Burkholderia cepacia complex (Bcc) is a closely related group of bacteria, composed of at least 20 different species, the accurate identification of which is essential in the context of infectious diseases. In industry, they can contaminate non-food products, including home and personal care products and cosmetics. The Bcc are problematic contaminants due to their ubiquitous presence and intrinsic antimicrobial resistance, which enables them to occasionally overcome preservation systems in non-sterile products. Burkholderia lata and Burkholderia contaminans are amongst the Bcc bacteria encountered most frequently as industrial contaminants, but their identification is not straightforward. Both species were historically established as a part of a group known collectively as taxon K, based upon analysis of the recA gene and multilocus sequence typing (MLST). Here, we deploy a straightforward genomics-based workflow for accurate Bcc classification using average nucleotide identity (ANI) and core-gene analysis. The workflow was used to examine a panel of 23 Burkholderia taxon K industrial strains, which, based on MLST, comprised 13 B. lata, 4 B. contaminans and 6 unclassified Bcc strains. Our genomic identification showed that the B. contaminans strains retained their classification, whilst the remaining strains were reclassified as Burkholderia aenigmatica sp. nov. Incorrect taxonomic identification of industrial contaminants is a problematic issue. Application and testing of our genomic workflow allowed the correct classification of 23 Bcc industrial strains, and also indicated that B. aenigmatica sp. nov. may have greater importance than B. lata as a contaminant species. Our study illustrates how the non-food manufacturing industry can harness whole-genome sequencing to better understand antimicrobial-resistant bacteria affecting their products.The  The authors confirm that all supporting data, code and protocols have been provided within the article. Illumina raw sequence reads and associated genomes have been deposited in the European Nucleotide Archive (ENA) under ENA project accession number PRJEB42964, with the exception of BCC1315, which was previously submitted under accession number ERA2359236.Burkholderia cepacia complex (Bcc) non-food product industrial contaminants, the genomes of 23 strains were sequenced and a genomic classification workflow was developed. We provide evidence to support the reclassification of misidentified industrial Bcc bacteria as the recently assigned B. aenigmatica sp. nov. The study provides an insight into how genomics may be utilized to improve the identification of Bcc bacteria, for which identification has historically relied upon the differential analysis of one (recA) or several (multilocus sequence typing) housekeeping genes. The findings of this study are beneficial to industry and medicine alike, due to the inherent difficulty in accurately identifying Bcc species, and the prevalent nature of named species Burkholderia contaminans and Burkholderia lata as cystic fibrosis pathogens and problematic contaminants of industrial products.Industrial microbiology is an area where microbial misidentification is problematic, and the application of genomics is currently limited. To resolve the identification of problematic Burkholderia cepacia complex (Bcc) is a group of closely related Gram-negative bacteria with a continuously evolving taxonomy, and currently comprises 20 named species [Pseudomonas cepacia up until their taxonomic reclassification in 1997 [recA gene sequencing [The  species . They ha species  and plan species , and, in species  and bein species . Members in 1997 . Bcc bacquencing , and mulquencing  for accuquencing .Burkholderia pose a contamination risk due their intrinsic antimicrobial and biocide resistance [Burkholderia isolates caused 3 % of European recalls due to microbial contamination of non-sterile non-food products between 2005 and 2018 [Burkholderia cenocepacia [B. cenocepacia III-B by recA and MLST analysis [Burkholderia servocepacia [MLST schemes have been developed for a multitude of bacteria of interest, including the Bcc . MLST wasistance . For exaand 2018 . Industrand 2018 , or moleand 2018 . Howeverand 2018 , industrcepacia , where ganalysis  as the pocepacia .recA gene and MLST, and within which two novel species were formally named as Burkholderia contaminans and Burkholderia lata [B. lata was identified as one of the predominant Bcc species found as an industrial contaminant [B. lata type strain 383T was shown to be capable of adapting its tolerance to a range of in-use preservatives, such as the isothiazolinones [et al. [B. lata and B. contaminans, but also revealing Burkholderia aenigmatica sp. nov. as a new taxon with sufficient genetic and phenotypic differences to support its proposal as a new species. Going forward, the accurate identification of this preservative-tolerant species group in the industrial environment is crucial. In relation to Bcc industrial contaminants, the performance of existing molecular techniques such as recA and MLST that had been used to identify them [Taxon K  is a multi-species grouping within the Bcc that was proposed in 2009 based on analysis of the ia lata . Subsequtaminant , and theolinones . In a 20 [et al.  observedify them  needs toify them .To date, few studies of non-food product microbial contaminants have made use of genomics to determine the species identity of problematic contaminants . Herein,B. lata, 4 were classified as B. contaminans and 6 were identified as a novel species subgroup within taxon K [Isolates were revived via plating onto tryptic soya agar, followed by inoculation of 3 ml tryptic soya broth for overnight culture, and were pelleted before genomic DNA was extracted. DNA extraction was performed on a panel of 23 historical contaminant isolates stored in the BCC collection at Cardiff University, using a Maxwell 16 Tissue DNA Purification kit and instrument . These isolates were identified as a part of taxon K  by MLST  taxon K . After Nhttps://github.com/enasequence/enaBrowserTools). This database included the complete genomes for type strains of B. contaminans (LMG 6992T), B. lata (383T), and the newly sequenced type strain of B. aenigmatica (LMG 13014T) [Illumina adaptors were trimmed from 150-nucleotide paired-end reads using the TrimGalore script, and genomes were assembled using SPAdes v3.14.0  and/or U 13014T) .https://github.com/widdowquinn/pyani), using both blastn  and MUMmer  for alignment [. As defined previously, ANIb refers to pairwise ANI values calculated using the blastn algorithm, whilst ANIm refers to pairwise ANI results calculated with the MUMmer algorithm. Heatmaps were also generated using PyANI to provide a visualization of the level of identity between taxon K genomes. An ANI threshold of 95 % was used for analyses [ANI analysis was conducted upon the taxon K genomes using the Python script PyANI v0.2.7 , which uses blastn to compare a query genome to PubMLST databases. All 58 genomes in the taxon K database were annotated using Prokka (v1.14.5) [MLST profiles were generated using MLSTcheck  (availabv1.14.5)  under dev1.14.5)  was usedv1.14.5) . A GAMMAv1.14.5)  and editv1.14.5) .B. contaminans by MLST  in comparison to the B. lata 383T species reference. Interestingly, the most closely related complete genome by ANI was that of the B. lata A05, a strain of industrial origin, isolated from a mouthwash containing 0.2 % of the preservative chlorhexidine [B. lata 383T gave an ANIm of 94.6 % (Table S2), but strain A05 displayed ANIm values of 98.6–99.7 % (Table S2) in comparison to the 13 industrial isolates, suggesting that it had been incorrectly designated as B. lata. ANI also revealed the existence of several other incorrectly classified/species borderline taxon K genomes, originating from non-industrial sources and labelled as B. contaminans or B. lata within the database . This was also supported by the MLST sequence types matching those of known B. aenigmatica types . Using a 95 % ANI threshold, these isolates were designated as B. aenigmatica. It is important, however, to note that that this ANI value is set arbitrarily based upon previous analyses of prokaryotic genomes [Recently, the novel Bcc species isolates . Compari genomes , and thain silico gene extraction from the genomes of all industrial isolates corroborated their historical ST identification (B. contaminans industrial isolates [B. contaminans type strain LMG 23361T (B. lata [B. aenigmatica [B. lata industrial isolate A05 [B. lata species type strain 383T (MLST analysis based on fication . Phylogeisolates  alongsidG 23361T . This MLB. lata , the B.gmatica  type strlate A05 , as adjaain 383T .B. contaminans isolates still placed alongside the type strain in this analysis. All 13 industrial isolates defined as B. lata by MLST (B. lata 383T. A number of isolates that clustered within this clade possessed STs (ST98, ST119, and ST333, B. aenigmatica sp. nov [B. aenigmatica LMG 13014T forming a subclade with the six previously unclassified taxon K isolates, and the remaining isolates forming a closely related subclade, with B. lata A05 being the most phylogenetically similar complete genome. This split is supported by both ANIb (B. aenigmatica and B. aenigmatica-like isolates form two distinct alignment clusters. Overall, core-gene phylogenetic analysis was concordant with both ANIm and ANIb, and the designation of 6 taxon K isolates and 13 taxon K isolates [B. aenigmatica. This dataset also revealed a unique intraspecies sub-clading, for which a limited amount of evidence was observed in previous datasets [To improve the phylogenomic resolution, core-gene analysis was used to generate an alignment of 255 core-genes for all taxon K genomes examined . The cor by MLST  were clu by MLST , which d sp. nov . Moreoveoth ANIb  and ANImoth ANIb , where isolates  as B. adatasets .B. lata or as unidentified by MLST into B. aenigmatica sp. nov. This distinction is not shown by blast or phylogenetic analysis of MLST genes, both of which place these isolates alongside the B. lata type strain, 383T, or do not provide species-level identitification at all.Overall, the data presented illustrate a clear workflow for the identification of Bcc species as important industrial contaminants and clinical bacteria. This genomic workflow expands on past minimum taxonomic criteria  and presB. aenigmatica-like isolates belong the B. aenigmatica sp. nov., ANI thresholds should be chosen as appropriate for a given dataset, as it is also known that there is genetic discontinuity around this boundary [B. aenigmatica. However, examining higher or lower ANI boundaries for different groups of bacteria may aid classification (B. lata, B. contaminans and novel species isolates within the taxon K contaminant collection. This is clearly shown in our analyses, which demonstrate a strong concordance between core-gene phylogenetic clading (see Although a 95–96 % ANI threshold  is accepboundary . In the fication . In the ding see , and ANIding see , where AB. lata industrial isolates sequenced in this study and the mislabelled deposited industrial strain A05 genome (B. aenigmatica [B. lata should be re-evaluated. This is an important identification correction in relation to industrial microbiology, as from 2013 B. lata was observed to be the most common species present in a collection of 60 contaminant strains [B. aenigmatica sp. nov. in relation to preservative tolerance and survival within industrial products remains to be determined. However multiple members of the taxon K group are intrinsically resistant to antimicrobials and have been identified as problematic contaminants [B. aenigmatica playing a role in contamination, and the contribution of a large panel of industrial genomes to public databases, will be invaluable in the further development of the taxonomy of Burkholderia species.To conclude, techniques that analyse differences throughout the genome, as opposed to between a limited number of conserved genes such as MLST, are essential to accurately identify Bcc species going forward. To this end, core-gene analysis  and ANI 5 genome  and S1 agmatica , the ide strains . The fitaminants . The eluClick here for additional data file.


Neonatal sepsis is a global concern with increasing morbidity and mortality. The burden of neonatal sepsis is highest in developing countries, especially in those lacking proper surveillance systems. The causative pathogens and their drug-resistance levels vary between countries with emergence of multidrug resistance organisms. Thus, accurate records on the recent trends of organisms causing neonatal sepsis will provide vital information for appropriate intervention. We aimed to investigate neonatal sepsis, identify its associated factors and causative pathogens and to assess the antibiotic susceptibility patterns in Sana’a city, Yemen.A cross-sectional study was conducted on neonates admitted to intensive care units of six hospitals in Sana’a city, Yemen, in the period from January 15, to March 30, 2020. Natal and prenatal medical data were collected using well-structured questionnaire. Neonates were subjected to sepsis work-up including blood culture, complete blood count and C-reactive protein. Organisms were identified by Gram staining and analyzed by the VITEK II system for bacterial bio-typing and antibiotic susceptibility testing.p = 0.005. Majority of isolated bacteria (74.39%) were gram-negative with Burkholderia cepacia (39%) and Klebsiella oxytoca (13%) being the most common pathogens of EOS and LOS. The most common gram-positive pathogens were Staphylococcus haemolyticus (9.1%) and Staphylococcus epidermidis (7.1%). B. cepacia showed multidrug resistance except for cefepime. All Klebsiella species isolates (100%) and most Pantoea species (93%) were ESBL and carbapenemase positive. All Escherichia coli and Acinetobacter baumannii isolates were ESBL positive. A significant number of gram-positive bacteria showed resistance to vancomycin.Of the 199-neonates with suspected neonatal sepsis, 154 (77.38%) had culture-proven sepsis. Early-onset neonatal sepsis (EOS) was higher  than late-onset neonatal sepsis (LOS) . Multivariable analysis identified vaginal delivery as an independent risk factor for neonatal sepsis B. cepacia being the single most common pathogen causing EOS and LOS. Findings also emphasize the emerging threat of multidrug-resistant bacteria in neonatal units and will help develop evidence-based management of neonatal sepsis in Yemen.The study findings show a high proportion of neonatal sepsis among neonates admitted to hospitals in Sana’a city with antibiotic-resistant  Neonatal sepsis is a leading cause of neonatal morbidity and mortality worldwide. The global burden of neonatal sepsis and other neonatal infections was estimated at 22 million disability-adjusted life-years 95% UI: 18.9–28.0) 8.9–28.0 –6.Klebsiella species, Staphylococcus aureus, E. coli, and Group B Streptococcus [B. cepacia, a bacterial pathogen that has intrinsic drug resistance and ability to live inside drug products [Pathogens causing neonatal sepsis are either acquired from the maternal flora or postnatally from the hospital or community . Bacteritococcus , 9. Receproducts , 11, hasproducts –16.Yemen is suffering of an ongoing war that started in 2015. The war has destroyed the health system, rendering health facilities either not functioning or partially functioning . There iAll methods were carried out in accordance with the relevant guidelines and regulations.This was a cross-sectional study conducted on neonates admitted to neonatal units in six major hospitals in Sana’a city; Al-Kuwait, C-Plas, Al-Gumhory, Al-Sabeen, AL-Thawra, and Family hospitals, in the period from January 15 to March 30, 2020. Neonates who were admitted for at least 72 h with suspected sepsis during the study period were included. Neonates with congenital anomalies and hemolytic jaundice were excluded from the study.Neonatal sepsis was suspected based on the presence of clinical signs or risk factors according to the international recommendations  and confBio and socio-demographic data were collected using a standard questionnaire by face-to-face interviews with the neonate’s mothers. The health condition of the mothers and clinical manifestations of the neonates were collected by physicians using standard tools. Written informed consent was obtained from guardians of all neonates after explaining the study objectives. All procedures described in this study were approved by the Research and Ethics Committee of the Faculty of Medicine and Health Sciences, University of Science and Technology, Sana’a .® GN ID identification card (lot 2410933203) was used to characterize gram-negative bacteria and VITEK® GP ID identification card (lot 2420938203) was used to characterize gram-positive bacteria. Different cards were used to perform antibiotic susceptibility tests; AST-GN 87 , AST-GN 72 and AST GN75  for gram-negative bacteria and AST-GP67  for gram-positive bacteria. All procedures were conducted for routine diagnostic and therapeutic reasons.Laboratory investigations were performed according to standard microbiological techniques . Under ap-value of < 0.05 was considered significant.Data were analyzed using IBM SPSS Statistics for Mac, version 23.0 . Categorical variables were presented in frequencies. The association between independent and dependent variables was tested using Pearson’s Chi-squared with reporting odds ratio (OR) and 95% confidence interval (CI). Fisher’s exact test was used when applicable. Multivariable analysis using the binary logistic regression model was conducted, including all variables, and the adjusted OR with its corresponding 95%CI was reported. A The study protocol was approved by the Research and Ethics Committee of the Faculty of Medicine and Health Sciences, University of Science and Technology, Sana’a, Yemen .A total of 199 neonates admitted to referral hospitals in Sana’a city, Yemen, were enrolled in this study. Nearly half of neonates  were preterm, majority of them  were moderates to late preterm. More than half of neonates  were low birth weight. A total of 113 neonates (56.8%) were born by vaginal delivery. The age of 133 neonates (66.8%) was less than 72 h on admission. Tachypnea, lethargy and poor feeding were the most common clinical manifestations observed among neonates (Table p = 0.002) than those whose deliveries were through Cesarean section. Although most neonates born at home acquired neonatal sepsis , the significance of association was in the borderline (Table p = 0.005).Majority of neonates 77.38% (154/199) had positive blood culture. Of them, 50.25% (100/199) had EOS. Neonates born by vaginal delivery were at three-times higher risk of neonatal sepsis  and convulsion (p = 0.01). Poor feeding, jaundice, irritability, and lethargy were not significantly associated with neonatal sepsis. Elevated C-reactive protein was significantly associated with neonatal sepsis (p = 0.001). One-third (32%) of neonates with proven sepsis had normal white cell counts and two-thirds (59%) had normal platelets counts  were gram-negative while 42 (26%) were gram-positive. B. cepacia (37%) was the most common organism causing neonatal sepsis with higher prevalence among EOS (38%) than LOS (35%), followed by K. oxytoca (11.6%), which was higher among LOS (14.8%) than EOS (10.0%). Pantoea agglomerans caused neonatal sepsis in nine neonates (5.8%). The main gram-positive pathogens were S. haemolyticus (9.1%), S. epidermidis (7.1%) and Staphylococcus hominis (5.1%). Coagulase-negative Staphylococci (CONS) was the most common gram-positive organisms. S. haemolyticus emerged as the most frequently CONS.Table B. cepacia, the most common pathogen caused neonatal sepsis in this study, was highly resistant to ampicillin/sulbactam, gentamicin, tobramycin, tetracycline, amoxicillin/clavulanic acid, cefalotin, cefazolin, cefuroxime, cefoxitin, cefpodoxime, imipenem, and amikacin. However, it was 100% susceptible to cefepime. K. oxytoca was susceptible to gentamicin, ciprofloxacin, levofloxacin, tetracycline, nitrofurantoin, and trimethoprim/sulfamethoxazole. Klebsiella species (100%) and Pantoea species (93%) were carbapenemase positive and ESBL positive. All isolates of E. coli and A. baumannii were ESBL positive (Table S. haemolyticus isolates were resistant to ampicillin and gentamicin (Table Staphylococcus species showed resistance to fluoroquinolones (ciprofloxacin).Most isolated pathogens showed antimicrobial resistance (AMR) to the commonly used antibiotics , cephalosporins and carbapenems Tables  and 6. Bve Table . A signiB. cepacia emerged as the predominant causative organism of both EOS and LOS. Majority of the isolated pathogens were resistant to commonly used antibiotics.In this multicentric cross-sectional study we report a high proportion of culture-confirmed neonatal sepsis, accounting for two-thirds (77.38%) of admitted neonates in the referral hospitals in Sana’a city in Yemen. EOS was more common than LOS and Culture confirmed sepsis was high in our study. However, the proportion of culture-confirmed neonatal sepsis varied between studies from developing countries; 62.8% in Pakistan , 57% in In this study, EOS was about two times higher than LOS, which is consistent with other reports from developing countries , 26, 27.Similar to previous studies neonates born by vaginal delivery were at a higher risk of neonatal sepsis compared with those delivered through Cesarean section , 27. FurB. cepacia emerged as the most common cause of neonatal sepsis. It was found in all neonatal units. Moreover, it was resistant to aminoglycosides, fluoroquinolones and imipenem. B. cepacia has been reported to cause multiple hospital outbreaks and significant neonatal septicemia in different countries [Pseudomonas species [B. cepacia from Yemen.In this study, gram-negative bacteria were the most common cause of EOS and LOS, which is consistent with previous reports from developing countries , 22, 31.ountries –16. Alsoountries . This or species , 34. To Staphylococcus haemolyticus was the most frequently isolated CONS in this study. Most isolated S. haemolyticus showed variable degrees of antibiotic resistance, which is the nature of S. haemolyticus to acquire antibiotic resistance due to unusual genome plasticity [asticity . So, thiOur results of antimicrobial resistance profile are consistent with other findings. Though some of the previous reports were based on cultures from different kinds of medical specimens and performed by conventional methods , 36–39. Proven sepsis was associated with elevated CRP. Elevated CRP indicates activation of the immune system and is commonly used as an indicator of bacterial sepsis , 19. TheB. cepacia followed by K. oxytoca and S. haemolyticus were the most common causes of both EOS and LOS. Majority of the isolated bacterial pathogens showed a high level of resistance to commonly used antibiotics. Vaginal delivery held high risk for developing neonatal sepsis. The study results emphasize the emergence of multidrug-resistant bacteria in the NICUs and might serve as a baseline for proper medical treatment of neonatal sepsis in Sana’a city.Culture-positive neonatal sepsis is high in Sana’a city with EOS representing two-thirds of the cases.


Clostridium perfringens type A/G that impacts the global poultry industry by compromising the performance, health, and welfare of chickens. Coccidiosis is a major contributing factor to NE. Although NE pathogenesis was believed to be facilitated by α-toxin, a chromosome-encoded phospholipase C enzyme, recent studies have indicated that NE B-like (NetB) toxin, a plasmid-encoded pore-forming heptameric protein, is the primary virulence factor. Since the discovery of NetB toxin, the occurrence of NetB+ C. perfringens strains has been increasingly reported in NE-afflicted poultry flocks globally. It is generally accepted that NetB toxin is the primary virulent factor in NE pathogenesis although scientific evidence is emerging that suggests other toxins contribute to NE. Because of the complex nature of the host-pathogen interaction in NE pathogenesis, the interaction of NetB with other potential virulent factors of C. perfringens needs better characterization. This short review will summarize the primary virulence factors involved in NE pathogenesis with an emphasis on NetB toxin, and a new detection method for large-scale field screening of NetB toxin in biological samples from NE-afflicted commercial broiler flocks.Necrotic enteritis (NE) is a devastating enteric disease caused by  The world population is rapidly expanding and is expected to reach over nine billion people by the year 2050 . Most ofEimeria spp. or Clostridium perfringens, both of which are ubiquitous in the environment and gastrointestinal tract of animals, compromise the health, performance, and welfare of chickens. Coccidiosis caused by Eimeria spp. results in an economic loss of about 13 billion US dollars per year due to mortality and the use of in-feed anticoccidials . Si. SiC. pefringens ,49. C. pnecrosis . Findinges of NE .cnaA, has recently been reported in NE-producing C. perfringens strains [C. perfringens strains harboring cnaA were all virulent and bound to collagen type IV, V, and gelatin in vitro. In a follow-up study by Wade et al., cnaA-deleted C. perfringens lost the ability to bind to collagen in vitro, did not induce NE lesions in vivo, and showed a reduced ability to colonize on the ileal mucosa in vivo [C. perfringens adherence to extracellular matrix proteins as a crucial virulence factor in NE pathogenesis [In addition to the toxins and enzymes that we discussed above, collagen adhesion protein, encoded by  strains . C. perf in vivo . These rogenesis .netB gene is encoded in the 42-kb NELoc-1 region on an ca. 85-kb plasmid [C. perfringens chromosome. NetB is secreted as 33-kDa β-barrel toxin that forms 1.6- to 1.8-nm pores in susceptible cells. It shares some structural similarity (less than 40%) with β-toxin from C. perfringens, α-hemolysin, γ-toxin, and leukocidin from Staphylococcus aureus, and cytotoxin K from Bacillus cereus [plc- and netB-deletion mutants of the virulent C. perfringens EHE-NE18 strain [plc-deletion mutant of the virulent C. perfringens JGS4143 strain induced NE in chickens [plc-deletion mutant. In addition, Zhou et al. developed a C. perfringens strain missing the NELoc-1 virulence plasmid from the virulent C. perfringens CP1 strain, which was then complemented with netB. While the virulent C. perfringens strain CP1 induced NE, the CP1 strain missing the NELoc-1-encoding plasmid did not [netB-deficient CP1 variant strain with netB gene restored its intermediate virulence. Collectively, these studies [netB-positive and -negative C. perfringens isolates.Recent molecular and proteomic studies have increased our understanding of the structure and function of NetB in NE pathogenesis ,52,53,54 plasmid . NetB to plasmid  and an as cereus ,39. The 8 strain . Furtherchickens . However did not . However studies ,57,58 prnetB-positive C. perfringens isolates in NE-afflicted broiler flocks has been reported over the last decade  or quantitative PCR. C. perfringens can be directly isolated by plating gut or environmental samples on selective agar  or by a liquid culture  followed by plating on solid agar . The function of selective media for C. perfringens has been documented elsewhere [Since the discovery of the NetB toxin as an essential virulence factor in NE, the prevalence of reported . In genelsewhere  althoughC. perfringens isolation from NE-afflicted or healthy chickens  netB-positive C. perfringens is more abundant in NE-afflicted vs. healthy chickens, (4) netB-positive C. perfringens may not be isolated from confirmed NE clinical cases, (5) netB-negative C. perfringens can be isolated from NE-afflicted chickens, and (6) netB-positive C. perfringens can be detected in retail broiler meats. Thus, these findings indicate that NE is a complex disease caused by multiple contributing and predisposing factors  although the NetB toxin is considered to be the primary virulent factor.Several conclusions can be drawn from studies that describe chickens : (1) FienetB-positive C. perfringens strains by Western blot analysis using polyclonal anti-recombinant NetB antiserum [C. perfringens isolates from NE chickens were NetB positive whereas only 4 out of 14 C. perfringens strains from healthy chickens produced NetB [C. perfringens isolated from NE-afflicted chickens secreted much higher amounts of NetB toxin in vitro compared to those from normal chickens. Allaart et al. reported that 9 out of 43 netB-positive C. perfringens strains isolated from hens with subclinical NE produced NetB protein in vitro [netB-negative C. perfringens. In one study, all 22 netB-positive C. perfringens strains isolated from chickens with clinical NE produced NetB [netB-positive C. perfringens may secrete NetB in vitro. The conflicting results may be related to the difference in netB gene copy number among netB-positive C. perfringens isolates. Indeed, Gu et al. isolated 18 netB-positive C. perfringens from NE-afflicted chickens and showed that only half of the netB-positive C. perfringens strains had higher netB gene copy numbers relative to their 16S rDNA levels [netB gene in C. perfringens does not guarantee the secretion of the NetB toxin in vitro.Until recently, native NetB protein was only detected in vitro in culture supernatants from ntiserum ,53,60,85ntiserum ,94. Abilin vitro . There aA levels . Gu’s obA levels  suggestsnetB-positive C. perfringens is related, (2) whether in vivo NetB protein can be detected by netB-negative C. perfringens in NE models, and (3) whether routine screening of biological samples  can predict virulent C. perfringens NE outbreaks on farms for timely management. The recent development of a NetB antigen-specific ELISA system [Escherichia coli NetB protein and native NetB protein secreted by netB-positive C. perfringens isolates [netB-positive C. perfringens exhibited cytopathic effects in LMH cells in vitro [A detection method to measure native NetB protein in biological samples  is not currently available. With increasing restriction of antibiotic-free poultry production, there is a need to develop a high throughput, reliable analytical tool to detect native NetB protein in biological samples to adequately address several questions related to fundamental and applied research on NE: (1) whether in vitro and in vivo production of NetB protein by A system  to detecisolates . Furtherin vitro . In addiin vitro . Similarin vitro . Howeverin vitro . Until rin vitro . The latin vitro  concludein vitro  are likeC. perfringens toxins and enzymes that can potentiate NE pathogenesis with or without NetB. Although the presence of netB in C. perfringens isolates can be typed with PCR and the NetB protein can be detected using Western blots, there is a need for a more defined large-scale immunoassay that can detect toxins associated with NE pathogenesis in the field samples from NE-afflicted farms. Recent availability of NetB-specific mouse monoclonal antibodies and a high throughput ELISA detection system will now allow sensitive NetB detection in samples from poultry farms. These NetB-specific monoclonal antibody-based detection assays will be a valuable tool in addressing fundamental questions of host-pathogen immunobiology in NE and as a screening tool for validating the efficacy of novel alternative-to-antibiotics feed additives or vaccines to mitigate the negative effects of NE.NE is not only an emerging threat to the global broiler industry but also compromises the welfare and health of commercial poultry in the era of antibiotic-independent poultry production ,97. This


Streptococcus (S.) intermedius-associated pleuropulmonary infection (PPI) is a rare presentation. Cases have been reported in the last decade in which S. intermedius was identified in pleural fluid cultures and lung biopsies in patients presenting with complicated pleural effusions and empyema. However, there are data lacking on predisposing factors, mode of dissemination, and treatment strategies for these patients. Moreover, these patients can present with a spectrum of radiographic findings, which can help with prompt diagnosis and effective patient management. However, the radiological findings in this patient and the rapidly worsening clinical symptoms make the diagnosis difficult.A  Streptococcus (S.) intermedius is a facultative anaerobe belonging to the S. anginosus group. This group includes three organisms: S. anginosus, S. intermedius, and S. constellatus. S. intermedius is commonly involved in causing abdominal and brain abscesses. S. constellatus causes pulmonary and blood infections, and S. anginosus causes gastrointestinal and genitourinary infections. Streptococcus intermedius rarely is associated with pleuropulmonary infection (PPI), which includes pneumonia, pleural effusion, and empyema [ empyema -2. Risk  empyema . ThroughThe patient was a 54-year-old male, chronic smoker, who presented with dyspnea and productive cough with yellowish-green sputum for a duration of two weeks. These symptoms were associated with loss of taste, weight loss, and generalized fatigue. He had no known history of aspiration episodes, chest trauma, or surgeries. He initially presented to an urgent care center for the above symptoms with an oxygen saturation percentage in the 80s. He presented to the emergency department on 3 liters/min of oxygen with an oxygen saturation of 96%. Abnormal labs included white cell count (WBC) 26 k/cumm (reference range 3.6-10.6); hemoglobin (Hb) 8.6 gm/dl (reference range 13.4-17); platelets 753 k/cumm (reference range 150-450); and C-reactive protein 26.6 mg/dl (reference range <1). Coronavirus disease 2019 (COVID-19) and influenza swabs were negative. A chest X-ray was done . Return of spontaneous circulation (ROSC) was achieved in five minutes, and the patient was intubated. He was put on pressor support. The patient underwent a bedside diagnostic bronchoscopy with washing, which showed red and inflamed mucous membranes of right-sided airways. A right-sided apical chest tube was placed, and 1500 ml of reddish-brown fluid was drained. Broncho-alveolar lavage (BAL) fluid was sent for cultures. On day two, bloody secretions were noticed in the nasogastric tube, and there was a suspicion for fistula, neoplasm, or esophageal trauma. Another chest tube was inserted posteriorly later to improve drainage of right lower lobe effusion since the patient was unstable for surgery/decortication. Subsequently, labs in the morning showed white blood cells 65 k/cumm; Hb 6.9 g/dl; lactate 21.5 mmol/l ; anaplastic lymphoma kinase (ALK) 340 units/l , alanine aminotransferase 4240 units/l , and aspartate aminotransferase, 4161 units/l (reference range: 13-39 units/L). Arterial blood gas analysis showed pH 6.86, HCO3 7 mmol/l, and Pco22 40 mmHg. Antibiotics were switched to broad-spectrum vancomycin and piperacillin-tazobactam. Pleural fluid culture results came back positive for S. intermedius. Testing for blood cultures, urine Streptococcus antigen, Legionella antigen, and Methicillin-resistant Staphylococcus aureus (MRSA) were negative. The patient received fresh frozen plasma for possible disseminated intravascular coagulation and blood transfusions for dropping hemoglobin levels. He continued to worsen and was in profound septic shock on three vasopressors and stress dose steroids to maintain mean arterial pressure greater than 65 mm Hg. The plan was to do video-assisted thoracoscopic surgery (VATS) by cardiothoracic surgery when the patient became hemodynamically stable. However, the patient deteriorated and went into asystole. Eventually, the family intervened and asked to avoid any further efforts to revive the patient. Unfortunately, the patient expired.S. intermedius is a bacterial organism well-known for causing brain and liver abscesses. However, it is a resident of the oral cavity and can be aspirated in patients at risk, causing pleuropulmonary disease. This is a rare phenomenon but there have been cases documented with S. intermedius PPI and varied radiological findings. A literature review was performed utilizing PubMed with the keywords “S. intermedius pneumonia” and “pleural effusion,” and articles published from the year 2010 onwards pertinent to S. intermedius PPI were reviewed. The emphasis was on the various radiological presentations, diagnostic techniques, and treatment strategies. We reviewed 10 articles in total published from the year 2010 onwards (Table The radiological findings seen in the patient population diagnosed with S. intermedius infection broadly include consolidation, loculated pleural effusion, cavitary lesions, nodules, empyema, and lung abscess -10. In oAs per the review of literature, more studies are needed to determine the virulence factors that mediate disease pathogenesis and predilection for specific tissues in S. intermedius pneumonia. Moreover, a few cases have been reported in the literature that mention the presence of multiple abscesses in patients positive for S. intermedius. Hence, looking for abscesses at other sites can help avoid missing any infection source .The ideal treatment approach in these patients includes a combination of both surgical and medical management. Antibiotics employed to treat this infection mostly included vancomycin, levofloxacin, and ceftriaxone. Moreover, surgical decortication has proven to be efficacious in these patients. This case report highlights the fact that with a radiological presentation indicating a loculated pleural effusion, empyema, or lung abscess, physicians should keep “S. intermedius PPI” as a differential, particularly in high-risk individuals. Timely management can reduce mortality and ensure high-value care.The radiological findings in this case are unique, in the sense that it is hard to predict if it is just an abscess, empyema, or malignancy with superimposed infection. Also, the patient did not have significant risk factors for S. intermedius infection, thus it makes us think if we still need studies to determine the pathophysiology of the disease and the population at risk. It stresses how rapidly an S. intermedius infection can worsen, hence there should be a high suspicion for this infection whenever clinicians see a similar radiological picture. Early decortication might help with faster recovery; however, unfortunately, this patient was not stable enough to undergo this procedure.


Coxiella burnetii, the causative agent of Q fever, is known to cause acute and persistent infection, but reactivation of infection is rarely reported. This case demonstrates reactivation of a distant, untreated Q fever infection after a relatively innocuous soft tissue injury in an adjacent joint without pre-existing pathology. A 52-year-old male abbatoir worker sustained an adductor muscle tear in a workplace injury. He was unable to walk thereafter, and developed a chronic, progressive, destructive septic arthritis of the adjacent hip with surrounding osteomyelitis of the femur and acetabulum. He had evidence of prior Q fever infection, with a positive skin test and serology 15 years beforehand. He was diagnosed with chronic osteoarticular Q fever on the basis of markedly elevated phase I antibodies, and symptomatic and serological response to prolonged antibiotic treatment with doxycycline and hydroxychloroquine. He required a two-stage hip arthroplasty. This case illustrates reactivation of latent C. burnetii infection at the site of a soft tissue injury. Clinicians need to be aware of this possibility in patients with previous Q fever infection, and in the setting of undiagnosed osteoarticular pathology following soft tissue injury. Coxiella burnetii, a phagolysosome-dwelling organism that can persist within the host [C. burnetii develop symptomatic persistent focalized infection [Osteomyelitis and septic arthritis are rare manifestations of Q fever. The causative agent is the host . Betweennfection , more confection . ClinicaA 52-year-old abbatoir employee from northern New South Wales, Australia, injured his right thigh while attempting to stop bovine intestines falling with his foot in November 2011. He had sudden onset of pain in the adductor insertion, radiating down the medial thigh. Over the next week, pain developed in the buttock and lateral thigh to the knee. Pain and swelling continued over the next few months.−1.Ultrasound 10 days post-injury demonstrated a tear of the adductor magnus, and plain X-ray was normal. Magnetic resonance imaging 2 months later showed extensive marrow oedema in the femoral head and neck, acetabulum and adjacent hemi-pelvis, with partial collapse of the femoral head, oedema of the gluteal and adductor muscles, and a tear in obturator externus. There was hip synovitis and fluid. C-reactive protein was 98.9 mg l>3200 by immunofluorescence , consistent with chronic Q fever. At age 40 at pre-immunization screening, the patient’s Q fever skin test had been positive, with an induration diameter of 15 mm, while the Q fever IgG enzyme immunoassay index was 1.4 (<1.0) and the phase 2 complement fixation titre was 2.5 (8–32). After that time, he had not taken antibiotics that are active against Q fever.Subsequent X-rays demonstrated a progressive, destructive process in the femur and acetabulum. Bone scan demonstrated avid focal tracer uptake in the right femoral head and neck. Thirteen months post-injury, the Q fever phase I IgG and total antibody titre was C. burnetii culture and PCR were negative after two doses of doxycycline. Cell culture and PCR were processed at the Australian Rickettsial Reference Laboratory . QuantiFERON-TB Gold and brucellosis serology were negative. Echocardiography was normal.Seventeen months post-injury, he attended the Infectious Diseases service. The hip was mildly tender, in fixed flexion, with severely limited passive movements, and active movements limited by pain, with non-pitting oedema of the leg. There was quadriceps wasting with no muscle tenderness. He commenced doxycycline and hydroxychloroquine. Histopathology from synovial core biopsy demonstrated non-granulomatous chronic inflammation. Bacterial, fungal, mycobacterial and Pain and mobility were much improved 7 weeks later, at 19 months post-injury. The fixed flexion deformity resolved, with nearly normal range of hip motion. C-reactive protein levels had returned to normal and phase I Q fever antibody titres were much reduced .C. burnetii joint tissue culture and PCR were negative.The patient underwent a successful two-stage total hip arthroplasty in February 2014 at 27 months post-injury, taking doxycycline throughout. Standard and The patient received 36 months of doxycycline and hydroxychloroquine, with no relapse in symptoms or radiological change 18 months post-treatment cessation, despite transient elevations in Q fever antibody titres during treatment.The patient’s illness was consistent with a reactivation of latent Q fever causing septic arthritis and osteomyelitis due to injury, based on serologically confirmed distant Q fever infection, recent illness, sequential imaging, high-titre phase I Q fever serology, and rapid clinical and serological response to treatment.There are 36 reported cases of osteoarticular infection in adults . OsteomyC. burnetii has been documented after resolution of acute infection: after a woman with laboratory-acquired infection was treated, C. burnetii was isolated from placenta and milk 6 months later; and C. burnetii was isolated from the placentas of four women who had had acute Q fever 6 months to 3 years beforehand [C. burnetii can persist in a viable state after acute infection. However, definite clinical reactivation in adults has rarely been reported. One case report describes a febrile illness post-aortic valve replacement, evolving into frank Q fever endocarditis, representing quiescent infection with clinical reactivation after surgical tissue injury [Reactivation of Q fever is not the typical paradigm of human disease, although it occurs in animals during gestation  or underforehand . This ile injury .C. burnetii antibodies post-cardiac surgery, all with valve replacements [Serological reactivation occurred in 7 of 42 patients with pre-existing acements . A case acements . Four ofacements .C. burnetii infection in injured, previously healthy tissue represents a new paradigm in Q fever. This principle of locus minoris resistentiae has been reported in other infections [This case of apparent reactivation of latent fections . Clinici


Ganoderma boninense, the fungal pathogen causing basal stem rot in oil palms. In this study, we focused on the effects of salicylic acid (SA) on the growth of three G. boninense isolates with different levels of aggressiveness. In addition, study on untargeted metabolite profiling was conducted to investigate the metabolomic responses of G. boninense towards salicylic acid. The inhibitory effects of salicylic acid were both concentration- (P < 0.001) and isolate-dependent (P < 0.001). Also, growth-promoting effect was observed in one of the isolates at low concentrations of salicylic acid where it could have been utilized by G. boninense as a source of carbon and energy. Besides, adaptation towards salicylic acid treatment was evident in this study for all isolates, particularly at high concentrations. In other words, inhibitory effect of salicylic acid treatment on the fungal growth declined over time. In terms of metabolomics response to salicylic acid treatment, G. boninense produced several metabolites such as coumarin and azatyrosine, which suggests that salicylic acid modulates the developmental switch in G. boninense towards the defense mode for its survival. Furthermore, the liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis showed that the growth of G. boninense on potato dextrose agar involved at least four metabolic pathways: amino acid metabolism, lipid pathway, tryptophan pathway and phenylalanine pathway. Overall, there were 17 metabolites that contributed to treatment separation, each with P<0.005. The release of several antimicrobial metabolites such as eudistomin I may enhance G. boninense’s competitiveness against other microorganisms during colonisation. Our findings demonstrated the metabolic versatility of G. boninense towards changes in carbon sources and stress factors. G. boninense was shown to be capable of responding to salicylic acid treatment by switching its developmental stage.Various phenolic compounds have been screened against  Ganoderma boninense Pat., the causal agent of basal and upper stem rot of oil palm (Elaeis guineensis), is a major threat to the oil palm industry in Southeast Asia  p pG. boni enzymes . AdditioG. boninense when challenged with SA through untargeted metabolite profiling analysis. In addition, the inhibitory effects of SA on the growth of three G. boninense isolates with different pathogenicity levels were also investigated.Thus, our primary objective was to investigate the metabolomics responses of G. boninense isolates were tested in this study, i.e., G8 (least aggressive), PER71 (moderately aggressive), and G10 (most aggressive), each with different levels of aggressiveness in causing disease in the nursery screening . Th. ThG. boGanoderma-inoculated plates (G0 and G200). These metabolites include: difructose anhydride (325.111 m/z) (Ganoderma-inoculated plates (G0 and G200 treatments). Similarly, D-phenylalanine L-valine, L-leucine, dimethyl fumarate and glutamate, which could be secondary products from amino acid content of potato infusion in the PDA medium, were also depleted in the Ganoderma-inoculated plates. Similar to our results, Ahmad et al. (2020) [G. boninense and Scytalidium parasiticum. Together, these imply the utilization of these sugars and nitrogen sources for the fungal growth.A number of metabolites were detected in higher abundance in control groups (C0 and C200) compared to 111 m/z) , 1,5-anh111 m/z) , D-pheny111 m/z) , L-valin111 m/z) , L-leuci111 m/z) , dimethy111 m/z) , and glu111 m/z) . Based o. (2020)  reportedMycobacterium spp., and Saccharomyces cerevisiae, respectively    detectedoninense . Unlike nd G200) . Penipanur study , 86.Ganoderma-inoculated plate (G0). The metabolites were eudistomin I, L-phenylalanine, nitric acid and chorismatic acid. The presence of SA, which may act either as stress factor, or carbon source, may have altered the metabolic pathways of G. boninense. This implies the metabolic versatility of G. boninense in responding to the surrounding stimuli, which is important in fungal physiology and pathogenicity, for nutrient assimilation and adaptation to host-imposed stress [G. boninense utilized this phenylalanine to produce coumarin in the presence of SA treatment. Earlier findings on the ability of G. boninense to catabolize a range of phenols, namely syringic acid, caffeic acid, 4-hydroxybenzoic acid, and catechin, have also hinted the metabolic versatility of G. boninense [G. boninense is undeniably a fungal pathogen that is metabolically versatile, which allows it to mitigate various environmental stresses for survival.Noticeably, several metabolites were detected at lower concentrations in the presence of SA (G200), compared to non-amended d stress . The absd stress . It is loninense , 81. On G. boninense that enable consequent modulation and switching of its developmental growth stage to combat salicylic acid stress. The production of coumarin and azatyrosine, which are the pre-cursors of melanin and/or fruit bodies, probably reduces the negative impacts of salicylic acid over time. In the absence of salicylic acid, G. boninense utilizes carbohydrate and amino acid for its growth and undergoes at least four metabolic pathways, namely amino acid metabolism, lipid pathway, tryptophan pathway and phenylalanine pathway. The capability to produce antimicrobial metabolites may benefit the survival of G. boninense during colonisation of oil palm tissues through successfully competing with other microorganisms. At lower concentrations of the treatment, the least aggressive G. boninense isolate could utilize salicylic acid as a carbon source to promote its growth. Together, these findings imply the pronounced metabolic versatility of G. boninense in mitigating changes to its growing environment, which is by switching its developmental stage as illustrated in this study.This study provides the first evidence detailing the metabolic responses of S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.


The MERINO trial has seemingly laid to rest the question: ‘Are carbapenems the preferred therapy for ESBL-producing infections?’ It has, however, brought another important question to the forefront: ‘How do we know when we have an ESBL-producing infection?’ A commonly used approach is the interpretation that non-susceptibility to third-generation cephalosporins (e.g. ceftriaxone MICs of ≥2 mg/L) is an accurate proxy for ESBL production. We believe that relying on antibiotic susceptibility results alone to predict ESBL production in clinical isolates is fraught with issues. Rather, we believe accurate molecular assays that detect a comprehensive range of ESBL genes, along with other relevant β-lactamase genes, are well within the reach of existing technology and necessary to optimize patient care. Herein, we elaborate on why the current approach for determining whether an organism is likely to be an ESBL producer (i) is inaccurate; (ii) encourages carbapenem overuse; (iii) ignores the potential for ESBL production in other Enterobacterales species; and (iv) promotes the silent epidemic of ESBL transmission. Current methods available to clinical laboratories for the detection of ESBL expression in bacterial isolates are fraught with limitations. However, these limitations should not prohibit further refinement to more accurately and rapidly detect ESBLs—a task that is within easy reach of the technologies available to today’s clinical microbiology laboratories. ESBL detection is integral to the management of patients with infections caused by Enterobacterales and should not be hamstrung by technologies that were developed a quarter of a century ago.Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Proteus mirabilis.Our colleagues suggest that third-generation cephalosporin MIC criteria (e.g. ceftriaxone MICs ≥2 mg/L) is a sufficient and suitable proxy for ESBL production among E. coli, Klebsiella spp. and P. mirabilis producing ESBLs have ceftriaxone MICs of ≥2 mg/L, not all E. coli, Klebsiella spp. and P. mirabilis with MICs ≥2 mg/L are ESBL producers. A portion of organisms with ceftriaxone MICs ≥2 mg/L (which remain carbapenem susceptible) will harbour plasmid-mediated ampC genes (p-ampC) or even no identifiable β-lactamase genes—and this has important treatment implications. As an example, a study including 5723 clinical E. coli, Klebsiella spp. and P. mirabilis isolates from 72 US hospitals in 2012 found that 87% of isolates with ceftriaxone MICs ≥2 mg/L carried blaCTX-M genes, 7% ampC genes, and 6% had only narrow-spectrum β-lactamase genes.E. coli, Klebsiella spp. and P. mirabilis ceftriaxone-non-susceptible isolates from 2014–15 found that ESBL genes (including blaCTX-M and ESBL variants of blaSHV and blaTEM) were identified in 64% of isolates, p-ampC genes in 0.5% of isolates, both ESBL and p-ampC genes in 13% of isolates, and narrow-spectrum β-lactamase genes alone were identified in 22% of the 376 organisms.E. coli and K. pneumoniae international ceftriaxone-non-susceptible isolates from 2014–17 that underwent WGS in the MERINO trial, 85% of isolates harboured ESBL genes, 10% possessed p-ampC genes and 2% had both ESBL and p-ampC genes.ampC-producing and narrow-spectrum β-lactamase-producing infections differ, greater specificity than simply a ceftriaxone MIC ≥2 mg/L is needed to accurately identify and manage ESBL-producing infections.The use of a ceftriaxone cut-off of ≥2 mg/L to signal presumed ESBL production is sensitive, but not specific. While virtually all ,A second concern bolstering the inaccuracy of inferring ESBL production based on ceftriaxone MICs is the uncertain supposition that ceftriaxone MIC values are reliably determined by automated susceptibility systems used in clinical laboratories. Breakpoints defined by organizations like the CLSI and FDA are based on reference broth microdilution (BMD), against which automated susceptibility test systems are calibrated. FDA criteria for clearance generally requires essential agreement (agreement of MIC) and categorical agreement of >89.9% with BMD. Data on the performance of automated systems for ceftriaxone MIC determination with contemporary isolates are not widely available; most systems were developed well before ESBL production was a concern. Over- and underestimation of accurate β-lactam MICs with automated susceptibility systems is likely common. This was demonstrated in two prominent studies—MERINO and CRACKLE-2—which highlight the under calling of piperacillin/tazobactam MIC and over calling of carbapenem MICs by the use of non-BMD-based susceptibility testing approaches.® (bioMérieux) for E. coli, K. pneumoniae, K. oxytoca and P. mirabilis clinical isolates with ceftriaxone MICs ≥2 mg/L (and carbapenem susceptible) collected from 668 unique patients at The Johns Hopkins Hospital over a 6 week period in 2018.Reporting ceftriaxone resistance alone, in the absence of information on ESBL production, provides insufficient data to clinicians as to when to consider alternatives to carbapenem therapy for Enterobacterales infections. We explored the hypothesis that ESBL reporting might lead to more responsible carbapenem use in a prior study. ESBL production was identified (but not reported to clinicians) using an ESBL ETESTE. coli, K. pneumoniae, K. oxytoca and P. mirabilis. This notion is reinforced by the limitations of current phenotypic ESBL tests, which are only validated for these four species.Serratia spp., Citrobacter spp., Enterobacter spp.) has led to virtually no North American ESBL prevalence estimates for these pathogens. ESBL production in other Enterobacterales may not be trivial. Investigators in Pittsburgh found that 33% of consecutive Enterobacter cloacae bloodstream isolates from 2003–04 produced SHV-type ESBLs.Enterobacter spp., Citrobacter spp. and Serratia spp., respectively, produced ESBLs.E. coli, Klebsiella spp. or P. mirabilis carried ESBL genes.The species-specific prevalence of ESBL production across the Enterobacterales order is unclear. While any Gram-negative organism has the potential to harbour ESBL genes, it is generally accepted that their presence is mostly limited to E. cloacae between 2008 and 2012 found that 63% of isolates were ESBL producing.E. coli, Klebsiella spp. and P. mirabilis, clinicians may have a false sense of security that ESBL production does not contribute to ceftriaxone resistance, leading to suboptimal selection of cefepime for the management of these infections.Understanding ESBL prevalence in these organisms is necessary to guide optimal antibiotic decision-making. For example, a study from Taiwan that evaluated 217 patients with bloodstream infections caused by E. coli producing ESBLs, sometimes exceeding half of all E. coli infections.The CDC estimate that the incidence of ESBL-producing Enterobacterales (ESBL-E) infections in the USA increased by 53% from 2012 through 2017.blaCTX-M, which, while the most common ESBL gene, is not the only ESBL gene. As such, expansion of molecular tests to include additional targets—at the very least, SHV and TEM ESBL variants are well within the ability of available platforms. Molecular panels commonly used in the USA include targets such as blaNDM, which, while epidemiologically valuable, remain exceedingly rare in the USA compared with non-CTX-M ESBLs. As an example, in a US surveillance study including 701 E. coli, Klebsiella spp. and P. mirabilis isolates with elevated third-generation cephalosporin MICs, less than 0.5% harboured blaNDM genes, whereas 20% harboured a SHV-type ESBL.blaNDM should be removed, but that relevant ESBL and p-ampC targets should be added. Inclusion of ESBL targets into these systems could be of tremendous clinical value. We believe settling for ceftriaxone non-susceptibility as a proxy for ESBL production is unacceptable. Rather, we believe accurate molecular assays that detect a comprehensive range of ESBL genes, along with other relevant β-lactamase genes, are necessary to optimize patient care.The question, therefore, is not whether to perform ESBL testing, but how. While many varieties of phenotypic tests for ESBL detection have been explored, directly testing for the presence of ESBL genes (versus relying on a surrogate) will invariably be more accurate. Similarly, molecular tests have focused solely on The work was also supported in part by a National Institutes of Health award K23-AI127935 to P.D.T.R.M.H. has received stock and salary from Accelerate Diagnostics Inc. Both P.D.T. and R.M.H. were past or present voting members of the CLSI Subcommittee on Antimicrobial Susceptibility Testing, but neither have received any financial compensation for these efforts. Neither author reports any conflicts of interest.


Brassica rapa (B. rapa) and Brassica oleracea (B. oleracea), and identified 468 putative PSGs. Our data show that, (1) PSGs are enriched in plant hormone signal transduction pathway and the transcription factor family; (2) PSGs are significantly lower expressed than randomly selected non-PSGs; (3) PSGs with tissue specificity are significantly higher expressed in the callus and reproductive tissues (flower and silique) than in vegetable tissues ; (4) the proportion of PSGs is positively correlated with the number of retained triplication gene copies, but the expression level of PSGs decay with the increasing of triplication gene copies; (5) the CG and CHG methylation levels of PSGs are significantly higher in introns and UTRs than in the promoter and exon regions; (6) the percent of transposable element is in proportion to the methylation level, and DNA methylation  has the tendency to reduce the expression of PSGs. This study provides insights into the characteristics, evolution, function, expression and methylation of PSGs in B. rapa.It is believed that positive selection is one of the major evolutionary forces underlying organism phenotypic diversification. Nevertheless, the characteristics of positively selected genes (PSGs), have not been well investigated. In this study, we performed a genome-wide analysis of orthologous genes between  The adaptive evolution of genes and genomes determine the morphology, behavior, physiological adaptation, species divergence and evolutionary innovation . TherefoBy comparing the ratio of non-synonymous substitution to the synonymous substitution (Ka/Ks), one could be able to detect many candidate genes under different selective modes. For example, the ratio of Ka/Ks greater than one  could be indicative of positive selection acting on. Nevertheless, the majority of genes in a genome, in fact, could be detected to have a ratio of Ka/Ks lower than one, providing the evidence that purifying selection (or negative selection) had occurred. Previous reports had indicated that there is a significant negative correlation between Ka/Ks and expression level ,9,10. ThDNA methylation usually refers to adding an extra methyl to the 5’-end of a nuclear acid, and it has been regarded as an important epigenetic modification in the regulation process of gene expression and transposon silencing . In mostB. rapa is one of the important vegetable crops, and is cultivated worldwide. It is a member of the family Brassicaceae  [Arabidopsis thaliana [B. rapa belongs to three diploid Brassica species in the classical triangle of U [Brassica species  have been completed [species) , includithaliana . B. rapagle of U , and undgle of U ,23. Due ompleted –26.Brassicaceae is clear now, we are wondering whether PSGs in Brassica have some specific features, such as functional importance, unique expression patterns, and different methylation levels. If so, they may play an important role in Brassica evolution and diversification. To do this, we first identified 468 PSGs candidates by comparing the ratio of Ka/Ks of orthologous genes between B. rapa and B. oleracea, and then further analyzed their distribution along chromosomes, their enrichment in pathways, expression level, and methylation properties. Our analysis provides evidence that PSGs have some unique characteristics, and may contribute to Brassica functional and phenotypic diversification. Our data also facilitate future utilization of PSGs and improvement of Brassica crop breeding.Considering the origin, relationship and genomic information in B. rapa and B. oleracea were downloaded from BRAD (http://brassicadb.cn/#/) [B. rapa (/download_genome/data/rapa/Brassica_rapa.20100830.cds.tar) and B. oleracea (/download_genome/Brassica_Genome_data/BOL11/Scaffold.seq.110729_check.cds) was downloaded and extracted from the website (http://brassicadb.cn/#/Download/) for further analysis, respectively.Gene and protein data sets for b.cn/#/) . Particuhttp://mapinspect.apponic.com/).We used MapInspect to map each positively selected gene to its corresponding chromosome location. The free soft was downloaded from the website  were downloaded from Phytozome (https://jgi.doe.gov/data-and-tools/phytozome/). Gene ontology and functional annotation was performed using the WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.pl). The GO enrichment analysis for the 468 PSGs, were performed by using hypergeometric tests and BLAST2GO with the 23817 B. rapa-B. oleracea orthologous gene pairs as the background. The GO term interaction was achieved using REVIGO (http://revigo.irb.hr/).The gene GO annotation data of http://www.kegg.jp/kegg/pathway.html).The KEGG pathway annotation was performed using the method reported , and thehttp://brassicadb.cn/#/) [A. thaliana were used to identify the B. rapa homologs by BLASTP search with the lowest E-value as the best hit. Then the identification of protein kinase related genes were obtained based on the protein kinase superfamily classification in A. thaliana [The resistance, transcription factors, flower, auxin and glucosinolate related genes were identified based on the classification of BRAD (b.cn/#/) ,33. The thaliana .https://cran.r-project.org/bin/windows/base/old/3.0.3/).The whole genome transcriptomic data from six tissues  of Chiifu-401-42 were collected and were downloaded to estimate the expression level of each gene . Venn DiB. rapa were collected and were downloaded to estimate the methylation level of PSGs in this study [The DNA methylation data from the whole genome of is study .B. rapa and B. oleracea, and then calculated the values of Ka, Ks and Ka/Ks [P = 0.0179) and Fisher exact probability test (P = 0.0150) . We fur 0.0150) , indicatB. rapa chromosomes  and PSGs with constitutive expression . The data show that, relative to PSGs with constitutive expression, PSGs with tissue specificity are significantly non-randomly distributed, indicating that tissue specific PSGs are highly unevenly located along B. rapa by WEGO (http://revigo.irb.hr/), including three different GO categories, such as cellular component, molecular function and biological process , protein kinase genes (1), and flower related genes (1) also contain PSGs, but no PSGs were detected in glucosinolate genes (P = 1.624e-05) and Fisher exact probability test (P = 5.00e-04) indicate that PSGs in B. rapa are significantly associated with gene types . The tissues with the highest expression FPKM values for PSGs were found to be in the flower and root, and these genes are mainly located at Chr03, Chr05, Chr06 and Chr09 (P = 0.2868). The expression levels of different tissues in each chromosome were shown in detail  and Fisher exact probability test (P = 0.3843)  of PSGs was not associated with chromosomal distribution according to both Chi-square test ( 0.3843) . HoweverB. rapa genome had undergone a whole genome triplication (WGT) event after splitting with Arabidopsis thaliana from their common ancestor, following by a rediploidization process [P = 0.0665; P = 0.2434; The  process ,36,40. TB. rapa, we divided a gene into five parts , and calculated the gene methylation levels using the data previously published [P = 1.43e-03 for CG, P = 6.59e-04 for CHG, and P = 1.72e-04 for CHH context, according to the KW test; P = 3.20e-04) and significantly lower methylation was detected at CG in exon (P = 4.00e-04), at CHG in UTR5 (P = 9.719e-05), and at CHH context both in promoter (P = 3.49e-02) and in UTR5 (P = 9.00e-04), according to one-side Welch Two Sample t-test ; At CHG context with insertions, the gene methylation level increases quickly in UTR5, promoter, exon, and UTR3 regions, but decreases a lot in intron regions ; at CHH context with insertions, the gene methylation level increases in promoter, but reduces much in intron regions  frequently insert into or close to functional genes, and thus can regulate genes epigenetically ,41. To ur = -0.1070, P = 0.0333), indicating that methylation level may be associated with gene expression.As we mentioned earlier, PSGs in reproductive organs  are usually higher expressed than those in vegetative organs  . To undeBecause some PSGs can only be expressed in one tissue (tissue specific), and some can express in every tissue (tissue constitutive), we are wondering if methylation levels are different between the two types of genes. Interestingly, for tissue specific PSGs, DNA methylation was only detected in flower, silique, and callus, and in these tissues, PSGs were highly methylated . For exaB. rapa and B. oleracea, has revealed some structural, evolutionary, and expression features of PSGs [B. rapa, including the chromosomal locations, biased distribution in different gene families, expression level and numbers of tissue specific expression in different tissues, correlations between WGT copies and expression levels, methylation levels in different genic regions, and the effects of insertions on gene expression levels. We also particularly investigated the difference between the two sets of PSGs in B. rapa , regarding their genomic distribution in different tissues, methylation levels at different contexts, and functional annotation. These analyses have revealed more comprehensive characteristics of PSGs in B. rapa. For example, PSGs are found to be biased distribution in different gene families, tissues, and genomic locations; PSGs expressed lower with the increase of WGT copies; PSGs are differentially methylated depending on different genic regions, methylation contexts and insertions (with or without); tissue specific PSGs and tissue constitutive PSGs differ in chromosomal locations, expression levels and methylation levels  and negatively selected genes (NSGs), the characteristics of these two set of genes have not be comprehensively investigated. Our previous analysis of orthologous genes between  of PSGs . For ins of PSGs . In thisels Figs –S16. TheBrassica species [Previous study has indicated that some particular genes, such as auxin-related genes, have been over-retained after WGT event, but the reasons and outcomes remain unclear . In this species .p53 related pathways were found more to be PSGs in mammalian genomes [The enrichment of PSGs were also been detected in other particular pathways. For example, the genes participating in the immunity and  genomes ,44. Anot genomes –45. AlthArabidopsis [Several studies had showed that significant negative correlations between Ka/Ks and expression level (abundance) were observed in bidopsis , yeast [bidopsis , and sixbidopsis . Our earbidopsis . These obidopsis , proteinbidopsis  and selebidopsis . We are P << 0.0001, student t-test; In this study, we also show that tissue specific PSGs can serve as an indicator for expression abundance, as well as methylation level. For instance, the expression levels of PSGs with specific expression are significantly lower than those PSGs with constitutive expression (B. rapa. Our data show that (1) CG context is the major DNA methylation  are three sequence contexts of the cytosine (5mC) ,14. In tion Figs  and S14;ion Figs ; (3) PSGion Figs ; (4) the B. rapa ,48–52. IIt is particularly interesting to find that the promoter regions without insertions are free of methylation, and those regions with insertions have high levels of methylation , providiBrassica species, B. rapa and B. oleracea, and then further analyzed their distribution along chromosomes, the enrichment in pathways, expression patterns, and methylation properties. Our data support that PSGs are biased distributed depending on the families, tissues, and genomic locations; the methylation levels of PSGs vary in different genic regions, methylation contexts and status of insertions; tissue specific PSGs and tissue constitutive PSGs differ in chromosomal locations, expression levels and methylation levels. Our analysis provides evidence that PSGs have some unique properties, and may contribute to Brassica functional and phenotypic diversification. Our data may also facilitate gene functional study and future utilization of PSGs in Brassica crop breeding.We have initially identified 468 positively selected gene (PSGs) candidates by comparing the orthologous genes between two S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigThe 468 PSGs can be classifiable into three main categories as follows: Biological process, cellular component, and molecular function. In some cases, one PSG may have multiple terms.(TIF)Click here for additional data file.S6 FigThe interactive graph view of GO terms in three main categories, including biological process (a), cellular component (b) and molecular function (c).(TIF)Click here for additional data file.S7 FigThe PSGs were assigned into 29 KEGG pathways within 13 clades under five major categories: Metabolism (I), genetic information processing (II), environmental information processing (III), cellular processes (IV), and organismal systems (V).(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 FigThe band in the box is the median value, and the bottom and top of each box indicates the first (lower) and third (higher) quartiles. The ends of the whiskers indicate 1.5 interquartile range of first and third quartiles, respectively. Points outside the range are mild outliers.(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 FigThe p values of a paired T-test between PSGs and random non-PSGs by the heat map at CG (a), CHG (b) and CHH (c) contexts in B. rapa.(TIF)Click here for additional data file.S13 FigThe methylation level of genes inserted by transposable elements (a) or not (b).(TIF)Click here for additional data file.S14 FigThe methylation (a) and expression (b) level of the PSGs in six tissues at three different methylation contexts.(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S16 FigComparisons of DNA methylation level of the PSGs with constitutive expression in six B. rapa tissues  at CG (a), CHG (b) and CHH (c) contexts.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Programmed cell death is an active and orderly form of cell death regulated by intracellular genes that plays an important role in the normal occurrence and development of the immune system, and pyroptosis has been found to be involved in tumorigenesis and development. However, compressive analysis and biological regulation of pyroptosis genes are lacking in cancers.Using data from The Cancer Genome Atlas, we established a score level model to quantify the pyroptosis level in cancer. Multiomics bioinformatic analyses were performed to assess pyroptosis-related molecular features and the effect of pyroptosis on immunotherapy in cancer.In the present study, we performed a comprehensive analysis of pyroptosis and its regulator genes in cancers. Most pyroptosis genes were aberrantly expressed in different types of cancer, attributed to the CAN frequency and differences in DNA methylation levels. We established a pyroptosis level model and found that pyroptosis had dual roles across cancers, while the pyroptosis levels were different among multiple cancers and were significantly associated with clinical prognosis. The dual role of pyroptosis was also shown to affect immunotherapeutic efficacy in several cancers. Multiple pyroptosis genes showed close associations with drug sensitivity across cancers and may be considered therapeutic targets in cancer.Our comprehensive analyses provide new insight into the functions of pyroptosis in the initiation, development, progression and treatment of cancers, suggesting corresponding prognostic and therapeutic utility.The online version contains supplementary material available at 10.1186/s12967-022-03313-x. Programmed cell death is an active and orderly form of cell death regulated by intracellular genes that plays an important role in the normal occurrence and development of the immune system . PyroptoIn this study, we first performed a comprehensive analysis of molecular patterns, including somatic copy number alterations, mutations, deoxyribonucleic acid (DNA) methylation, pathway enrichment, the immune microenvironment, patient survival, effects on immunotherapy and drug resistance, across cancers. We also established a model of pyroptosis-related gene levels across 33 cancer types. Our results highlight the important role of pyroptosis in cancer and provide new insight into the functions of pyroptosis and therapy in cancer.https://xenabrowser.net/datapages/): marked copy number segment, DNA methylation (Illumina human methylation 450), gene expression RNAseq (HTSeq), somatic mutation , and survival data. The lists of cancer types are presented in Additional file https://www.cancerrxgene.org/) and Cancer Therapeutics Response Portal  identifying and targeting cancer dependencies with small molecules. Immune-associated data, including immune cells and immunophenoscores, were downloaded from ImmuCellAI (Immune Cell Abundance Identifier) (http://bioinfo.life.hust.edu.cn/ImmuCellAI#!/). Three immunotherapy datasets  were from the Group on Earth Observations (GEO) dataset (https://www.ncbi.nlm.nih.gov/gds). Thirty-three pyroptosis-related genes were obtained from a previous publication . The correlations between methylation and gene expression levels were evaluated using Spearman correlation. P < 0.05 was considered significant.DNA methylation (Illumina Human Methylation 450) data of 33 cancers were downloaded from the UCSC database. Some cancers do not have normal methylation data. The differential methylation of 14 cancerous tissues and normal tissues was determined using the Wilcoxon rank test. Genes were defined as hypomethylated or hypermethylated according to the adjusted http://www.gsea-msigdb.org/gsea/index.jsp).To evaluate the pyroptosis level in cancer, we calculated the pyroptosis score using single sample gene set enrichment analysis (ssGSEA) in the R ‘performed gene set variation analysis (GSVA)’ package . The enrWe evaluated the effect of pyroptosis levels on cancer survival prognosis. The following four survival outcomes were included: overall survival (OS), disease-specific survival (DSS), progression-free interval (PFI), and disease-free interval (DFI). We calculated the hazard ratio of the pyroptosis score in each cancer using Cox regression. The pyroptosis score was categorized into high and low groups according to the median. Kaplan–Meier analysis was used to compare the survival curves of the high and low pyroptosis groups. P < 0.05 was considered significant.To investigate the association between pyroptosis and the immune microenvironment, we calculated the Pearson correlation coefficients between the pyroptosis score and immune parameters, including the immune score, stromal score, estimated score, and tumour purity.Immune cells included B cells, T cells, myeloid dendritic cells, endothelial cells, NK cells, macrophages, haematopoietic stem cells, and immune cell subsets. Some immune-related pathways, matrix/metastasis-related pathways, and DNA damage repair pathways were also evaluated.We also investigated the effect of the pyroptosis level on survival prognosis in three GEO datasets .To evaluate the association between the pyroptosis level and small-molecule drugs, we calculated the Pearson correlation coefficients between the pyroptosis score and drug sensitivity percentage calculated by the percent viability curve approach. We identified and targeted cancer dependencies with small molecules using the Genomics of Drug Sensitivity in Cancer and Cancer Therapeutics Response portal dataset.In this study, we identified thirty-three genes that play critical roles in regulating pyroptosis by reviewing previous studies Fig. . The lisThe alteration rates of GSDMD and NLRP3 were similar (6.3% and 6.0%). The alteration rates of other genes ranged from 01 to 3%, and CASP6 had the lowest alteration level (0.7%). We further analysed the alteration patterns among all cancer types and found differences in alterations among different cancer types. UCEC showed alterations in all pyroptosis genes, while the alteration levels of all genes related to kidney chromophobe (KICH) and mesothelioma (MESO) were very low Fig. A. IndiviThe gene mutations mainly consisted of missense mutations, nonstop mutations and multihit mutations. The mutation levels of these genes ranged from 0 to 3%. NLRP3 showed the highest mutation level (3%), and NLRP7, NLRP1, CASP8 and NLRC4 showed the same mutation frequencies 2%). The remaining genes had mutation frequencies of only 1%  is known to play a role in tumorigenesis development, and we further evaluated the association between CNV and pyroptosis gene expression. Pearson correlation analysis indicated that most pyroptosis genes were correlated with CNV in most cancers Fig. B. For exWe also assessed the methylation levels of pyroptosis genes in tumour and normal tissues. We found that pyroptosis genes exhibited complex methylation patterns in the 14 types of cancers Fig. C, and onTo further explore the role of pyroptosis in the development of tumours and understand pyroptosis-related biological processes, we built an estimated model of pyroptosis levels in all cancers based on enrichment scores by single-sample GSEA. We observed that LAML had the highest pyroptosis level, while pheochromocytoma and PGL had the lowest pyroptosis levels Fig. A. We furP < 0.001), SKCM (P < 0.001), LGG (P < 0.001), PAAD (P = 0.002), UVM (P = 0.006), BLCA (P = 0.029), THYM (P = 0.043), and SARC (P = 0.048), while the pyroptosis score was significantly associated with DSS in seven types of cancers . A significant correlation between the pyroptosis score and PFI was also observed in seven cancers , LGG (P < 0.001), GBM (P < 0.001), THYM (P = 0.002), PAAD (P = 0.002), SKCM (P = 0.017), and BLCA (P = 0.029). Among these cancers, the elevated pyroptosis score was associated with poor survival outcomes for patients with twelve types of cancer  and the tumour mutation burden (TMB), which were suggested to be associated with the prognosis of multiple cancers after immunotherapy. We observed that the pyroptosis level was positively associated with MSI in COAD, STAD, THCA, and PRAD, while negative relationships were observed in LIHC, KIRP, OV, PADD, TGCT and DLBC . The results showed that a high pyroptosis level was associated with poor OS in primary bladder cancer  using the CTRP and GDSC datasets. We selected the top 30 compounds targeting pyroptosis-related genes (|r|> 0.3). The results showed that the expression levels of IL-6, IL18 and GSDME were positively associated with these compounds, while CASP4 was positively correlated with sensitivity to 26 cancer drugs Fig. D. AIM2, Pyroptosis is an inflammatory programmed cell death method that is characterized by the activation of inflammatory caspases  in inflammasomes and the secretion of inflammatory cytokines such as interleukin-1β and interleukin-18 . ResearcThe molecular mechanism of pyroptosis in tumorigenesis and development remains unclear. However, the correlations of pyroptosis with some functions and pathways may provide some important clues. The GSVA results indicated that the pyroptosis genes were mainly enriched in two components. The first was inflammation-related pathways, such as the IL6/JAK/STAT3 signalling, inflammatory response, IL2/STAT5 signalling, TNF-alpha signalling via NFKB, and KRAS signalling pathways, which have been proven to be associated with tumours. The inflammatory response can promote the occurrence and progression of tumours . SustainWe further analysed the correlation between drug sensitivity and pyroptosis gene expression. A previous study reported that GSDMD is activated by the cleavage of Caspase-3 and induces pyroptosis in response to tumour chemotherapy drugs . Human nOur study has several advantages over previous studies on certain cancers. First, previous studies focused on the development and validation of prognostic models involving certain cancer types –35. Our Some study limitations should be addressed. The effect of the pyroptosis level on immunotherapy in several cancers was not assessed due to a lack of available immunotherapy data. In addition, this study did not explore the molecular mechanisms of pyroptosis in cancers. Future studies should validate the effect of the pyroptosis level on immunotherapy in more cancers, and the potential molecular mechanisms should also be explored to identify potential treatment targets in certain cancers.In the present study, we performed a comprehensive analysis of pyroptosis and genes regulating pyroptosis in cancers. Our study found that (1) aberrantly expressed pyroptosis genes are mainly attributed to CAN frequency and differences in DNA methylation levels in cancer. (2) Moreover, the established pyroptosis level model based on the ssGSEA method uncovers the dual roles of pyroptosis in different cancers, and (3) the pyroptosis level is associated with clinical prognosis in multiple cancers, especially LGG, GBM, KIRC, PAAD, SKCM, UVM, BLCA, COAD, THYM, and SARC. (4) The dual role of pyroptosis also affects the immunotherapeutic efficacy in several cancers, including bladder cancer, urothelial carcinoma, and non-squamous non-small cell lung cancer, and (5) six pyroptosis genes  are closely correlated with drug sensitivity across cancers and may be considered therapeutic targets in cancer. Our comprehensive analysis highlighted the possibility of pyroptosis-based cancer therapeutic strategies.Additional file 1: Table S1. Lists of cancer types. Table S2. The 33 pyroptosis-associated genes used for classification. Table S3. Association between pyroptosis genes mutation and prognosis among cancers. Table S4. Methylation differences for pyroptosis genes in cancers. Table S5. Correlation of DNA methylation with pyroptosis genes expression. Table S6. CTRP drug sensitivity for pyroptosis genes in cancers. Table S7. GDSC drug sensitivity for pyroptosis genes in cancers.Additional file 2: Fig. S1. Protein–protein interaction network of pyroptosis genes. Fig. S2. Gene alteration of pyroptosis genes in each cancer. A: heterozygous copy number variation in each cancer. B: homozygous copy number variation in each cancer C: The gene mutation frequency of pyroptosis genes in overall cancer. Fig. S3. Heatmap showed the mutation frequency of pyroptosis in each cancer. Fig. S4. Aberrant expression of pyroptosis-related genes among cancers. Fig. S5. Pyroptosis and survival prognosis based on Kaplan–Meier analysis. A: elevated pyroptosis level favors survival in ESCA, GBM, HNSC, KIRC, LAML, LGG, LUSC, PAAD, THYM, UCES, UCS, UVM. B: Elevated proptosis level disfavors survival in BRCA, KICH, MESO, SARC, SKCM, STAD, THCA, BLCA. Fig. S6. Risky score of pyroptosis genes in each cancer. Fig. S7. KEGG pathway enrichment of pyroptosis in several cancer. A–F: BRCA, GBM, HNSC, KIRC, LUSC, PAAD. Fig. S8. Correlations of pyroptosis level with immune cells infiltration. A: B cell, T cell, myeoid dendritic cell, endothelial cell. B: immune cell cibersort. Fig. S9. Correlations of pyroptosis level with microsatellite Instability (A) and tumor mutation burden (B).


Trypanosoma cruzi infection in people with HIV (PWH). The reported prevalence of T. cruzi infection in PWH living in endemic countries ranges from 1–28% and is likely similar in at-risk US populations. While classic cardiac and gastrointestinal presentations of chronic Chagas disease occur in PWH, PWH are additionally at risk for a severe and often fatal form of T. cruzi-mediated disease called reactivation disease. T. cruzi reactivation typically occurs in PWH with low CD4 counts and poor virologic control. National HIV guidelines in several endemic South American countries recommend that all PWH be screened for T. cruzi infection at the time of HIV diagnosis; however, this recommendation is not widely implemented. The early detection of T. cruzi infection in PWH is critical as the sequelae of Chagas disease, including T. cruzi reactivation, may be preventable through the restoration of robust cellular immunity via the initiation of antiretroviral therapy and the appropriate use of antitrypanosomal therapy.Many questions remain unanswered regarding the epidemiology, pathophysiology, diagnosis, treatment, and monitoring of  Trypanosoma cruzi. More than six million people are estimated to be infected in the Americas , or other fluids or tissue [T. cruzi infection [r tissue ,37. Hower tissue ,20. Evennfection .T. cruzi-endemic regions, Del Castillo et al. published the first recognized case of reactivated T. cruzi infection in a PWH in 1990 [T. cruzi antibodies in the CSF of PWH in 1988 [T. cruzi trypomastigotes in the CSF of two PWH in 1989 [T. cruzi reactivation describe PWH naïve to ART; however, no studies evaluating risk of reactivation in PWH taking versus not taking ART exist.Cases of symptomatic reactivation are reported in patients with long-standing suppressed cellular immunity. A 1982 publication first reported a case of Chagas disease reactivation in a patient who was immunocompromised due to therapy for a hematological malignancy . Others  in 1990  (though  in 1988  and T. c in 1989 ). NearlyT. cruzi reactivation typically manifests as nodular lesions  comprising macrophages, neutrophils, microglia, astrocytes, and perivascular lymphocytes (see “Diagnosis of CNS Reactivation” section below) [T. cruzi infection of and multiplication in astrocytes [3) and is considered to be an opportunistic infection in people with AIDS. IL-18 serum concentrations may influence or predict the risk of T. cruzi reactivation in PWH [Reactivation disease most frequently presents in the CNS as space-occupying cerebral lesions and/or meningoencephalitis (75–80% of cases) ,45. CNS n below) . Astrocytrocytes . CNS reatrocytes   [Reactivation manifesting as cardiac involvement  also occurs (10–55% of cases) ,14. Dists differ . Less cos differ , gastrois differ ,50, peris differ , or skins differ ,52. The h (>75%) ,44,45, wScreening for chronic Chagas disease in PWH: HIV guidelines in several South American countries where Chagas disease is endemic, such as in Argentina and Brazil, recommend that all PWH be screened for T. cruzi infection at the time of HIV diagnosis or entry to care through the use of sensitive T. cruzi serologic testing [ testing . However testing ,54,55. D testing .Diagnosis of chronic Chagas disease in PWH: Current recommendations for initial diagnostic testing for chronic Chagas disease among PWH are the same as for the general population, i.e., confirmed diagnosis requires positive results by two or more serologic tests based on different antigens or techniques [T. cruzi serology may be negative in co-infected PWH with significant immunocompromise; negative serology in the setting of a high index of suspicion should prompt further testing [T. cruzi parasitemia than HIV-negative people with chronic T. cruzi infection [T. cruzi infection is variable but is generally higher in the coinfection setting because of the higher parasite load distribution [chniques . Importa testing ,44. Evennfection ,32. The nfection ,32. PCR ribution ,58,59.Diagnosis of T. cruzi reactivation disease in PWH: The early diagnosis of reactivated T. cruzi infection is critical. The differential diagnosis of meningoencephalitis and the cerebral space occupying the lesions  in PWH is broad, and direct microscopy is often negative in the setting of CNS reactivation [T. cruzi serologic tests can be negative in severely immunocompromised PWH. Traditional microscopic diagnosis is inexpensive but is also highly operator dependent and has relatively low sensitivity [T. cruzi infection cannot be absolutely ruled out by negative serology and microscopy, especially when reactivation disease is suspected [tivation . As mentsitivity ,35. Thususpected ,44,45,60uspected ,61. A hiuspected .T. cruzi-coinfected patients. Although investigators have speculated about the utility of the pre-emptive antitrypanosomal treatment of asymptomatic HIV–T. cruzi-coinfected patients based on high peripheral blood parasite load, there is no consensus on the treatment criteria in the absence of symptomatic reactivation [In transplant recipients with pre-existing chronic Chagas disease, recommendations include routine monitoring with quantitative molecular methods as well as testing during febrile episodes; the finding of rising parasite loads in serial specimens is accepted as a sufficient indicator of reactivation necessitating antitrypanosomal therapy ,64. Simitivation . When efNew developments in T. cruzi infection diagnosis in PWH: One new molecular assay with many of the advantages of PCR but without the requirement of expensive equipment is called the loop-mediated isothermal amplification method (LAMP). Argentine investigators recently developed such a test to detect T. cruzi genetic material, the T. cruzi Loopamp kit (Tc LAMP) [T. cruzi antigen test  [T. cruzi serologic tests, Chunap detected 100% (7/7) of PWH with reactivation, 91.7% (11/12) of PWH with moderate parasitemia, and 41.7% (5/12) of PWH with negative parasitemia. Finally, for PWH who are suspected of having reactivation disease and who are being evaluated in high-resource settings, clinicians can consider tissue sample evaluation via the sequencing of the internal transcribed spacer 2 and D2 regions of the 28S rRNA gene, particularly in PWH with an unrevealing microscopic evaluation of blood and CSF specimens [T. cruzi but also other pathogens that share the D2 subunit and are often involved in the differential diagnosis of meningoencephalitis in PWH, such as Toxoplasma gondii, Cryptococcus spp., Histoplasma spp., and Leishmania spp. The ability to identify one of many potential pathogens with a single test is advantageous for the timely institution of the appropriate treatment and improved outcomes.Tc LAMP) . Compare Chunap) . Of 31 Ppecimens . The D2 Diagnosis of CNS reactivation: CNS reactivation in PWH is usually suspected due to presentation with neurologic symptoms in conjunction with abnormalities on head imaging in the setting of Chagas disease risk factors. Importantly, the absence of lesions on a computed tomography of the head (CTH) does not rule out CNS involvement. If available, magnetic resonance imaging (MRI) of the brain is the preferred imaging study [T. cruzi reactivation. For instance, cerebral lesions due to Toxoplasma encephalitis are generally indistinguishable from those caused by T. cruzi reactivation, though some experts believe that Toxoplasma more often causes cortical or basal ganglia lesions, while T. cruzi tends to cause white matter or subcortical lesions [Toxoplasma and T. cruzi CNS disease can occur simultaneously [ng study . Rim-enhng study . Because lesions . Toxoplaaneously ,69. The aneously ,45. CSF aneously ,45. If laneously . If the aneously .T. cruzi reactivation disease is likely preventable with timely, sustained immune reconstitution via the administration of ART to coinfected patients. Whether pre-emptive antitrypanosomal therapy further decreases reactivation risk is unclear. With no proven test of a parasitological cure, T. cruzi-infected PWH must be considered to remain at risk for reactivation, even after receiving a course of antitrypanosomal therapy.T. cruzi infection. Currently there are no specific guidelines for treatment regimens for co-infected PWH, though some experts recommend longer therapy derations [If reactivation occurs, immediate antitrypanosomal therapy plus the initiation or optimization of ART has been shown to reduce mortality ,44,45. Ierations .Benznidazole: Oral benznidazole (5–7 mg/kg/day in 2 doses for 60–90 days) is the most used antitrypanosomal agent and is usually chosen as the first-line treatment for chronic or reactivated Chagas disease in adult patients. However, our understanding of benznidazole pharmacokinetics and pharmacodynamics is limited, particularly in PWH. Consequently, the treatment of T. cruzi CNS reactivation disease is guided by limited empirical knowledge. The usual benznidazole dose may be suboptimal in PWH with T. cruzi meningoencephalitis. Investigators measured CSF and plasma benznidazole levels in one Argentine case series of six patients, from whom they obtained 6 CSF and 19 plasma samples [T. cruzi from the CSF [T. cruzi reactivation disease at 32 weeks of pregnancy [ samples . Only th the CSF ). Import the CSF , just asregnancy .Nifurtimox: Clinical experience with oral nifurtimox (8–10 mg/kg/day in 2 or 3 doses for 60–120 days) is even more limited than that with benznidazole [nidazole ,73. NifuNew/repurposed therapies under study: Benznidazole remains the first-line drug for the treatment of all forms of Chagas disease (regardless of HIV status). However, several new and re-purposed medications are under study. In vitro studies indicate that protease inhibitors such as lopinavir and nelfinavir may have some direct activity against T. cruzi [T. cruzi . ReportsT. cruzi ,76,77, fT. cruzi , and allT. cruzi ,80. No rStarting ART and risk of immune reconstitution inflammatory syndrome: Restoring robust cellular immunity is critical to survival in symptomatic reactivation disease, but data are lacking regarding the optimal timing of ART initiation and the choice of specific agents. Because benznidazole and nifurtimox are thought to be primarily metabolized in the liver by the cytochrome P450 system [T. cruzi co-infection ; thus, T. cruzi secondary chemoprophylaxis in PWH and low CD4 counts: The utility of secondary prophylaxis to prevent the reactivation of T. cruzi infection in co-infected PWH with low CD4 counts remains unclear. Secondary prophylaxis for PWH treated for T. cruzi infection is often recommended in countries with relatively high T. cruzi prevalence [T. cruzi CNS reactivation disease, two PWH were successfully treated with benznidazole secondary prophylaxis at 5 mg/kg/d for three days a week until achieving CD4 counts higher than 200 cells/mm3 and an undetectable HIV viral load [evalence . In one ral load .T. cruzi co-infection. A better understanding of these issues can help reduce morbidity and mortality related to co-infection in both endemic and non-endemic countries. Specific topics include (1) better defining the prevalence of and risk factors for HIV–T. cruzi co-infection to target interventions to those who are most at risk; (2) elucidating the immunologic effector mechanisms underlying reactivation; (3) determining whether parasite factors, such as specific genotype or polyclonality, alter the risk or location of reactivation; (4) the optimization of algorithms for T. cruzi screening and diagnosis, particularly in PWH with low CD4 counts; (5) placing therapeutics of coinfected patients on a more solid footing, including best practices for therapeutic drug level monitoring, identifying factors leading to low CSF drug levels, and developing better treatment regimens; (6) evaluating criteria and regimens for secondary chemoprophylaxis; and (7) ensuring that candidate test-of-cure assays are evaluated and function acceptably in coinfected PWH with significant immunosuppression.Many gaps remain in our knowledge of the epidemiology, pathophysiology, diagnosis, treatment, and monitoring of HIV–