers, and a hairy strap-shaped lip firmly fused to a column [Trichoglottis exhibits a range of rich variations in colors and shapes of flower and is a valuable ornamental orchid. To date, approximately 75 artificial interspecific hybrids have been produced and registered with the Royal Horticultural Society . Pacific . Many sp Pacific ,4. SpeciTrichoglottis, the intergeneric phylogenetic relationships of this genus have remained unclear and are generally considered to be one of the most complicated groups in Aeridinae. Molecular phylogenetics, based on two markers (matK and ITS), indicated that Trichoglottis is sister to Staurochilus, and both together are sisters to Ceratochilus and Ventricularia [Trichoglottis has been expanded by the inclusion of Ceratochilus, Staurochilus, and Ventricularia [Staurochilus versus Trichoglottis is still debated. Based on three combined markers , Staurochilus was revealed as a sister to Acampe [atpI-H, matK, psbA-trnH, and trnL-F), the results showed that Trichoglottis was polyphyletic, Staurochilus (including S. luchuensis and S. ionosma) was paraphyletic, and T. latisepala was embedded in Staurochilus [Trichoglottis. Furthermore, the use of DNA barcoding for analysis has limitations in terms of resolution, making it difficult to differentiate between Trichoglottis and Staurochilus.Since the establishment of icularia . Trichogicularia . Howevero Acampe . Additiorochilus . NeverthTrichoglottis species. These new sequences are compared with the two previously described Trichoglottis plastomes [Trichoglottis s.l.) include two species (T. ionosma and T. philippinensis) formerly placed in Staurochilus. Simple sequence repeats (SSRs) were used to investigate the genetic diversity of these plastomes. Codon usage analysis was conducted to find the codon bias. The sequence divergence was calculated for barcoding investigation. Further, a total of 25 previously sequenced plastomes representing all previously recognized major lineages of Aeridinae were integrated for phylogenetic analysis with the aim of understanding the generic delimitation of Trichoglottis s.l. The present results provide a useful genetic resource for molecular identification and evolutionary studies of Trichoglottis and its related genera.One traditional approach for increasing the resolution in molecular phylogenetic data sets is to sample more loci ,10. Plaslastomes , which  to 149,841 bp (T. ionosma), which fell within the typical range observed for common angiosperm plastome sizes. The GC content of whole plastomes exhibited minimal variation. The T. ionosma had a content of 36.6%, and the remaining six species exhibited a content of 36.7%.The plastomes of tructure , consisttructure . The sizTrichoglottis encoded 120 genes, comprising 74 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes (ndh genes (T. ionosma possessed seven pseudogenes (ndhB/C/D/E/G/J/K), the other six species possessed eight pseudogenes (ndhB/C/D/E/G/I/J/K). The result of Mauve analysis revealed no significant rearrangements among these plastomes (The plastomes of A) genes . All thedh genes , Table 1lastomes .Trichoglottis , the ycf1 gene of T. latisepala and T. philippinensis was entirely located within IRb, while, for the other five species, the ycf1 gene spanned from IRb to SSC, with a range of 11 to 41 bp. In all species, the ycf1 gene spanned the junction between the SSC and IRa (JSA), and the trnH and psbA genes were found adjacent to the junction between the IRa and LSC (JLA).Visualization of boundary genes in the plastomes revealed a highly conserved distribution pattern among oglottis . The rplTrichoglottis. A total of 98 (T. philippinensis) to 134 (T. ionosma) SSRs were detected (T. subviolacea (2). Additionally, it was observed that A/T (48–90) dominated over C/G (0–2) in mononucleotide repeats. In dinucleotide repeats, the number of AT/AT (10–16) exceeded that of AG/CT (4). Furthermore, trinucleotide repeats exclusively contained A/T.The different types of SSR  were analyzed in the seven plastomes of detected . MononucTrichoglottis plastomes. The number of long repeats ranged from 33 (T. subviolacea) to 46 (T. ionosma) , the other six species possessed all four types of long repeats. The length of long repeats in all species mostly ranged from 30 bp to 40 bp. For long repeats above 50 bp, only T. ionosma had one, while the other species possessed two. Complement repeats were only detected below 40 bp in length.Four types of long repeats  were also detected in ionosma) . Except Trichoglottis plastomes. These protein-coding genes encoded 19,285–19,351 codons. The heatmap showed that the codon usage bias was highly conserved . The results showed Pi values ranging from 0 to 0.12857 (GUUtrnN-rpl32)  . The IR ervation . Among tTrichoglottis formed a well-supported monophyletic group  and was revealed as a sister to Acampe. The phylogenetic relationship inferred by complete plastomes showed strong support within Trichoglottis , but relatively moderate support was shown in trees inferred by 68 protein-coding genes . The phylogenetic trees showed that Trichoglottis could be divided into four diverging lineages. T. ionosma (formerly treated as Staurochilus ionosmus) was supported as the first lineage. T. philippinensis (formerly treated as S. philippinensis) was supported as the second lineage. T. subviolacea was supported as the third lineage. Four species, T. lanceolaria, together with T. latisepala, T. cirrhifera, and T. orchidea, were supported as the fourth lineage. Extremely short branch lengths were observed within the fourth lineage.The phylogenetic trees constructed based on complete plastomes and 68 protein-coding genes revealed similar topological structures, and the main clades were recovered with high-level support . The speTrichoglottis and provided a valuable opportunity to further understand plastome evolution in this complex taxon. All plastomes of Trichoglottis  to 149,841 bp (T. ionosma), falling entirely within the reported range of plastomes in Orchidaceae, which ranged from 19,047 bp (Epipogium roseum) to 212,688 bp (Cypripedium tibeticum) [Gastrodia flexistyla) to 37.8% (Cypripedium macranthos) [In this study, we expanded plastome sampling in oglottis  displayelastomes  ranged fbeticum) ,17. The ranthos) ,18.Trichoglottis encoded a total of 120 genes, including 74 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes, with the pseudogenization or loss of all ndh genes  were completely lost and the other seven genes (ndhB/C/D/E/G/J/K) were pseudogenes; in T. ionosma, three genes (ndhA/F/H) were completely lost and the other eight genes (ndhB/C/D/E/G/I/J/K) were pseudogenes in the other six species. Species of Trichoglottis are usually epiphytic on tree trunks or lithophytic on rocks. Therefore, the results provide support for a close association between pseudogenization or loss of ndh genes and epiphytic habitats.In terms of gene content, dh genes , Table 1hidaceae ,20,21. Ihidaceae . Epiphytndrobium , Polystaystachya , and Aerystachya ,15. For Trichoglottis plastomes was extremely conserved; except for a slight difference at JSB, the ycf1 in T. latisepala and T. philippinensis was entirely located within the IRb region, while in the remaining species, the ycf1 crossed over JSB. These results indicated that no significant expansion or contraction occurred in the IR regions of Trichoglottis, which may be one of the primary factors contributing to the high conservation of plastome structure.IR/SC boundary shift is a common phenomenon in the evolutionary process of angiosperms and serves as the main factor for the difference in plastome length and gene content ,26. In tT. philippinensis) to 134 (T. ionosma) SSRs were detected, which is more than previous results [T. subviolacea, making it a specific molecular marker for identifying T. subviolacea. The number of long repeats ranged from 33 (T. subviolacea) to 46 (T. ionosma), which is similar to previous studies [Trichoglottis.Simple sequence repeats (SSRs), widely distributed in plastomes, are effective molecular markers used for detecting genome rearrangement, species identification, and analyses of genetic differences among individuals ,28. A to results ,29,30,31 results . Most SS results . Additio studies ,29,30. TTrichoglottis. Leucine (Leu) had the highest amino acid frequency, while cysteine (Cys) had the lowest frequency. Knill et al. [Relative synonymous codon usage (RSCU) is an important measure to calculate the preference for synonymous codon usage. When the RSCU value is greater than 1, it indicates a preferred selection of that codon, whereas when it is less than 1, there is no preference . The codl et al.  proposedl et al. . Our resl et al. ,29,30,31Trichoglottis s.l. has remained unresolved for a long time. Based on nuclear ribosomal ITS and a number of plastid sequences, a conflicting relationship between Staurochilus and Trichoglottis s.s. was revealed, but weak support and unstable topology were found [Trichoglottis s.l. was strongly supported as a monophyletic group, and two species, T. ionosma and T. philippinensis, formerly treated as Staurochilus, were nested as successively basal lineages  were identified as mutation hotspots and could be used in the phylogenetic and identification of Trichoglottis and its related genera.Nucleotide diversity (Pi) was used to measure the degree of polymorphism. Similar to other Orchidaceae taxa, the non-coding regions of  regions ,29,31. ATrichoglottis were selected, among which five plastomes were newly sequenced in this study. Plant materials were collected from Fujian Agriculture and Forestry University . Based on previous molecular systematic studies [Calanthe, Calypso, Cattleya, Masdevallia, and Tridactyle) as the outgroups. The taxa with voucher information and GenBank accession numbers are provided in Seven species of  studies , a totalTotal DNA was extracted from fresh leaves with the Plant Mini Kit  based on the manufacturer’s protocol, and DNA degradation and contamination were evaluated on 1% agarose gels. Next-generation sequencing (NGS) was conducted using the Illumina Hiseq 4000 sequencing platform , and 150-bp paired-end reads were produced. Scripts were used to filter the Illumina data in the cluster with the default parameter. When the low-quality (Q ≤ 5) base number in sequencing reads surpassed 50% of the reads base number and the N content in reads exceeded 10% of the reads base number, paired reads were eliminated from the analysis. More than 10 Gb of clean data were obtained for each species.https://github.com/quxiaojian/PGA, accessed on 10 July 2023) [Trichoglottis philippinensis (MN124404) was used as a reference. The annotated plastomes were manually examined using Geneious 11.1.5 [The paired-end sequencing reads were filtered and assembled into complete plastid genomes by using the GetOrganelle pipe-line  with defly 2023) , and thesis MN1244 was uses 11.1.5 .ggplot2 [Trichoglottis plastomes were identified by Mauve [The Perl script MISA  was emplggplot2 . The reaby Mauve . The IRsby Mauve  was usedTrichoglottis plastomes were extracted using PhyloSuite v1.2.2 [Trichoglottis using DAMBE [A total of 68 protein-coding genes from e v1.2.2 . These png DAMBE . Finallyng DAMBE .Trichoglottis using the Shuffle-LAGAN [Thrixspermum centipeda (MW057769) plastome was used as a reference. The nucleotide variability (Pi) of complete plastomes and 68 protein-coding genes of seven Trichoglottis species was calculated using DnaSP 6 [The online program mVISTA was used to analyze the diversity of plastome sequences of le-LAGAN  alignmen DnaSP 6  with a wndh genes were widespread, pseudogenized, or lost in Aeridinae species) were extracted using PhyloSuite v1.2.2 [In this study, phylogenetic trees were constructed using a concatenated matrix of 68 protein-coding genes and a matrix of complete plastomes. The 68 protein-coding genes , maximum parsimony (MP), and Bayesian inference (BI) on the website CIPRES Science Gateway web server  . For ML Trichoglottis  were newly assembled. Our results showed that the structure and gene content of Trichoglottis plastomes are highly conserved. All ndh genes were lost or pseudogenized. The Trichoglottis s.l. (including Staurochilus) was strongly supported as a monophyletic group, and two species formerly treated as Staurochilus were revealed as successively basal lineages. Five non-coding regions (GUUtrnN-rpl32 > UAAtrnL > GCUtrnS-UCCtrnG > rbcL-accD > GGUtrnT-psbD) were identified to further elucidate the phylogeny of Trichoglottis. These findings provide valuable insights into plastome evolutionary patterns and phylogenetic relationships for Trichoglottis and its related genera and even extend to the orchid family.In this study, five plastomes of


Staphylococcus aureus (S. aureus) disproportionately cause skin and soft tissue infections (SSTI) among African Americans (AA). Recurrence of S. aureus SSTI can be seen in >50% of pediatric cases. Risk factors associated with recurrent infections in children are not well reported. We hypothesize that recurrence of S. aureus SSTI in AA children living in Atlanta, Georgia may be related to location-based risks.Community onset S. aureus infections from 2011-2019 were included in this retrospective study, using electronic health records (EHR). We included patients who had community-onset S. aureus SSTI, were < 19 years old, and were Georgia resident. Sociodemographic variables collected included race, ethnicity, age, sex, and health insurance. Area-level variables from US Census Bureau included proxies for poverty, education, unemployment, and household crowding. Infections were stratified into MRSA and MSSA by oxacillin susceptibility. Recurrent SSTI was defined as an initial SSTI with one or more subsequent SSTI documented more than 14 days from initial infection within a calendar year. Home address was geocoded and assigned a census tract. Statistical analyses included chi-squared tests and multiple logistic regression to detect differences between patients with recurrent SSTI from non-recurrent SSTI using SPSS. Hotspot analyses were performed at the census tract level to identify hot and cold spots for recurrent SSTI stratified by methicillin susceptibility using ArcGIS Pro.Patients with S. aureus SSTIs; 64 patients had recurrent SSTI (42 MRSA and 22 MSSA). Females had high proportion (64.1% ) of recurrent SSTIs compared to 48.0% of females with single SSTI (p = 0.011). Females also were significantly more likely to have recurrent MSSA (81.8%) compared to 54.8% of females with recurrent MRSA (p = 0.032). Spatial analysis demonstrated hotspots of recurrent SSTIs in the northern counties of metropolitan Atlanta, whereas single episode SSTIs did not demonstrate this type of spatial distribution.5,784 unique children presented with SSTI Recurrence by County LevelIn Georgia, children who are female are at increased risk for recurrent SSTI, and hotspots of patients with recurrent SSTIs are distinct from those with single episodes of SSTIs.Lilly Immergluck, MD,MS, Melinta: Grant/Research Support|Melinta: Grant/Research Support


Terminalia catappa leaf aqueous crude extract (TCLE) on hematological and coagulation disturbances in a Type 2 diabetic rat model.Diabetes mellitus is a leading cause of mortality worldwide associated with hyperglycemia-induced hematological aberrations and thromboembolic complications. This study aimed to explore the modulatory effect of High-fat diet streptozotocin-induced diabetic rats were treated orally with 400 and 800 mg/kg body weight TCLE daily for 28 days. Full blood count, coagulation parameters, plasma calcium (Ca), and erythrocyte glycogen (GLYC) levels were assessed using standard procedures.Terminalia catappa leaf aqueous crude extract treatment had a significant (p < 0.05) prolonging effect on clotting and bleeding times while increasing Ca, GLYC and mean corpuscular volume in diabetic rats. On the other hand, lymphocytes (LYM), platelet (PLT) count, mean PLT volume, neutrophil-LYM ratio (NLR), and PLT-LYM ratio (PLR) of TCLE-treated diabetic animals were significantly reduced (p < 0.05) compared with untreated diabetic animals. Lymphocyte, PLT count, NLR, and PLR correlated positively (p < 0.05) with plasma glucose, while a significant positive association was observed between Ca and GLYC. On the other hand, a strong negative association (p < 0.05) was observed between clotting time and fasting plasma glucose.T. catappa leaf extract may be useful in reversing diabetic-mediated hematological anomalies due to its anticoagulant and anti-anemic activities.These findings suggest that  Diabetes mellitus (DM) is a leading cause of mortality worldwide, with an abnormal rise in blood glucose levels as the hallmark , 2. TypeDiabetics usually present altered immune systems and hematological characteristics on clinical evaluation, which correlates with various hematological abnormalities . These hTerminalia catappa L. is a medicinal plant with antidiabetic properties );Group II, diabetic rats treated with distilled water (1 mL/kg BW);Group III, diabetic rats treated with glibenclamide (10 mg/kg BW);Group IV, diabetic rats treated with TCLE (400 mg/kg BW); andGroup V, diabetic rats treated with TCLE (800 mg/kg BW).et al. [et al. [The experimental period and dosage determination were similar to the study of Iheagwam et al. . Type 2  [et al. . The treClotting time was evaluated following Ivy’s method . The aniet al. [Bleeding time was performed following the method reported by Ayodele et al. . A proxiet al. [Plasma Ca was analyzed according to the Randox diagnostic kit  instructions. Erythrocyte glycogen (GLYC) concentration was assessed following the protocol reported by Iheagwam et al. . White bpost hoc analyses. Indices significantly altered by the diabetic state were assessed for association with parameters of hyperglycemia using Pearson’s correlation.Data obtained were analyzed using IBM statistical package for the social sciences statistics 23  with results presented as mean ± standard error of the mean (SEM). Values were considered statistically different at a 5% probability level using a one-way analysis of variance and Duncan’s multiple range test for The results of TCLE treatment on coagulation parameters are illustrated in Figures-Compared to normal rats, Ca concentration and GLYC stores were depleted (p < 0.05) in diabetic rats. Treatment with TCLE and glibenclamide significantly increased Ca and GLYC concentration in diabetic rats compared with untreated diabetic rats. The values of Ca and GLYC in the treatment groups were significantly (p < 0.05) lower than those in the normal group Figures- and 4.In Among all evaluated erythrocyte indices, only MCV was significantly (p < 0.05) reduced in the diabetic group compared with the normal group. Diabetic rats treated with TCLE exhibited a marked increase (p < 0.05) in MCV when compared to untreated diabetic rats, with values similar to those obtained for normal and glibenclamide groups .Platelet and MPV were significantly raised (p < 0.05) in the diabetic groups, while other thrombocyte indices were not changed by the diabetic state and treatments compared with the normal group . IncreasAdministration of 400 and 800 mg/kg BW TCLE in diabetic rats significantly p < 0.05) reduced NLR and PLR compared to the diabetic group. These indices initially increased significantly (p < 0.05) after induction of diabetes when compared with normal rats , PLT (0.83), NLR (0.81), and PLR (0.79) exhibited a significant (p < 0.05) strong positive correlation with fasting blood glucose (FBG), while a strong negative correlation was observed for CT . In relation to erythrocyte GLYC, only Ca (0.86) was associated significantly (p < 0.05) in a positive manner .et al. [et al. [Terminalia catappa leaf aqueous crude extract’s ability to increase CT and BT while reducing PLT and MPV might suggest a possible anticoagulant and fibrinolytic activity. Terminalia catappa leaf aqueous crude extract contains active principles such as benzofuranone, flavonoids, and octadecanoic acid that decrease the production of thromboxane A2, slowing down PLT aggregation and thereby increasing BT and CT [Diabetic conditions, especially T2DM and obesity, are associated with a procoagulant state that induces thromboembolic complications, increasing the mortality rate of diabetics and patients with CVD , 20. Micet al. , and Fid [et al. , who rep [et al. . Higher  [et al. . TerminaT and CT , 25. ThiT and CT , 25, 26.T and CT , 27.et al. [Calcium is required in blood coagulation for tenase and prothrombinase complex formation to activate prothrombin and transform fibrinogen into fibrin. The observed decrease in diabetic animals suggests that hypercoagulant signaling utilizes a large amount of systemic Ca, depleting Ca reserve in the process . An incret al. . Erythroet al. [et al. [et al. [et al. [et al. [et al. [et al. [Streptozotocin possesses a cytotoxic effect in the bone marrow, leading to a suppressed immune system . This cyet al.  and Rash [et al. , LYM lev [et al. , who rep [et al.  reported [et al. , 33. The [et al. , Chinedu [et al. .The association between T2DM and chronic inflammation induces diabetic microangiopathy . During T. catappa leaf extract leaf may be useful in reversing diabetic-mediated hematological anomalies. Thus, TCLE may possess anticoagulant and anti-anemic activities useful for managing thrombotic disorder in the diabetic state. Prospective studies can be conducted to ascertain the anticoagulant and anti-anemic mechanism of action and the phytoprinciple(s) responsible.The findings signify that FNI, OOO, and SNC: Conceptualized the study. FNI: Designed the methodology, analyzed, and interpreted the result. FNI and PAG: Carried out the experiments and wrote the first draft of the manuscript. All authors have read, reviewed, and approved the final manuscript.


K. pneumoniae ST147 cluster encoding NDM- and OXA-48 carbapenemases and one VIM-encoding P. aeruginosa ST823 cluster. In the current study, we investigated if NDM- and OXA-48-encoding K. pneumoniae and VIM-encoding P. aeruginosa isolates recovered between 2014 and 2021 from oncological patients belonged to those same clusters.Hospitals with their high antimicrobial selection pressure represent the presumably most important reservoir of multidrug-resistant human pathogens. Antibiotics administered in the course of treatment are excreted and discharged into the wastewater system. Not only in patients, but also in the sewers, antimicrobial substances exert selection pressure on existing bacteria and promote the emergence and dissemination of multidrug-resistant clones. In previous studies, two main clusters were identified in all sections of the hospital wastewater network that was investigated, one The 32 isolates were re-cultured, whole-genome sequenced, phenotypically tested for their antimicrobial susceptibility, and analyzed for clonality and resistance genes in silico.Among these strains, 25 belonged to the two clusters that had been predominant in the wastewater, while two others belonged to a sequence-type less prominently detected in the drains of the patient rooms.Patients constantly exposed to antibiotics can, in interaction with their persistently antibiotic-exposed sanitary facilities, form a niche that might be supportive for the emergence, the development, the dissemination, and the maintenance of certain nosocomial pathogen populations in the hospital, due to antibiotic-induced selection pressure. Technical and infection control solutions might help preventing transmission of microorganisms from the wastewater system to the patient and vice versa, particularly concerning the shower and toilet drainage. However, a major driving force might also be antibiotic induced selection pressure and parallel antimicrobial stewardship efforts could be essential. Antibiotic resistance is a major global concern, a complex public health issue and is accelerated by improper use of antibiotics as well as a growing population and increased networking and travelling. It depends on many interconnected factors and is far from limited to the clinical setting –3. ResisKlebsiella pneumoniae and Pseudomonas aeruginosa are two of the most important opportunistic and nosocomial pathogens worldwide and are known for the ability to produce biofilms to escape treatment with antibiotics [K. pneumoniae and VIM encoding P. aeruginosa isolates recovered from clinical specimens of patients in oncology wards of a tertiary care center to the highly resistant strains that most prominently colonized patient bathrooms or were recovered from different sampling points of the same sewer system. Environmental K. pneumoniae ST147 and P. aeruginosa ST823 were analyzed when encoding the mentioned carbapenemases [K. pneumoniae ST147 is a well-known high-risk clone that likely emerged in the 1990s and swiftly became a prominent global pathogen [P. aeruginosa ST823 is mostly associated with blaVIM and only few publications exist [ibiotics , 15, 16.enemases . K. pneupathogen –20. Diffpathogen . On the ns exist –23. Repons exist , 24.K. pneumoniae and P. aeruginosa isolates, or such with an unusual carbapenem-susceptibility profile (ertapenem/imipenem/meropenem) were routinely analyzed for the presence of common resistance genes, using the Allplex Entero-DR Assay .In the period under study Gram-negative bacterial pathogens recovered from clinical specimens were routinely identified via MALDI-TOF MS  and susceptibility-tested with the VITEK 2 system . Carbapenem-resistant P. aeruginosa isolates and all NDM- and OXA-48-encoding K. pneumoniae isolates recovered from patients of oncology wards between September 2014 and November 2021 were traced in the laboratory information system, thawed and sub-cultured twice on Columbia 5% sheep blood agar  prior to testing and DNA extraction. Only first isolates were selected for each pathogen–patient combination. The environmental strains used for comparison had been isolated between Nov. 2016 and Sept. 2018 [All VIM-encoding pt. 2018 Enterobacterales and Pseudomonas, respectively.Antimicrobial susceptibility of all isolates after re-cultivation from cryo stocks was determined thrice by broth microdilution. Susceptibility tests were employed strictly according to the manufacturer’s instruction. From each isolate, a bacterial suspension in 0.9% saline solution was prepared. The suspension was adjusted to a McFarland value of between 0.48 and 0.52 using a DensiCHEK plus photometer . For broth microdilution, Micronaut-S MDR MRGN-Screening MIC-Plates  were utilized . The isolation was performed according to the manufacturer’s instructions. Obtained DNA was qualitatively and quantitatively evaluated using the NanoDrop OneC from Thermo Fisher Scientific Inc. . Dual-indexed Illumina sequencing libraries were constructed from each sample using the Illumina Nextera XT DNA Library Preparation Kit, pooled, and sequenced on the Illumina MiSeq platform with the Illumina MiSeq Reagent Kit v3, 600 cycles . All steps were carried out following the manufacturer's instructions. Raw reads have been uploaded to the Sequence Read Archive (SRA); accession PRJNA845217.https://cge.cbs.dtu.dk/services/ResFinder/) and PlasmidFinder-2.0 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) [https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/AMRFinder/) . Analysis of the de novo assembled contigs was then performed with online tools of the CGE-server, ResFinder-4.0 (Finder/) –28 and t-04-2022 ), which -04-2022 . In addiP v1.3.0 .http://www.ridom.de/seqsphere) [K. pneumoniae sensu lato was employed as cgMLST template for the K. pneumoniae strains and the cgMLST template for P. aeruginosa was used for the P. aeruginosa strains (www.ridom.de/seqsphere/u/Task_Template_Sphere.html). Minimum spanning trees were calculated after ignoring pairwise missing values and after exclusion of genes that were present only in the template strain.For epidemiological clustering Ridom SeqSphere + (version 6.0.2)  (qsphere)  was usedK. pneumoniae and P. aeruginosa isolates were processed using ASA3P v1.3.0 [3P calculating 100 bootstraps using isolates CNK1 and A15448 [Raw sequencing reads for both P v1.3.0 . SNP-basd A15448  as referAll data relevant to the study are included in the article or uploaded as Appendix. The precise source of each environmental sample can be located in the corresponding publication . EnvironThe ethics committee of the University Hospital Bonn confirmed that no ethics approval was required for this study.P. aeruginosa isolates and ten NDM- and OXA48-encoding K. pneumoniae isolates from patients of the oncology clinic were included into this study. Typing revealed that the majority of P. aeruginosa isolates (17) belonged to ST823; two belonged to ST235, another two to ST111, and one to ST233 (see Table K. pneumoniae isolates (which belonged to ST78) belonged to ST147. In the majority of patients (18/32), the isolates were first detected after more than four weeks of hospitalization. In half of the patients (16/32), the isolates were obtained from stool samples. All but one, K. pneumoniae ST147 isolates, were recovered from the same ward.From September 2014 until November 2021, twenty-two VIM-encoding P. aeruginosa ST823 isolates, fell into two different groups as clustering showed (cluster distance threshold was set to 12) when clustered with environmental isolates from 2016 to 2018 from a study on hospital drains and wastewater [P. aeruginosa ST235 isolates, CVP4 did not cluster with environmental isolates but CVP19 was closely related to two environmental isolates . The P. aeruginosa isolates, on the other hand, even more markedly formed one large cluster with the environmental isolates.As Fig. K. pneumoniae isolates by PlasmidFinder-2.0 are listed in Table K. pneumoniae ST78 isolates and ST147 isolates did not share any plasmids. All ST147 isolates carried the same plasmids, except for the three latest ST147 isolates, recovered in 2020 and 2021, which carried an additional IncHI2-type plasmid. Tables P. aeruginosa genomes and on NDM- and OXA48-encoding K. pneumoniae genomes by AMRFinderPlus, respectively. There were only two internal stop codons found with INTERNAL_STOP (CVP2 fosA and CVP5 fosA) in resistance genes. The VIM-encoding gene that was detected in the course of routine diagnostics in CVP19 was not detected on its sequenced genome, but a repeated targeted PCR with the Allplex Entero-DR Assay  from the isolate and its extracted genomic DNA confirmed the presence of the resistance gene. Predicted antibiotic resistances by the ASA3P pipeline for P. aeruginosa ST823 and K. pneumoniae ST147 isolates are displayed in Fig. Plasmid incompatibility groups detected in P. aeruginosa isolate (CVP19) was susceptible at high dosing regimen to meropenem, nine to piperacillin, eleven to piperacillin/tazobactam, and 13 to ceftazidime, as well as 15 at standard dosing regimen to ceftazidime/avibactam. While all P. aeruginosa were still susceptible to colistin and 15/22 to ceftazidime/avibactam, 5/10 K. pneumonia isolates were resistant to both. Both K. pneumoniae ST78 isolates and the three latest ST147 isolates were resistant to colistin. Analysis of the contigs of the IncHI2-type plasmid showed that it did not carry colistin resistance genes; however, the full sequence of the plasmid was not available for analysis. Among these, two ST147 and one ST78 isolate were also resistant to tigecycline. Two ST147 isolates and one ST78 were resistant to chloramphenicol.According to EUCAST 2022 v12 breakpoints  all tested isolates were resistant to fluoroquinolones and imipenem  and hygienic measures , constant monitoring and strict hygiene precautions help to prevent infections, however, further technical solutions are needed to prevent biofilm formation and selection pressure at the sanitary inventory level, as antibiotics are necessary for therapy, but their metabolites in the drains cause undesirable effects.


Helicobacter pylori (H. pylori), which one of the notable pathogens in the stomach. Antibiotic-induced changes in the microbiota can negatively affect the host. This study aimed to determine the influence of H. pylori resistance on the diversity and abundance of the stomach microbiome.Inadequate antimicrobial treatment has led to multidrug-resistant (MDR) bacteria, including H. pylori positive from cultures and histology. DNA was amplified from the V3-V4 regions of the 16S rRNA gene. In-vitro E-test was used to detect antibiotic resistance. Microbiome community analysis was conducted through α-diversity, β-diversity, and relative abundance.Bacterial DNA was extracted from biopsy samples of patients presenting dyspepsia symptoms with H. pylori positive samples were eligible after quality filtering. Following resistance status to five antibiotics, samples were classified into 24 sensitive, 24 single resistance, 16 double resistance, 5 triple resistance. Samples were mostly resistant to metronidazole . Comparation of four groups displayed significantly elevated α-diversity parameters under the multidrug resistance condition . A notable change was observed in triple-resistant compared to sensitive (P <0.05) and double-resistant (P <0.05) groups. Differences in β-diversity by UniFrac and Jaccard were not significant in terms of the resistance . In the triple-resistant group, the relative abundance of Helicobacter genera was lower, whereas that of Streptococcus increased. Moreover, the linear discriminant analysis effect size (LEfSe) was associated with the presence of Corynebacterium and Saccharimonadales in the single-resistant group and Pseudomonas and Cloacibacterium in the triple-resistant group.Sixty-nine H. pylori in the triple-resistant samples decreased with increasing cohabitation of pathogenic bacteria, which may support antimicrobial resistance. However, antibiotic susceptibility determined by the E-test may not completely represent the resistance status.Our results suggest that the resistant samples showed a higher trend of diversity and evenness than the sensitive samples. The abundance of  Antibiotic resistance has been observed in healthcare-associated and community-acquired infections and has become a global public health challenge. The World Health Organization has acknowledged studies demonstrating a longer duration of hospitalization and increased 30-day mortality in patients with resistant bacteria . The estHelicobacter pylori (H. pylori) is one of the most common human pathogens, and 50% of the global population is estimated to be infected by it. H. pylori triggers numerous pathological alterations in the stomach, including peptic ulcer disease, primary gastritis, and gastric cancer [H. pylori eradication rates have decreased because of its increasing resistance to one or more antibiotics [H. pylori resistance rates were 17, 44, 18, 3, and 4% for clarithromycin, metronidazole, levofloxacin, amoxicillin, and tetracycline, respectively [H. pylori strains have been reported in China [H. pylori antibiotic resistance.c cancer . Thus, tc cancer . Howeveribiotics ,12. A syectively . Moreovein China . These dH. pylori and other gastric microbiota communities in the gastric microenvironment. Previous studies have linked changes in the gastric microbiome composition between H. pylori-negative and H. pylori-positive individuals [Helicobacter genus is associated with an increase in the abundance of other pathogenic populations. Additionally, the gastric microbiota composition is altered during the progression of gastritis to gastric cancer [The advancement of 16S rRNA sequencing over the years has helped reveal the composition of the gastric microbiota, which helps understand the complex interactions between ividuals ,16. The c cancer ,18. Althc cancer ,20, dataH. pylori-positive to five antibiotics in gastric mucosa samples. H. pylori-positive biopsy samples with a resistant status were analyzed for diversity and richness. Subsequently, linear discriminant analysis effect size (LEfSe) analysis was conducted to determine the abundance of microbiota.Here, we conduct the first microbiome community study on the resistance of H. pylori infection study was conducted from 2014 to 2016 in Indonesia; a total of 1074 samples were obtained through endoscopy and biopsy sampling combined with our previous results [H. pylori treatment prior to examination. We collected demographic data (age and sex) and a simple diagnosis of dyspepsia using a questionnaire, as described in our previous study [The  results . All subus study . PatientH. pylori culture, the specimens were homogenized in 500 μL of phosphate-buffered saline, and the remaining homogenized specimens then stored at a temperature of −80°C for DNA extraction later. Histological examination was conducted using biopsy specimens from the corpus and antrum. All the specimens were obtained using Radial Jaw 4 forceps (Boston Scientific).We investigated the microbiome on gastric mucosal specimens obtained from the lesser curvature of the antrum, approximately 3 cm from the pyloric ring. The biopsy samples were stored in a transport medium containing 10% glycerol. For the H. pylori infection, we included 80 specimens of H. pylori-positive from the culture, and the histological analysis involved DNA extraction and sequencing. The final analysis included 69 specimens after quality filtering  and the Oita University Faculty of Medicine . All protocols followed the principles of the Declaration of Helsinki involving human subjects.H. pylori culture and histological examination. H. pylori was isolated from the antral tissues, cultured, and incubated at 37°C for 10 days on a selective agar plate for H. pylori containing equine serum, soybean peptide, sodium chloride, and selective antibiotics  in a microaerophilic environment. Colonies were then sub-cultured in antibiotic-free agar medium (Mueller-Hinton II) supplemented with 10% horse blood (Becton Dickinson) in microaerophilic conditions; subsequently, the H. pylori stock was stored at −80°C in Brucella broth (Difco). All the biopsy specimens were preserved in 10% buffered formalin and embedded in paraffin for histological evaluation. Serial sections were stained with hematoxylin-eosin and May-Giemsa stains, and H. pylori was detected in samples with bacterial burdens of grade 1 or above.Using cultures of antral biopsy specimens, we determined the infection status based on the H. pylori cultures were sub-cultured thrice on a Brucella agar plate supplemented with 7% horse blood without antibiotics under microaerophilic conditions overnight. Subsequently, H. pylori colonies were collected in phosphate-buffered saline, customized to a 3.0 McFarland concentration, and embedded on 5% horse blood-supplemented Mueller Hinton agar. E-test strips containing five types of antibiotics were patched in the center. The antibiotic concentrations were as follows: 0.016–256 mg/L for clarithromycin, amoxicillin, tetracycline, and metronidazole, respectively, and 0.002–32 mg/L for levofloxacin. The area of inhibition was measured after 72 h. All sensitivities were determined according to the clinical breakpoint of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [Antibiotic sensitivity measurements were conducted by measuring the minimum inhibitory concentration (MIC) of five antibiotics , as described in our previous study . Procedu(EUCAST) .DNA extraction and PCR amplification were conducted as previously described . DNA washttps://qiime2.org). The α-diversity including the observed richness, Simpson and Shannon diversity indexes, and Pielou’s evenness were analyzed using the alpha significance QIIME diversity. The Wilcoxon signed-rank test was used to compare the diversity indexes. The plot was drawn in the “ggplot2” package of the R environment (ver 4.02).Reads obtained from the Illumina MiSeq platform were analyzed using the QIIME2 (Version 2021.2) pipeline . The demThe OTU tables, metadata, taxonomy, and phylogenetic tree were exported into the phyloseq object using biom-convert and “phyloseq” package of the R environment for beta diversity and abundance analysis. The β-diversity analysis measured the unweighted UniFrac and Bray-Curtis distances. Principal component analysis was conducted using the R software. The Adonis function from the “vegan” package was used to conduct permutational analysis of variance (PERMANOVA) with the Bonferroni correction. Using the “phyloseqCompanion” package in R, we conducted LEfSe to determine OTUs that are likely to explain differences between sensitive, single-, double-, and triple-resistant strains. We also used the Kruskal-Wallis test to determine the presence of any differences in the relative abundances of specific OTUs in the resistant strains.H. pylori eradication   and 39 (56.52%) were male and female, respectively. Most patients were diagnosed with gastritis  and had no history of  69.56%) . The druP = 0.54), Simpson index (P = 0.38), and Pielou’s evenness index (P = 0.3664). Bacterial community richness at the OTU level was also comparable between the groups (P = 0.5119).The analyzed data were set at a minimum sampling depth of 500 reads for diversity normalization, and no samples were excluded. To investigate the general condition, we analyzed the gastric microbiota community according to the susceptibility status. Overall, we observed a higher tendency for diversity and evenness, but this was not statistically significant in the resistant group compared with the sensitive group. P = 0.04) and Simpson (P = 0.032) indexes, and evenness of Pielou’s index (P = 0.025) among the groups. The pair-wise comparison of sensitive and triple-resistant strains showed an increasing trend of diversity richness for MDR strains, and double- and triple-resistant strains showed significant results in the Shannon index (P <0.01) and the comparison between double- and triple-resistant groups (P <0.01) were also conducted. The α-diversities showed that triple-MDR strains had the highest diversity and evenness trends. In contrast, double-resistant group displayed a lower trend in the diversity and evenness compared with the sensitive and other resistant groups. Generally, the observed OTUs were comparable between groups , as indicated by PERMANOVA  at the genus level was conducted for taxa with an abundance of at least 2% of the population. Several taxa characterizing each disease group were observed with an LDA score cutoff of 5.0 . In addive group .H. pylori broke the paradigm and subsequently changed perspectives regarding the microbiota community in the gastric niche [H. pylori is a class I carcinogen associated with gastric cancer. This infection alters the physiological conditions of the gastric environment to serve other bacterial habitats [H. pylori resistance status.The stomach was thought to be sterilized by commensal bacteria because of the low pH of gastric acid. The discovery of ic niche . H. pylohabitats . As the To the best of our knowledge, microbiome analysis of MDR strains remains limited, and this exploration is the first of its kind for gastric. This study highlights a few findings related to the resistance status. Overall, the resistant strains showed a higher trend in diversity and community evenness compared to the sensitive group. Subsequently, based on the resistance type, there was an increase in the α-diversity index among the MDR. The diversity and richness significantly increased in triple-resistant strains compared with sensitive and single-resistant strains . NotablyH. pylori infection in the stomach [Enterobacteriaceae spp. with extended-spectrum beta-lactamase (ESBL) gene potential [The virulence factors CagA and VacA can alter the gastric microbiota and immune phenotypes previously attributed to  stomach . Moreoveotential .Bacteroidetes spp. [H. pylori resistance to treatment in Indonesia revealed a high distribution of metronidazole and levofloxacin resistance. The 23S rRNA A2143G mutation for metronidazole is associated with clarithromycin resistance [In the recent study, patients were mostly diagnosed with gastritis with no prior eradication history. We could not specify deeper type of any previous dyspeptic therapy nor antibiotic treatment of patients. Antibiotic activity plays a role in the microbiota composition. Strains showed major antibiotic resistant to metronidazole, followed by clarithromycin and levofloxacin in the E-test. Clinically, metronidazole has been extensively used to target anaerobic bacteria, such as tes spp. , whereastes spp. . Investisistance . This masistance . The conH. pylori infection occupies most of the gastric niche [Pseudomonas was the most differentially abundant genus in triple-resistant group. A higher abundance of Streptococcus was also observed. There is a possibility of subjects that possessed MDR H. pylori could also be infected with other MDR bacteria and their augmented abundance repressed the growth of H. pylori, as shown in our results. A previous study reported that the abundance of gram-negative bacteria, such as Escherichia coli, Enterobacter cloacae, and Salmonella enterica increased in patients with MDR bacterial infections [Streptococcus and various Proteobacteria is a marker of persistent dysbiosis, which subsequently increases bacteremia [H. pylori [H. pylori to form biofilms, which are known to reduce susceptibility compared with the planktonic phase [The abundance of phyla in all groups showed that the core microbiome phyla found in healthy individuals (Firmicutes and Bacteroidetes) were suppressed by Campylobacterota dominance, as shown in ic niche ,37. Notafections . The domcteremia ,39. Thoscteremia , wherein. pylori . This isic phase . Moreoveic phase .H. pylori in Indonesia [This study had several limitations. First, the findings presented here were analyzed using 16S rRNA sequencing; thus, we could not determine the presence of drug resistance in other organisms. However, this can serve as a preliminary view of the gastric microbial community in patients with MDR strains. Further investigation using the shotgun metagenome is necessary to confirm the presence of antimicrobial resistance genes possessed by all bacteria present in the environment and help better understand the reciprocal effect. Second, our previous prospective population-based study demonstrated the low prevalence of ndonesia ,44. MoreH. pylori was correlated with the enriched diversity of the gastric microbiome composition, where the abundance of non-H. pylori pathogens increased, especially in triple-resistant strains. Given this possibility, clinicians should be mindful of antibiotic combination treatments because drug resistance in the gut environment could affect the gastrointestinal niche and probably induce drug resistance in other bacteria.In conclusion, recent data indicate that dysbiosis due to multidrug use and cohabitation compositions may be correlated. The resistance status of