WGS Analysis Research Articles

Discovery and characterization of novel lipopeptides produced by Virgibacillus massiliensis with biosurfactant and antimicrobial activities

The study aimed to evaluate the biosurfactants (BSs) production by SM-23 strain of Virgibacillus identified by phenotypical and WGS analysis as Virgibacillus massiliensis. We first demonstrated the lipopeptides production by Virgibacillus massiliensis specie and studied their biochemical and molecular analysis as well as their biological potential. The GC–MS analysis indicated that methyl.2-hyroxydodecanoate was the major fatty acid compound with 33.22%. The maximum BSs production was obtained in LB medium supplemented by 1% olive oil (v/v) at 30 °C and 5% NaCl with 1.92 g/l. The obtained results revealed the significant biosurfactants/bioemulsifier potential compared to triton X100 with E24 of 100%, and an emulsification stability SE of 83%. The lipopeptides types were identified by FTIR analysis. A strong antimicrobial action was observed by the produced lipopeptides by the agar diffusion method against E.coli, K. pneumoniae, S. aureus, Fusarium sp, Alternaria sp, and Phytophtora sp. The complete genome sequencing showed genes involved in the synthesis of multiple compounds identified as amphipathic cyclic lipopeptides such as locillomycin/locillomycin B/locillomycin C and bacillibactin. Our results highlighted significant lipopeptides properties displayed by V. massiliensis that can be exploited to develop a novel strategy in the formulation of natural biocidal and fungicidal agents.

Whole genome sequence analysis of population structure and insecticide resistance markers in Anopheles melas from the Bijagós Archipelago, Guinea-Bissau

BackgroundAnopheles melas is an understudied malaria vector with a potential role in malaria transmission on the Bijagós Archipelago of Guinea-Bissau. This study presents the first whole-genome sequencing and population genetic analysis for this species from the Bijagós. To our knowledge, this also represents the largest population genetic analysis using WGS data from non-pooled An. melas mosquitoes.MethodsWGS was conducted for 30 individual An. melas collected during the peak malaria transmission season in 2019 from six different islands on the Bijagós Archipelago. Bioinformatics tools were used to investigate the population structure and prevalence of insecticide resistance markers in this mosquito population.ResultsInsecticide resistance mutations associated with pyrethroid resistance in Anopheles gambiae s.s. from the Bijagós were absent in the An. melas population, and no signatures of selective sweeps were identified in insecticide resistance-associated genes. Analysis of structural variants identified a large duplication encompassing the cytochrome-P450 gene cyp9k1. Phylogenetic analysis using publicly available mitochondrial genomes indicated that An. melas from the Bijagós split into two phylogenetic groups because of differentiation on the mitochondrial genome attributed to the cytochrome C oxidase subunits COX I and COX II and the NADH dehydrogenase subunits 1, 4, 4L and 5.ConclusionsThis study identified an absence of insecticide-resistant SNPs common to An. gambiae in the An. melas population, but did identify structural variation over insecticide resistance-associated genes. Furthermore, this study presents novel insights into the population structure of this malaria vector using WGS analysis. Additional studies are required to further understand the role of this vector in malaria transmission.Graphical abstract

Genetic basis of macrolide resistance in porcine Pasteurella multocida isolates from the German national resistance monitoring program GERM-Vet 2008-2021.

To analyse porcine Pasteurella multocida isolates obtained from the national resistance monitoring program GERM-Vet 2008-2021 in Germany for phenotypic and genotypic macrolide resistance. The antimicrobial susceptibility of the isolates was determined by broth microdilution according to CLSI standards. Closed genomes were obtained by WGS via Illumina MiSeq and MinION platforms followed by a hybrid assembly. Of 1114 porcine P. multocida isolates, only four isolates (0.36%), one each from 2008, 2010, 2019 and 2021, exhibited resistance to at least one macrolide tested. The isolate from 2010 was only resistant to erythromycin and WGS analysis neither revealed a macrolide resistance gene nor a macrolide resistance-mediating mutation. The isolates from 2008 and 2019 were resistant to erythromycin, tilmicosin, tildipirosin, tulathromycin and gamithromycin and showed either only the A2058G mutation in all six 23S rRNA operons or the chromosomally located macrolide resistance genes msr(E) and mph(E), respectively. The isolate from 2021 was resistant to erythromycin, tulathromycin, gamithromycin and tylosin and carried a novel integrative and conjugative element of 64 966 bp, designated Tn7730, in its chromosomal DNA. It harboured the macrolide resistance genes mef(C), mph(G) and estT, the lincosamide resistance gene lnu(H), and the tetracycline resistance gene tet(Y), the last two were detected for the first time in P. multocida. Macrolide resistance in German porcine P. multocida can be due to resistance-mediating mutations or resistance genes. The presence of the novel Tn7730 carrying three different macrolide resistance genes is alarming and should be monitored.

Genomic insights into prevalence of virulence and multi drug resistance genes in milk borne Klebsiella pnuemoniae: Face of emerging resistance to last resort antibiotics

Spread of hypervirulent and multi-drug resistant Klebsiella pneumoniae in raw milk is public health concern due to its potential impact on food safety and public health. Therefore, this study investigated antibiotic susceptibility test (AST), antibiotic resistance genes (ARGs), mutations conferring ARGs, virulence factor and plasmid replicons to check prevalence of fosfomycin resistant MDR K. pneumoniae isolated from raw milk samples collected from Saurashtra region of Gujarat, India. K. pneumoniae isolated from raw milk and subjected to disk diffusion assay. From that, MDR along with fosfomycin resistant isolates were analysed for multi locus sequence typing, presence of ARGs, mutations conferring resistance, virulence factors and plasmid replicon types by using its whole genome sequence. Results shows that, among 32 K. pneumoniae, 8 were phenotypically resistant to fosfomycin. As per WGS analysis, 8 MDR isolates were assigned into different sequence types such as ST3321, ST37, ST2715, ST1087, ST3157, ST299 and ST29. Among that, ST37 is well recognized MDR high risk clone reported worldwide and first time reported from raw milk of Saurashtra region of Gujarat, India. ARGs responsible for resistance to fosfomycin (fosA) were found in all 8 isolates. Other ARGs such as blaSHV, kdeA, OqxA, OqxB, dfrA1, sul1, qnrB4, aadA2 and ere(A) were also detected. High diversity of virulence factors was also identified by detection of genes encoding virulence factors related to iron uptake such as entE, fepD, entA, entB, Irp2, fepG, ybtU, ybtP, fepC, ybtA, ybtE, fepB, ybtS, fyuA, ybtQ, ybtT, ybtX, Irp1, adherence such as yagZ/ecpA, yagV/ecpE, yagX/ecpC, yagV/ecpE, ykgK/ecpR and invasion such as fimA, pla, fimC, fimH, fimB, fimE were detected in eight genomes. Mutations in murA, uhpT and glpT conferring a fosfomycin resistance were also present in genomes of 8 K. pneumoniae. IncF was the most common plasmid replicon type detected in all 8 genomes. The study reports high diversity of virulent and multidrug resistant K. pneumoniae in raw milk. Hence, genomic surveillance plans are urgently required for food borne pathogens.

Two outbreaks and sporadic occurrences of Candida auris from one hospital in China: an epidemiological, genomic retrospective study.

To investigate the clinical relevance, origin, transmission, and resistance of Candida auris (C. auris) isolates from two outbreaks and sporadic occurrences from one hospital in China. A total of 135 C. auris isolates were collected. Clinical characteristics were obtained and antifungal susceptibility testing (AFST) was performed using the method of broth microdilution. Phylogenetic tree, WGS analysis, and single nucleotide polymorphisms (SNPs) were used to determine the origin, transmission, and resistance mechanisms. A total of 31 patients (91.2%, 31/34) received invasive medical procedures and 13 patients (38.2%, 13/34) had antifungal agents before C. auris infection/colonization, except one patient whose clinical information was missing. Only 4 cases of C. auris candidemia were observed. 18 patients died, 13 patients recovered, and the outcomes of 3 patients were not available. A total of 35 C. auris isolates, which were successfully cultivated and the first isolated or harbored specific drug-resistant phenotype from each patient, were selected to be sequenced and further analyzed. C. auris isolates presented low genetic variability and belonged to clade I, possibly originating from BJ004-H7 in Beijing. All 35 isolates were resistant to Fluconazole (FCZ) and amphotericin B (AMB), and 3 isolates were resistant to caspofungin (CAS). Mutations in ERG11 and FKS1 were linked to reduced azole and echinocandin susceptibility, respectively. Two outbreaks of highly clonal, multidrug-resistant C. auris isolates within the medical facility were reported. The intensive performance of disinfection measures helped block in-hospital transmission. Understanding the epidemiology, drug resistance and management of C. auris will be helpful for implementing effective infection control and treatment strategies.

Insights into macrolide resistance in Arcobacter butzleri: potential resistance mechanisms and impact on bacterial fitness and virulence.

Macrolides are recommended for treating the emerging enteropathogen Arcobacter butzleri; nonetheless, this bacterium often exhibits highly variable resistance rates, and the mechanisms behind this resistance phenotype remain largely unexplored. To understand the phenotypic and genotypic consequences associated with the acquisition of erythromycin resistance in A. butzleri, as well as the effects on the fitness of this species. Resistant strains resulting from spontaneous mutations and adaptive laboratory evolution under increasing erythromycin concentrations were examined regarding their cross-resistance and collateral susceptibility profiles. Genetic causes of phenotypic antibiotic resistance were analysed by sequencing and bioinformatics, with functional correlation through ethidium bromide accumulation assays. Growth profiles in the presence and absence of erythromycin, motility and biofilm formation abilities were assessed to detect potential changes in fitness and virulence. Clones from spontaneous mutation rate evolution demonstrated decreased susceptibility to erythromycin and other classes of antibiotics, associated with mutations in the transcriptional repressor areR, causing overexpression of the AreABC efflux pump. In turn, WGS analysis of the evolved strain showed additional mutations in the ribosomal proteins L4 and L22 and in the areR gene. Furthermore, the acquisition of macrolide resistance altered A. butzleri virulence and entailed a high biological cost. The findings of this study have proved that efflux activity contributes synergistically with mutations in the ribosomal proteins L4 and L22 to A. butzleri's high-level macrolide resistance. The results further suggest an impact on the bacterial physiology and virulence, with the increased fitness cost justifying the low worldwide prevalence of high-level resistant circulating strains.

The novel 2024 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations and superseded WHO N. gonorrhoeae reference strains-phenotypic, genetic and reference genome characterization.

MDR and XDR Neisseria gonorrhoeae strains remain major public health concerns internationally, and quality-assured global gonococcal antimicrobial resistance (AMR) surveillance is imperative. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) and WHO Enhanced GASP (EGASP), including metadata and WGS, are expanding internationally. We present the phenotypic, genetic and reference genome characteristics of the 2024 WHO gonococcal reference strains (n = 15) for quality assurance worldwide. All superseded WHO gonococcal reference strains (n = 14) were identically characterized. The 2024 WHO reference strains include 11 of the 2016 WHO reference strains, which were further characterized, and four novel strains. The superseded WHO reference strains include 11 WHO reference strains previously unpublished. All strains were characterized phenotypically and genomically (single-molecule PacBio or Oxford Nanopore and Illumina sequencing). The 2024 WHO reference strains represent all available susceptible and resistant phenotypes and genotypes for antimicrobials currently and previously used (n = 22), or considered for future use (n = 3) in gonorrhoea treatment. The novel WHO strains include internationally spreading ceftriaxone resistance, ceftriaxone resistance due to new penA mutations, ceftriaxone plus high-level azithromycin resistance and azithromycin resistance due to mosaic MtrRCDE efflux pump. AMR, serogroup, prolyliminopeptidase, genetic AMR determinants, plasmid types, molecular epidemiological types and reference genome characteristics are presented for all strains. The 2024 WHO gonococcal reference strains are recommended for internal and external quality assurance in laboratory examinations, especially in the WHO GASP, EGASP and other GASPs, but also in phenotypic and molecular diagnostics, AMR prediction, pharmacodynamics, epidemiology, research and as complete reference genomes in WGS analysis.

IPS cell generation-associated point mutations include many C > T substitutions via different cytosine modification mechanisms

Genomic aberrations are a critical impediment for the safe medical use of iPSCs and their origin and developmental mechanisms remain unknown. Here we find through WGS analysis of human and mouse iPSC lines that genomic mutations are de novo events and that, in addition to unmodified cytosine base prone to deamination, the DNA methylation sequence CpG represents a significant mutation-prone site. CGI and TSS regions show increased mutations in iPSCs and elevated mutations are observed in retrotransposons, especially in the AluY subfamily. Furthermore, increased cytosine to thymine mutations are observed in differentially methylated regions. These results indicate that in addition to deamination of cytosine, demethylation of methylated cytosine, which plays a central role in genome reprogramming, may act mutagenically during iPSC generation.

Emergence of an IncX3 plasmid co-harbouring the carbapenemase genes blaNDM-5 and blaOXA-181.

The spread of transmissible plasmids with carbapenemase genes has contributed to a global increase in carbapenemase-producing Enterobacterales over the past two decades, with blaNDM and blaOXA among the most prevalent carbapenemase genes. To characterize an Escherichia coli isolate co-carrying blaNDM-5 and blaOXA-181 (JBEHAAB-19-0176) that was isolated in the Japan Antimicrobial Resistant Bacterial Surveillance in 2019-20, and to evaluate the functional advantage of carrying both genes as opposed to only one. The whole-genome sequence of the isolate was determined using long- and short-read sequencing. Growth assay and co-culture experiments were performed for phenotypic characterization in the presence of different β-lactam antibiotics. WGS analysis showed that blaNDM-5 and blaOXA-181 were carried by the same IncX3 plasmid, pJBEHAAB-19-0176_NDM-OXA. Genetic characterization of the plasmid suggested that the plasmid emerged through the formation of a co-integrate and resolution of two typical IncX3 plasmids harbouring blaNDM-5 and blaOXA-181, which involved two recombination events at the IS3000 and IS26 sequences. When cultured in the presence of piperacillin or cefpodoxime, the growth rate of the transformant co-harbouring blaNDM-5 and blaOXA-181 was significantly higher than the transformant with only blaNDM-5. Furthermore, in co-culture where the two blaNDM-5-harbouring transformants were allowed to compete directly, the strain additionally harbouring blaOXA-181 showed a marked growth advantage. The additional carriage of blaOXA-181 confers a selective advantage to bacteria in the presence of piperacillin and cefpodoxime. These findings may explain the current epidemiology of carbapenemase-producing Enterobacterales, in which bacteria carrying both blaNDM-5 and blaOXA-48-like genes have emerged independently worldwide.

Prevalence and genetic basis of Mycobacterium tuberculosis resistance to pretomanid in China

BackgroundPretomanid is a key component of new regimens for the treatment of drug-resistant tuberculosis (TB) which are being rolled out globally. However, there is limited information on the prevalence of pre-existing resistance to the drug.MethodsTo investigate pretomanid resistance rates in China and its underlying genetic basis, as well as to generate additional minimum inhibitory concentration (MIC) data for epidemiological cutoff (ECOFF)/breakpoint setting, we performed MIC determinations in the Mycobacterial Growth Indicator Tube™ (MGIT) system, followed by WGS analysis, on 475 Mycobacterium tuberculosis (MTB) isolated from Chinese TB patients between 2013 and 2020.ResultsWe observed a pretomanid MIC distribution with a 99% ECOFF equal to 0.5 mg/L. Of the 15 isolates with MIC values > 0.5 mg/L, one (MIC = 1 mg/L) was identified as MTB lineage 1 (L1), a genotype previously reported to be intrinsically less susceptible to pretomanid, two were borderline resistant (MIC = 2–4 mg/L) and the remaining 12 isolates were highly resistant (MIC ≥ 16 mg/L) to the drug. Five resistant isolates did not harbor mutations in the known pretomanid resistant genes.ConclusionsOur results further support a breakpoint of 0.5 mg/L for a non-L1 MTB population, which is characteristic of China. Further, our data point to an unexpected high (14/475, 3%) pre-existing pretomanid resistance rate in the country, as well as to the existence of yet-to-be-discovered pretomanid resistance genes.

Abstract GS03-04: Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics

Abstract Background: Invasive lobular carcinoma (ILC) of the breast is the second most common histologic subtype of breast cancer (BC), following invasive ductal carcinoma of no special type (IDC-NST). The hallmark histologic feature of ILC is cellular discohesiveness, the result of bi-allelic inactivation of CDH1, and represents an important genotypic-phenotypic correlation in BC. Although most ILCs harbor CDH1 loss-of function mutations associated to loss-of-heterozygosity (LOH) of the wild type-allele, a subset of ILCs lack these alterations despite displaying a typical lobular phenotype. Here, we sought to identify alternative molecular mechanisms converging on CDH1 inactivation by employing an integrative artificial intelligence (AI) and genomics approach. Materials and Methods: A genomics-driven AI-based algorithm using hematoxylin and eosin (H&E) whole slide images (WSIs) as input, previously developed to detect bi-allelic CDH1 mutations (inactivating mutation associated to LOH) in BC was employed. WSIs of 1,057 BCs including ILCs (n=187) and non-lobular BCs (n=870) previously subjected to FDA-cleared tumor/normal targeted sequencing were subjected to analysis with the AI-based algorithm. Cases predicted to harbor CDH1 bi-allelic mutations by the AI-model but lacking CDH1 bi-allelic mutations by targeted sequencing were assessed through targeted sequencing data re-analysis, CDH1 gene promoter methylation evaluation and/or whole genome sequencing analysis. Results: AI-based analysis WSIs corresponding to 1,057 BCs resulted in the identification of 34 cases found to lack CDH1 bi-allelic mutations by targeted sequencing but predicted to harbor these genetic alterations by the AI-based model. CDH1 gene promoter methylation assessment revealed CDH1 promoter methylation in 18 cases. Targeted sequencing data reanalysis revealed other genetic mechanisms of CDH1 inactivation including CDH1 homozygous deletions (n=3), intragenic deletion with LOH (n=1), and likely pathogenic non-coding CDH1 alterations associated with LOH (n=2). WGS analysis of an ILC revealed a novel deleterious CDH1 fusion stemming from translocation t(13;16), resulting in loss of the 5’UTR, transcription start site and exons 1 and 2 of CDH1, associated with complete loss of E-cadherin protein expression. Taken together, we identified alternative/novel mechanisms of bi-allelic CDH1inactivation in 74% (25/34) cases analyzed. Conclusions: By applying an AI-based algorithm trained to detect a genetic alteration (i.e., CDH1 bi-allelic mutations), we were able to identify alternative epigenetic and genetic molecular mechanisms of CDH1 inactivation in ILCs, including novel non-coding CDH1 genetic alterations and a new inactivating CDH1 fusion gene. These findings indicate that molecular mechanisms affecting a single gene or process converging on the same phenotype can be unveiled by the integration of AI and genomics, highlighting the robustness of this approach for the discovery of novel biology. Citation Format: Fresia Pareja, Higinio Dopeso, Yikan Wang, Andrea Gazzo, David Brown, Pier Selenica, Jan Bernhard, Fatemeh Derakhshan, Edaise M. da Silva, Lorraine Colon-Cartagena, Thais Basili, Antonio Marra, Jillian Sue, Qiqi Ye, Arnaud Da Cruz Paula, Selma Yeni, Xin Pei, Hunter Green, Kaitlyn Gill, Yingjie Zhu, Matthew Lee, Ran Godrich, Adam Casson, Britta Weigelt, Nadeem Riaz, Hanna Y Wen, Edi Brogi, Matthew Hanna, Diana Mandelker, Jeremy Kunz, Brandon Rothrock, Sarat Chandarlapaty, Christopher Kanan, Gerard Oakley III, David Klimstra, Thomas Fuchs, Jorge Reis-Filho. Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-04.

WGS analysis of hypervirulent and MDR Klebsiella pneumoniae from Vietnam reveales an inverse relationship between resistome and virulome

The emergence of Klebsiella (K.) pneumoniae as a leading cause of nosocomial infections in Southeast Asia is of concern. Vietnam has an outstanding position with regard to antimicrobial-resistant (AMR) pathogens and is a hotspot for carbapenem-resistant K. pneumoniae. In the current study, 19 clinical K. pneumoniae strains isolated from patients in Vietnam were tested for antibiotic susceptibility, and their genome sequences were analyzed to investigate potential resistance profiles, genotypes, AMR determinants, and virulence-associated genes. More than half of the isolates were multidrug-resistant (MDR), displaying resistance to ciprofloxacin, levofloxacin, chloramphenicol, piperacillin, cefotaxime, ceftazidime, and cefepime. Carbapenem- resistance was detected in 47% (n=9) of isolates. Five isolates were assigned to sequence type (ST) 23 by multi-locus sequence typing (MLST). These ST23 strains exhibited determinants characteristic of hypervirulent strains and were sensitive to all antibiotics tested except for one strain, which was resistant to chloramphenicol and trimethoprim/sulfamethoxazole. All isolates had AMR determinants that conferred resistance to aminoglycosides and ß-lactams. Single nucleotide variants of oqxA and oqxB conferring resistance to phenicol/quinolone, ompK37 and ompA conferring resistance to beta-lactams, and lptD conferring resistance to rifamycin, were found in all isolates. Additionally, 95% of the isolates (n=18) carried fosA genes; however, only two were resistant to fosfomycin. Five isolates carried genes conferring resistance to colistin; only one carried mcr-1.1 and showed resistance to colistin. One hundred thirty virulence-associated genes were identified. The current study demonstrated an inverse relationship between the number of detected virulence determinants and antibiotic resistance. Several well-known resistance genes have been identified but did not mediate resistance, which could be due to insufficient levels or lack of expression. A comprehensive study of the genotypic findings of AMR determinants and virulence with phenotypic data is required. The presence of hypervirulent K. pneumoniae carrying specific virulomes is a growing problem, and monitoring of virulence characteristics of K. pneumoniae is important.

Antimicrobial Properties and Cytotoxicity of Iron Oxide Nanoparticles Synthesized Using Melia azedarach Leaf Extract Against Diarrhoeal Pathogens

AbstractThe high mortality and morbidity resulting from an increasing incidence of antibiotic resistance among pathogens highlight a crucial need for the development of novel alternative therapy. In the quest for alternative therapy, this study was conducted to synthesise nanoparticles (NPs) from ferric chloride hexahydrate (FeCl3.6H2O) via Melia azedarach L. (Meliaceae) leaf extract and evaluate their antibacterial properties against multidrug resistant pathogenic Escherichia coli, Salmonella enterica, and Vibrio cholerae. Multidrug-resistant pathogenic bacteria were isolated from cattle faeces and characterised by whole genome sequence analysis. The extracts from M. azedarach leaves were used as reducing, capping, and stabilising agents on the precursor metal (FeCl3.6H2O) to produce NPs. The biosynthesised NPs were characterised by ultraviolent visible (UV–vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectrophotometry (FTIR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The NPs were tested against multidrug-resistant bacteria to determine the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and rate of kill. In this study, WGS analysis confirmed the bacteria (E. coli, V. cholerae, and S. enterica) isolates to be multidrug-resistant pathogen and NP that has a spherical shape with an average particle size of 49.75 nm was successfully biosynthesised. The NPs displayed significant antimicrobial activities with MIC of 62, 31, and 62 mg/mL, MBC of 500, 250, and 500 µg/mL, and the lowest killing times of 4, 2, and 3 h against E. coli, V. cholerae, and S. enterica, respectively. Interestingly, the concentrations of the NPs required to inhibit the growth of E. coli, V. cholerae, and S. enterica were not toxic to HEK293 cells. Based on the promising antimicrobial activities of the biosynthesised NPs in this study, they could be useful in important therapeutic applications aimed at combating multidrug-resistant pathogens.

Abstract 4637: National multidisciplinary tumor board improves diagnostic stratification and therapeutic management in cancers of unknown primary

Abstract Introduction: With the increasing complexity of current diagnostic investigations, the integration of clinical, pathological and genomic characteristics is crucial for the management of patients (pts) with cancers of unknown primary (CUP). A national multidisciplinary tumor board (NatCUPMTB) was created in July 2020 in France to discuss the diagnostic and therapeutic management of CUP pts. The objective of this study was to evaluate the diagnostic, prognostic and therapeutic impact of this NatCUPMTB after 39 months of activity. Methods: This was a multicenter retrospective study with prospective follow-up. All pts discussed at least once in the NatCUPMTB between July 2020 and October 2023 were included. Pts and tumors characteristics, pathological and genomic analyses including WGS, WES and transcriptome analysis performed on the two PFMG2025 (French Genomic Medecine Plan 2025) national sequencing laboratories, multidisciplinary tumor board (MTB) conclusions, and follow-up after MTB were collected. Results: 244 pts were included. The median age at diagnosis was 60 yo, 53% were female, and the majority of patients had an OMS status <2. The median number of metastatic sites at diagnosis was 2, with a majority located in the lymph nodes (62%). The median time between diagnosis and first MTB presentation was 3 months (1-20). At the time of analysis, NatCUPMTB conclusions were available for 129 pts with a second MTB presentation. MTB investigations enabled to identify a likely primary origin in 89/129 (69%) pts, the most frequent being breast carcinoma (N=15), renal carcinoma (N=13) and lung carcinoma (N=13). The most frequently molecular alterations found were in TP53 (37%), KRAS (19%), CDKN2A (18%), NF2 (12%), KMT2C (10%), CDKN2B (9%), PBRM1 (9%) genes. MTB diagnoses were based on the combination of clinical, pathological and genomic investigations in 51/89 (59%) of pts. Importantly, a personalized therapeutic strategy was recommended by NatCUPMTB in 91/129 (71%) of pts. Among these recommendations, 72/91 (79%) were based on the diagnosis of tissue of origin (TOO), 9/91 (10%) on an actionable molecular alteration validated in the TOO indication, 8/91 (9%) on an actionable molecular alteration not validated in the TOO indication and 2/91 (2%) based on a druggable alteration only independently from the TOO. After a median follow-up of 1.5 months, the median overall survival (OS) was 17.7 months from the 2nd MTB presentation. Conclusion: NatCUPMTB provides significant diagnostic in 69% of pts with CUP and a therapeutic strategy recommendation for 71% of pts. Citation Format: Célia Dupain, Nicolas Jacquin, Isabelle Guillou, Etienne Rouleau, Julien Masliah Planchon, Isabelle Soubeyran, Christelle De La Fouchardière, Camille Tlemsani, Hélène Blons, Laëtitia Marisa, Anna Patrikidou, Fabienne Escande, Pierre Blanc, Jennifer Wong, Pierre Saintigny, Sandrine Boyault, Adrien Buisson, Yves Allory, Anne Vincent-Salomon, Vincent Cockenpot, Janick Selves, Ivan Bièche, Maud Kamal, Christophe Le Tourneau, Sarah Watson. National multidisciplinary tumor board improves diagnostic stratification and therapeutic management in cancers of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4637.

Abstract 3551: Continued analysis of extensive data towards Genome in a Bottle benchmarks for a new tumor normal pair

Abstract Here, we detail in-progress genome-scale measurements from a variety of technologies of the first tumor normal benchmark from the Genome in a Bottle (GIAB) consortium. We created the first broadly-consented tumor cell line from a pancreatic ductal adenocarcinoma with matched normal pancreatic and duodenal tissue. Data is being collected from a large homogeneous batch of the tumor cell line and paired normal tissues. As we receive data we make it publicly available on the NCBI hosted GIAB FTP site, https://ftp.ncbi.nlm.nih.gov/ReferenceSamples/giab/data_somatic/. When complete, this dataset will include WGS measurements from Illumina, ONT, Hi-C, Bionano, single cell WGS, PacBio HiFi and Onso, and Element. Analysis and development of a variant calling benchmark using this data is ongoing in collaboration with an open working group of the GIAB consortium. Initial Hi-C and optical mapping data from the tumor cell line indicates substantial aneuploidy from translocations that cause large deletions. We found roughly 17 large inversions and translocations and 16 chromosomes with extensive loss of heterozygosity due to missing >30% of one copy, in addition to a few smaller duplications. Low coverage single cell sequencing that provides ploidy estimates across chromosomes showed that most of the large deletions appear in all or nearly all cells with some variation in a few cells. These observations are consistent with bulk WGS analyses from two batches of cells. Additionally, many of the observed large deletions correspond to deletions seen in the population of TCGA chromosomal Loss of Heterogeneity samples. Examining somatic SNVs in non-repetitive regions, we find that close to 60% occur in almost all cells in diploid regions, 30% occur in almost all cells in haploid regions, and 5% in only some cells in diploid and haploid regions, respectively. We take these results to indicate the cell line is relatively homogeneous and stable with most CNVs being deletions. As such, we plan to explore using long reads, ultralong reads, and Hi-C data to generate a near-complete genome assembly of the dominant tumor clone as well as a complete diploid assembly of the normal. With this personalized genome assembly, we will explore aligning tumor and normal reads to each haplotype of the normal to characterize somatic variants, including variants in minor clones. Building on the methods GIAB and T2T have developed to polish diploid assemblies of GIAB’s normal genomes and mosaic variant characterization, we will develop benchmarks for somatic variants against the normal assembly as well as standard references. Citation Format: Justin Wagner, Jennifer McDaniel, Gail L. Rosen, Nathan D. Olson, Vaidehi Patel, Chunlin Xiao, Andrew Liss, Justin Zook. Continued analysis of extensive data towards Genome in a Bottle benchmarks for a new tumor normal pair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3551.

Assessment of Colistin Heteroresistance among Multidrug-Resistant Klebsiella pneumoniae Isolated from Intensive Care Patients in Europe.

Heteroresistance (HR) to colistin is especially concerning in settings where multi-drug-resistant (MDR) K. pneumoniae are prevalent and empiric use of colistin might lead to treatment failures. This study aimed to assess the frequency of occurrence of colistin HR (CHR) among (MDR) K. pneumoniae (n = 676) isolated from patients hospitalized in 13 intensive care units (ICUs) in six European countries in a clinical trial assessing the impact of decolonization strategies. All isolates were whole-genome-sequenced and studied for in vitro colistin susceptibility. The majority were colistin-susceptible (CS) (n = 597, MIC ≤ 2 µg/mL), and 79 were fully colistin-resistant (CR) (MIC > 2 µg/mL). A total of 288 CS isolates were randomly selected for population analysis profiling (PAP) to assess CHR prevalence. CHR was detected in 108/288 CS K. pneumoniae. No significant association was found between the occurrence of CHR and country, MIC-value, K-antigen type, and O-antigen type. Overall, 92% (617/671) of the K. pneumoniae were MDR with high prevalence among CS (91%, 539/592) and CR (98.7%, 78/79) isolates. In contrast, the proportion of carbapenemase-producing K. pneumoniae (CP-Kpn) was higher among CR (72.2%, 57/79) than CS isolates (29.3%, 174/594). The proportions of MDR and CP-Kpn were similar among CHR (MDR: 85%, 91/107; CP-Kpn: 29.9%, 32/107) and selected CS isolates (MDR: 84.7%, 244/288; CP-Kpn: 28.1%, 80/285). WGS analysis of PAP isolates showed diverse insertion elements in mgrB or even among technical replicates underscoring the stochasticity of the CHR phenotype. CHR isolates showed high sequence type (ST) diversity (Simpson's diversity index, SDI: 0.97, in 52 of the 85 STs tested). CR (SDI: 0.85) isolates were highly associated with specific STs (ST101, ST147, ST258/ST512, p ≤ 0.003). The widespread nature of CHR among MDR K. pneumoniae in our study urge the development of rapid HR detection methods to inform on the need for combination regimens.

Novel tetracycline resistance gene tet(65) located on a multi-resistance Corynebacterium plasmid.

Corynebacterium (C.) sp. 22KM0430 related to C. oculi and isolated from a dog exhibited resistance to tetracycline, and its WGS analysis revealed a putative resistance gene on a 35 562-bp plasmid also harbouring the MLSB resistance gene erm(X). To characterize the novel tetracycline resistance gene tet(65) and demonstrate its functionality by expression in C. glutamicum and Escherichia coli and plasmid curing of the host strain. tet(65) was cloned with and without its repressor tetR(65) and expressed in C. glutamicum DSM20300 and E. coli DH5α. Plasmid was cured by non-selective passages. Minimal inhibitory concentrations (MICs) of tetracyclines were determined according to CLSI guidelines. Association of tet(65) with efflux was shown by the addition of reserpine to MIC assays. Phylogenetic position and transmembrane structure of Tet(65) were analysed using MEGA11 and DeepTMHMM. Tet(65) shows 73% amino acid identity with the closest related Tet(Z), contains 12 transmembrane domains and is structurally related to the Major Facilitator Superfamily. The tetracycline MICs decreased in the plasmid-cured strain and increased when tet(65) was expressed in C. glutamicum and in E. coli. The MICs of tetracycline decreased in the presence of reserpine indicating that tet(65) functions as an efflux pump. A GenBank search also identified tet(65) in C. diphtheriae and Brevibacterium (B.) casei and B. luteolum. A novel tetracycline efflux gene tet(65) was identified in a C. oculi related species and was also present in the human pathogen C. diphtheriae and in Brevibacterium species indicating broader potential for dissemination.

Isolation, Identification, Antimicrobial Resistance, Genotyping, and Whole-Genome Sequencing Analysis of Salmonella Enteritidis Isolated from a Food-Poisoning Incident.

Salmonella enterica is a common pathogen in humans and animals that causes food poisoning and infection, threatening public health safety. We aimed to investigate the genome structure, drug resistance, virulence characteristics, and genetic relationship of a Salmonella strain isolated from patients with food poisoning. The pathogen strain 21A was collected from the feces of patients with food poisoning, and its minimum inhibitory concentration against commonly used antibiotics was determined using the strip test and Kirby-Bauer disk methods. Subsequently, WGS analysis was used to reveal the genome structural characteristics and the carrying status of resistance genes and virulence genes of strain 21A. In addition, an MLST-based minimum spanning tree and an SNP-based systematic spanning tree were constructed to investigate its genetic evolutionary characteristics. The strain 21A was identified by mass spectrometry as S. enterica, which was found to show resistance to ampicillin, piperacillin, sulbactam, levofloxacin, and ciprofloxacin. The WGS and bioinformatics analyses revealed this strain as Salmonella Enteritidis belonging to ST11, which is common in China, containing various resistance genes and significant virulence characteristics. Strain 21A was closely related to the SJTUF strains, a series strains from animal, food and clinical sources, as well as from Shanghai, China, which were located in the same evolutionary clade. According to the genetic makeup of strain 21A, the change G > A was found to be the most common variation. We have comprehensively analyzed the genomic characteristics, drug resistance phenotype, virulence phenotype, and genetic evolution relationship of S. Enteritidis strain 21A, which will contribute towards an in-depth understanding of the pathogenic mechanism of S. Enteritidis and the effective prevention and control of foodborne diseases.

Prevalence and genomic characterization of the Bacillus cereus group strains contamination in food products in Southern China

The Bacillus cereus group, as one of the important opportunistic foodborne pathogens, is considered a risk to public health due to foodborne diseases and an important cause of economic losses to food industries. This study aimed to gain essential information on the prevalence, phenotype, and genotype of B. cereus group strains isolated from various food products in China. A total of 890 strains of B. cereus group bacteria from 1181 food samples from 2020 to 2023 were identified using the standardized detection method. These strains were found to be prevalent in various food types, with the highest contamination rates observed in cereal flour (55.8 %) and wheat/rice noodles (45.7 %). The tested strains exhibited high resistance rates against penicillin (98.5 %) and ampicillin (98.9 %). Strains isolated from cereal flour had the highest rate of meropenem resistance (7.8 %), while strains from sausages were most resistant to vancomycin (16.8 %). A total of 234 out of the 891 B. cereus group strains were randomly selected for WGS analysis, 18.4 % of which displayed multidrug resistance. The species identification by WGS analysis revealed the presence of 10 distinct species within the B. cereus group, with B. cereus species being the most prevalent. The highest level of species diversity was observed in sausages. Notably, B. anthracis strains lacking the anthrax toxin genes were detected in flour-based food products and sausages. A total of 20 antibiotic resistance genes have been identified, with β-lactam resistance genes (bla1, bla2, BcI, BcII, and blaTEM-116) being the most common. The B. tropicus strains exhibit the highest average number of virulence genes (23.4). The diarrheal virulence genes nheABC, hblACD, and cytK were found in numerous strains. Only 4 of the 234 (1.7 %) sequenced strains contain the ces gene cluster linked to emetic symptoms. These data offer valuable insights for public health policymakers on addressing foodborne B. cereus group infections and ensuring food safety.

Persistence and evolution of linezolid- and methicillin-resistant Staphylococcus epidermidis ST2 and ST5 clones in an Italian hospital

Staphylococcus epidermidis is a member of the human skin microbiome. However, in recent decades, multidrug-resistant and hospital-adapted S. epidermidis clones are increasingly involved in severe human infections associated with medical devices and in immunocompromised patients. In 2016, we reported that a linezolid- and methicillin-resistant S. epidermidis ST2 clone, bearing the G2576T mutation, was endemic in an Italian hospital since 2004. This study aimed to retrospectively analyse 34 linezolid- and methicillin-resistant S. epidermidis (LR-MRSE) strains collected from 2018 to 2021 from the same hospital. LR-MRSE were typed by Pulsed-Field Gel Electrophoresis and multilocus sequence typing and screened for transferable linezolid resistance genes. Representative LR-MRSE were subjected to whole-genome sequencing (WGS) and their resistomes, including the presence of ribosomal mechanisms of linezolid resistance and of rpoB gene mutations conferring rifampin resistance, were investigated. ST2 lineage was still prevalent (19/34; 55.9%), but, over time, ST5 clone has been widespread too (15/34; 44.1%). Thirteen of the 34 isolates (38.2%) were positive for the cfr gene. Whole-genome sequencing analysis of relevant LR-MRSE displayed complex resistomes for the presence of several acquired antibiotic resistance genes, including the SCCmec type III (3A) and SCCmec type IV (2B) in ST2 and ST5 isolates, respectively. Bioinformatics and polymerase chain reaction (PCR) mapping also showed a plasmid-location of the cfr gene and the occurrence of previously undetected mutations in L3 (ST2 lineage) and L4 (ST3 lineage) ribosomal proteins and substitutions in the rpoB gene. The occurrence of LR-MRSE should be carefully monitored in order to prevent the spread of this difficult-to-treat pathogen and to preserve the efficacy of linezolid.