Clonal Relationship Research Articles

Next-Generation Sequencing of Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated from Patients Hospitalized in the University Hospital Facilities.

Carbapenem-resistant (CR) Klebsiella pneumoniae represents an urgent worldwide threat. We focused on CR K. pneumoniae in selected facilities of the University Hospital Bratislava (UHB) to investigate sequence types (STs), clonal relatedness, and antimicrobial resistance. Suspected carbapenem-nonsusceptible K. pneumoniae strains were obtained from hospitalized patients. Further examination included carbapenemase confirmation, cgMLST, and quantitative susceptibility testing. Of the total 41 CR K. pneumoniae strains, 26 (63.4%) were determined as ST11 in hospital No. 1; of these ST11, 22 (84.6%) were found in the first internal clinic. Six (14.6%) ST258 and three (7.3%) ST584 occurred in hospital No. 2; the most, i.e., four (66.7%), ST258 were detected in the geriatric clinic. In hospital No. 3, we found two (4.8%) ST584 and one (2.4%) ST258. Of the ST11 set, 24 (92.3%) produced NDM-1. Furthermore, seven (87.5) ST258 and five (100%) ST584 strains generated KPC-2. Antimicrobial resistance was as follows: ertapenem 97.6%, meropenem 63.4%, tigecycline 7.3%, eravacycline 7.3%, and colistin 2.5%. We revealed a presumably epidemiological association of ST11 with transmission, particularly in the first internal clinic of hospital No. 1, while ST258 and ST584 were related to interhospital dissemination between medical facilities No. 2 and No. 3. It is essential to prevent the circulation of these pathogens within and between healthcare facilities.

Beta-lactamase determinants and molecular typing of carbapenem-resistant classic and hypervirulent Klebsiella pneumoniae clinical isolates from southwest of Iran.

This study investigated the molecular epidemiology of carbapenem-resistant classic Klebsiella pneumoniae (CR-cKp) and carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) isolates in southwestern Iran. From 2019 to 2021, 136 (88.9%) cKp and 17 (11.1%) hvKp isolates were identified using biochemical tests and polymerase chain reaction (PCR). Antibiotic resistance, beta-lactamases, and clonal relatedness of carbapenem-resistant isolates were investigated using disk diffusion, PCR, and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), respectively. The different markers of hvKp isolates were as follows: string test (35.3%, n = 6/17), magA (11.8%, n = 2/17), rmpA (11.8%, n = 2/17), rmpA2 (52.9%, n = 9/17), iucA (52.9%, n = 9/17), and peg344 (35.3%, n = 6/17). Also, 55.1% (n = 75/136) of cKp and 47.1% (n = 8/17) of hvKp isolates were CR-cKp and CR-hvKp, respectively. All CR-hvKp (100.0%, n = 8) isolates were MDR. Colistin, tetracycline, and tigecycline were the most effective antibiotics. The occurrence of beta-lactamase genes in 75 CR-cKp and 8 CR-hvKp isolates was as follows: bla NDM (41.3, 25.0%), bla IMP (4.0, 0.0%), bla VIM (8.0, 0.0%), bla GES (14.7, 25.0%), bla OXA-48-like (20.0, 0.0%), bla CTX-M (26.7, 12.5%), bla SHV (24.0, 12.5%), bla TEM (10.7, 0.0%), bla FOX (6.7, 0.0%), bla DHA (6.7, 0.0%), bla CMY (5.3, 0.0%), bla LAT (12.0, 0.0%), and bla ACT (8.0, 0.0%). ERIC-PCR showed a high diversity among isolates. In this study, the occurrence of MDR CR-hvKp isolates harboring bla NDM and bla GES was detected for the first time in southwestern Iran. To prevent the spread of CR-hvKp and reduce selection pressure, long-term surveillance and more effective treatment strategies should be implemented.

Antimicrobial susceptibility, virulence genes and genomic characterization of Trueperella pyogenes isolated from abscesses in dairy cattle

Trueperella pyogenes is an opportunistic animal pathogen mainly associated with various suppurative infections in wild and domestic animals. Limited studies have investigated the pathogenesis of diseases caused by this pathogen. The main objective of the current study was to investigate the prevalence, phenotypic properties, virulence genotypes, antimicrobial susceptibility and genetic characterization of T. pyogenes isolated from abscess lesions in different tissues of on-farm dairy cattle. The study was performed on 150 postpartum cattle with clinical abscess symptoms on 22 farms around Tehran, Iran. Classical and disk diffusion methods are used for phenotypic characterization and antibiotic susceptibility. Detection of virulence factor encoding genes and genomic characterization of the isolates also are carried out by conventional PCR and BOX-PCR assays, respectively. Sixty-eight T. pyogenes strains (45.3%) were isolated, 12 were identified as pure cultures and the other 56 strains were isolated from mixed cultures. Seven distinct biotypes were identified among the T. pyogenes isolates. The isolates were mostly resistance to trimethoprim-sulfamethoxazole (70.6%), erythromycin (36.7%), tetracycline (26.5%) and tylosin (23.5%) antibiotics. Also, the genes plo, nanH, nanP and fimA were detected in all isolates. Forty-two isolates (61.7%) carried all virulence factor genes detected in this study. Three isolates only carried plo, nanH, nanP and fimA genes were identified as the least frequent genotype. All sixty-eight isolates and the reference strain were categorized into seven main clusters (A-G). A strong association was observed between virulence factor encoding genes, pathogenicity and biochemical biotypes in some specific clonal relationships.

Clonal relationships of adjacent Gleason pattern 3 and Gleason pattern 5 lesions in Gleason Scores 3+5=8 and 5+3=8

Genomic studies have demonstrated a high level of intra-tumoral heterogeneity in prostate cancer. There is strong evidence suggesting that individual tumor foci can arise as genetically distinct, clonally independent lesions. However, recent studies have also demonstrated that adjacent Gleason pattern (GP) 3 and GP4 lesions can originate from the same clone but follow divergent genetic and morphologic evolution. The clonal relationship of adjacent GP3 and GP5 lesions has thus far not been investigated. Here we analyzed a cohort of 14 cases-11 biopsy and 3 radical prostatectomy specimens-with a Gleason score of 3+5=8 or 5+3=8 present in the same biopsy or in a single dominant tumor nodule at radical prostatectomy. Clonal and subclonal relationships between GP3 and GP5 lesions were assessed using genetically validated immunohistochemical assays for ERG, PTEN, and P53. 9/14 (64%) cases showed ERG reactivity in both GP3 and GP5 lesions. Only 1/14 (7%) cases showed a discordant pattern with ERG staining present only in GP3. PTEN expression was lost in 2/14 (14%) cases with perfect concordance between GP5 and GP3. P53 nuclear reactivity was present in 1/14 (7%) case in both GP5 and GP3. This study provides first evidence that the majority of adjacent GP3 and GP5 lesions share driver alterations and are clonally related. In addition, we observed a lower-than-expected rate of PTEN loss in GP5 in the context of Gleason score 3+5=8 or 5+3=8 tumors.

Distinct clonal identities of B-ALLs arising after lenolidomide therapy for multiple myeloma

AbstractPatients with multiple myeloma (MM) who are treated with lenalidomide rarely develop a secondary B-cell acute lymphoblastic leukemia (B-ALL). The clonal and biological relationship between these sequential malignancies is not yet clear. We identified 17 patients with MM treated with lenalidomide, who subsequently developed B-ALL. Patient samples were evaluated through sequencing, cytogenetics/fluorescence in situ hybridization (FISH), immunohistochemical (IHC) staining, and immunoglobulin heavy chain (IgH) clonality assessment. Samples were assessed for shared mutations and recurrently mutated genes. Through whole exome sequencing and cytogenetics/FISH analysis of 7 paired samples (MM vs matched B-ALL), no mutational overlap between samples was observed. Unique dominant IgH clonotypes between the tumors were observed in 5 paired MM/B-ALL samples. Across all 17 B-ALL samples, 14 (83%) had a TP53 variant detected. Three MM samples with sufficient sequencing depth (>500×) revealed rare cells (average of 0.6% variant allele frequency, or 1.2% of cells) with the same TP53 variant identified in the subsequent B-ALL sample. A lack of mutational overlap between MM and B-ALL samples shows that B-ALL developed as a second malignancy arising from a founding population of cells that likely represented unrelated clonal hematopoiesis caused by a TP53 mutation. The recurrent variants in TP53 in the B-ALL samples suggest a common path for malignant transformation that may be similar to that of TP53-mutant, treatment-related acute myeloid leukemia. The presence of rare cells containing TP53 variants in bone marrow at the initiation of lenalidomide treatment suggests that cellular populations containing TP53 variants expand in the presence of lenalidomide to increase the likelihood of B-ALL development.

Molecular characteristics of multifocal esophageal squamous cell carcinomas to discriminate multicentric origin from intramural metastasis.

Multifocal esophageal squamous cell carcinomas (ESCCs) can be diagnosed as of multicentric origin (MO) or intramural metastasis (IMM). We aimed here to accurately discriminate MO from IMM and explore the tumor immune microenvironment of multifocal ESCCs. Multifocal ESCCs were identified in 333 ESCC patients, and in 145 patients discrimination between MO and IMM was not possible by histopathological examination. Of the 145 patients, tissues of 14 were analyzed by whole-exome sequencing (WES) of 71 different tumor regions, and MO, IMM, and MO/IMM mixed groups were identified in three, ten, and one cases, respectively, based on the similarity of genomic architecture between or among different tumors from one patient. Further phylogenetic analyses revealed complex clonal evolution patterns in IMM cases, and tumor cells disseminated from the primary tumors to IMM tumors were independent of lymph node metastasis. The NanoString-based assay showed that immune cell infiltrates were significantly enriched, and that the immune and proliferation pathways were more activated, in large tumors than in small ones in MO but not IMM cases. Similarly, PD-L1 expression and the density of paratumoral CD8+ T cells were higher in large tumors than in small tumors in MO. Taken together, through analysis of the genomic and immune landscapes, our study has comprehensively characterized the heterogeneity and clonal relationship of multifocal ESCCs, which may be helpful in distinguishing MO from IMM, and for interpreting the immunotherapy responses for multifocal ESCC patients. © 2022 The Pathological Society of Great Britain and Ireland.

Emergence of a Novel Lineage and Wide Spread of a blaCTX-M-15/IncHI2/ST1 Plasmid among Nosocomial Enterobacter in Guadeloupe.

Between April 2018 and August 2019, a total of 135 strains of Enterobacter cloacae complex (ECC) were randomly collected at the University Hospital Center of Guadeloupe to investigate the structure and diversity of the local bacterial population. These nosocomial isolates were initially identified genetically by the hsp60 typing method, which revealed the clinical relevance of E. xiangfangensis (n = 69). Overall, 57/94 of the third cephalosporin-resistant strains were characterized as extended-spectrum-β-lactamase (ESBL) producers, and their whole-genome was sequenced using Illumina technology to determine the clonal relatedness and diffusion of resistance genes. We found limited genetic diversity among sequence types (STs). ST114 (n = 13), ST1503 (n = 9), ST53 (n = 5) and ST113 (n = 4), which belong to three different Enterobacter species, were the most prevalent among the 57 ESBL producers. The blaCTXM-15 gene was the most prevalent ESBL determinant (56/57) and was in most cases associated with IncHI2/ST1 plasmid replicon carriage (36/57). To fully characterize this predominant blaCTXM-15/IncHI2/ST1 plasmid, four isolates from different lineages were also sequenced using Oxford Nanopore sequencing technology to generate long-reads. Hybrid sequence analyses confirmed the circulation of a well-conserved plasmid among ECC members. In addition, the novel ST1503 and its associated species (ECC taxon 4) were analyzed, in view of its high prevalence in nosocomial infections. These genetic observations confirmed the overall incidence of nosocomial ESBL Enterobacteriaceae infections acquired in this hospital during the study period, which was clearly higher in Guadeloupe (1.59/1000 hospitalization days) than in mainland France (0.52/1,000 hospitalization days). This project revealed issues and future challenges for the management and surveillance of nosocomial and multidrug-resistant Enterobacter in the Caribbean.

Clonal relationship and alcohol consumption-associated mutational signature in synchronous hypopharyngeal tumours and oesophageal squamous cell carcinoma

BackgroundThe patients with dual oesophageal squamous cell carcinoma (ESCC) and hypopharyngeal cancer (HPC) have poor prognosis; their underlying genetic pathogenesis is unclear. We hypothesise that development of synchronous ESCC/HPC depends on multicentricity or independent origin, rather than multifocality due to local or lateral spreading.MethodMultiple region whole-exome sequencing (M-WES) and clonality analysis were used to assess clonal relationship and spatial inter- or intra-tumour heterogeneity (ITH) in 62 tumour regions from eight dual ESCC/HPC and ten ESCC patients.ResultsAll synchronous ESCC/HPC patients had COSMIC 16 mutation signatures, compared to only 40% ESCC in the current study (p = 0.013) and public data set (n = 165, p = 0.003). This alcohol consumption-related mutation signature 16, commonly involved in multiple alcohol-related cancers, was significantly associated with drinking and alcohol metabolism-related ADH1B rs1229984. The mutational landscape and copy number profiles were completely distinct between the two primary tumours; clonality analysis further suggested the two primary tumours shared no or only one clone accompanying independent subclone evolution. M-WES strategy demonstrated higher sensitivity and accuracy for detection of mutational prevalence and the late branch mutations among different regions in the ESCC tumours, compared to traditional sequencing analysis based on single biopsy strategy. Patients with high ITH assessed by cancer cell fraction analysis after M-WES were significantly associated with both relapse and survival.ConclusionsOur hypothesis-generating M-WES ITH assessment data have implications for prognostication. Collectively, our findings support multicentric independent clonal evolution, the field cancerisation theory, and suggest novel insights implicating an aetiologic role of alcohol metabolism in dual ESCC/HPC carcinogenesis.

Clonality relatedness and molecular characteristics of Richter transformation

Objective: To investigate the clinical, genetic, and clonality related aspects of individuals with Richter transformation (RT) . Methods: From January 2019 to December 2021, 18 RT patients with diagnoses at the First Affiliated Hospital of Nanjing Medical University (Pukou CLL center) were retrospectively examined. The immunoglobin heavy variable (IGHV) gene usage and IGHV-D-J rearrangement pattern of diagnosed CLL/SLL and transformed diffuse large B-cell lymphoma (DLBCL) were compared to determine the clonality relatedness. To investigate the risk factors of RT, Clinical and laboratory data from patients with newly diagnosed CLL/SLL and transformed DLBCL were gathered. Results: The median age of RT was 56.5 (41-75) years old. 17 patients transformed to DLBCL and 1 transformed to Hodgkin lymphoma (HL) . Of 17 individuals who had DLBCL transformation, 15 had CLL/SLL-related clonality and 2 had unrelated clonality. Next-generation sequencing (NGS) analysis of 11 paired initially diagnosed treatment-naive CLL/SLL and RT DLBCL found that EGR2、TP53 and NOTCH1 were among the most frequently mutated genes both in treatment-naive CLL/SLL and in RT DLBCL. In several cases, specific mutations were gained or lost throughout RT, indicating clonal evolution. Among 10 patients before exposure to BTK inhibitors before RT, four patients acquired BTK mutation. The aforementioned mutations should be considered high-risk variables for transformation; in addition, TP53 and EGR2 mutations could be linked to a poor prognosis following RT in patients receiving a cocktail of new medicines. Conclusion: Most RT DLBCL patients in our center are clonality related (15/17, 88.2% ) and we recommend all qualified centers to evaluate clonality relatedness of RT DLBCL patients. There was some variability in the mutational landscape between DLBCL that had undergone a transformation and initially diagnosed, treatment-naive CLL/SLL. The underlying molecular mechanism of RT needs more research.

Clonal relationship analysis of 4 cases of multifocal primary lung cancer diagnosed histologically based on next-generation sequencing

目的： 基于二代测序法，探讨经组织学诊断为同步多灶原发性肺癌（SMPLC）病例的分子病理学特征及克隆起源。 方法： 对4例SMPLC的8个病灶进行全基因组外显子测序，并进行基因突变、基因组稳定性、克隆性关系及克隆演化的分析。 结果： 突变分析显示例1、2及3的2个病灶间突变的一致性均为0，例4的一致性为3/（50+44）。例1的2个病灶基因组稳定性差别较大。例2的2个病灶基因组稳定性差别不大。例3、4的2个病灶基因组均稳定。用单核苷酸变异测试对样本突变信息进行分析，结果显示例1、2、3的2个病灶间相互独立。例4的2个病灶之间存在克隆关系。对样本拷贝数变异进行分析，认为例1、2的2个病灶彼此独立。对例4进行克隆演化分析，提示2个病灶来源于同一个克隆，可能为肺内转移。 结论： 二代测序分析是对多灶肺癌组织学诊断的重要补充。不同组织学类型，或含原位癌病灶是判断SMPLC较可靠的依据。但不同组织学亚型诊断价值有限。为保证肿瘤分期和术后治疗的精准性，有必要对多灶肺癌的每一个病灶进行基因突变检测。.

Single cell clonal analysis identifies an AID‐dependent pathway of plasma cell differentiation

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody‐secreting plasma cells. Here, we have addressed the role of activation‐induced deaminase (AID), which initiates somatic hypermutation and class switch recombination, in the terminal differentiation of GC B cells. By combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID‐experienced cells, we have identified a novel subset of late‐prePB cells (L‐prePB), which shares the strongest clonal relationships with plasmablasts (PBs). Mice lacking AID have various alterations in the size and expression profiles of transcriptional clusters. We find that AID deficiency leads to a reduced proportion of L‐prePB cells and severely impairs transitions between the L‐prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state.

Antibiotic resistance and carriage class I integron in clinical isolates of Acinetobacter baumannii from Al Muthanna, Iraq.

The goal of this work was to systematically characterize and detect class 1, 2, and 3 integrons with many antibiotic resistance A. baumannii strains collected from a clinical environment in Iraq's Al-Muthanna hospitals. In this investigation, 24 non-replicated clinical strains of A. baumannii were evaluated using Chrome agar as a selective medium and PCR of the rplB gene. The clonal relatedness of the isolates to class 1 integron was evaluated using a PCR technique. The prevalence of class 1 integron was detected by PCR in only 12 clones of A. baumannii followed by HinfI digestion analysis showing three identical bands at 160 bp, 1350 bp, and 870 bp. In addition, PCR sequencing confirmed the presence of gene cassette arrays consisting of aacA4-catB8-aadA1 (100%) in class 1 integron. The sequence analysis of the integron shows 97.87 identity with A. baumannii isolates from Australia (GenBank accession number CP054302) among A. baumannii isolates. The blast analysis of this class I integron showed that the presence of the intI1, aacA4-catB8-aadA1 genes can considerably boost the acquisition of MDR phenotypes in A. baumannii isolates. We concluded that antibiotics of many types are widely used. The presence of integrons in A. baumannii is concerning for public health. In the clinical setting, it appears that the class 1 integron can be used as a predictive biomarker for the presence of MDR phenotypes. In these bacteria, however, the integron does not possess carbapenemases genes.

Characterization of Escherichia coli Cefotaxime-Resistance in Al-Ahsa, KSA: Predominance of CTX-15 and First Report of blaCMY-42 Gene

We determined an antibiotic resistance mechanism in the eastern region, KSA, and the genetic factor clonal relatedness within Gram-negative bacteria. During our retrospective study, a total number of 29 E. coli ESBL producer strains were isolated for patients visiting King Fahad Hospital, Al-Ahsa, KSA. The bla genes were detected via PCR and identified via sequencing. Associated plasmid-mediated quinolone resistance genes, as well as int1 and int2 genes, were also studied. Phylogenetic groups, the ST131 clone, virulence factors, and PFGE were also checked. The blaCTX-M-9 (3.7%), blaCTX-M-27 (22.2%), and blaCTX-M-15 (77.8%) genes were identified; however, the blaCMY-42 (7.4%) gene was recorded for the first time in KSA. The qnrS1 gene was found in 44.4% of strains, and among them, 50% concomitantly harbored the aac(6′)Ib-cr. The int1 gene was detected in 25.9% strains; nonetheless, the int2 gene was found in 7.4% of isolates. The strains belonged mainly to the B2 and D phylogroups. PFGE showed unrelated patterns. Some isolates belonged to the pandemic clone ST131. We describe a large dissemination of antibiotic resistance to third-generation cephalosporins in the eastern region, KSA, with the occurrence of the blaCMY-42 gene. The clone ST131 seems to be the principal contributor for blaCTX-M-15 gene spread.

Investigation of the VISA and hVISA Presence in Methicillin-Resistant Staphylococcus aureus Isolates by Various Methods

Staphylococcus aureus is an important human pathogen that causes community and hospital-acquired infections. The role of vancomycin in the treatment of methicillin-resistant S.aureus infections is indisputable. However, vancomycin intermediate susceptible S.aureus (VISA) and heterogeneously VISA (hVISA) isolates, that cause treatment failures during the use of vancomycin, cannot be detected by routine laboratory methods. The gold standard method for the detection of these isolates is the population profile analysis-area under the curve (PAP-AUC) method. In this study, it was aimed to determine the presence of mecA and mecC gene regions that cause methicillin resistance, the clonal relationship between isolates, and the presence of VISA and hVISA. A total 68 methicillin-resistant S.aureus (MRSA) strains were included in this study which were isolated in the microbiology laboratory of the hospital between 2015- 2020. Identification of the isolates were determined by matrix assisted laser desorption ionization-time of flight mass spectrophotometry (VITEK MS, BioMérieux, France). Methicillin resistance was investigated by disk diffusion method using cefoxitin (30 µg, Bioanalyse, Türkiye) disk and vancomycin MIC values were determined by broth microdilution method. mecA and mecC gene regions were investigated by polymerase chain reaction (PCR) method. The presence of VISA and hVISA were investigated by modified agar screening, macro gradient diffusion test and confirmated by PAP-AUC methods, and the clonal relationship between isolates were investigated by pulsed field gel electrophoresis method. The mecA gene region was determined in all isolates, but the mecC gene region was not found in any of the isolates. The MIC50 value of the isolates was determined as 1 μg/mL and the MIC90 value was determined as 2 μg/mL by broth microdilution method. Six VISA and four hVISA suspected strains were detected by a modified agar screening method. Among the isolates identified as suspicious by the modified agar screening method, one isolate was evaluated as VISA and one isolate was evaluated as hVISA by the gold standard PAP-AUC method. No dominant epidemic isolate has been identified by PFGE. As a result, VISA and hVISA were determined in the hospital. The increase in these isolates is a serious concern. For this reason, it is believed that it would be beneficial to investigate the VISA/hVISA ratios in MRSA isolates at certain periods, and to take necessary infection control measures to implement measures and practices to prevent the spread of these isolates in the community and hospital environment.

Molecular Epidemiology of Clinical Carbapenem-Resistant Acinetobacter baumannii-calcoaceticus complex Isolates in Tertiary Care Hospitals in Java and Sulawesi Islands, Indonesia.

Carbapenem-resistant Acinetobacter baumannii (A. baumannii)-calcoaceticus complex (CRAb-cc) is an important pathogen causing nosocomial infections worldwide; however, molecular epidemiology of the A. baumannii-calcoaceticus complex in Indonesian hospitals is scarce. This study aimed to determine the clonal relatedness of CRAb-cc in two tertiary care hospitals in Malang and Manado in Indonesia. The CRAb-cc isolates from routine clinical cultures in two tertiary care hospitals in Malang and Manado were identified using the Vitek2® system (bioMérieux, Lyon, France). Multi-locus variable-number tandem-repeat analysis (MLVA) typing, multi-locus sequence typing (MLST), clonal complex (CC), and phylogenetic tree analysis were conducted for a subset of isolates. Seventy-three CRAb-cc isolates were collected. The CRAb-cc isolates were frequently found among lower-respiratory-tract specimens. We detected the MLVA type (MT) 1, MT3, and MT4 CRAB-cc isolates belonging to the sequence type (ST) 642, and CC1 was the predominant clone in this study. In conclusion, we identified the clonal relatedness of A. baumannii-calcoaceticus complex isolates in two tertiary care hospitals in Malang and Manado in Indonesia. Further study is required to investigate the clinical importance and distribution of ST642 in Indonesian hospitals for developing prevention and control measures.

279. CLONAL RELATIONSHIP AND ALCOHOL CONSUMPTION-ASSOCIATED MUTATIONAL SIGNATURE IN SYNCHRONOUS HYPOPHARYNGEAL TUMORS AND ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract The patients with dual esophageal squamous cell carcinoma (ESCC) and head and neck cancers have poor prognosis, but the underlying genetic pathogenesis is unclear. We hypothesize that independent origin rather than multifocality, which means local or lateral spreading involves the development of synchronous ESCC and hypopharyngeal cancer (HPC). We also hypothesize that multi-regional assessment provides better understanding of the evolution of tumor subclones and assessment of intra-tumor heterogeneity to guide personalized treatment with prognostic implications. Multiple region whole-exome sequencing (M-WES) and clonality analysis were used to assess clonal relationship and spatial inter- or intra-tumor heterogeneity (ITH) in 62 tumor regions, six normal tissues, and 15 blood samples from eight dual ESCC/HPC and ten ESCC patients. All synchronous ESCC/HPC patients had COSMIC 16 mutation signature compared to only 40% ESCC in the current study (p = 0.013) and public ESCC dataset (n = 165, p = 0.003). The mutational landscape and copy number variation (CNV) profiles were completely distinct between the ESCC/HPC; clonality analysis further suggested the two primary tumors shared no or only one clone accompanying independent subclone evolution. The M-WES strategy demonstrated higher sensitivity and accuracy for detection of mutational prevalence and the late branch mutations among different regions in the ESCC tumors compared to traditional single biopsy strategy. Patients with high ITH was significantly associated with both relapse and survival. Our hypothesis-generating M-WES ITH assessment data have implications for prognostication. We are the first to show a distinctive CNV and genetic mutation profile, the predominant alcohol-related COSMIC 16 mutational signature in synchronous ESCC/HPC, providing genetic evidence for a multi-centric independent origin for clonal evolution and the theory of field cancerization implicating an etiologic role of alcohol metabolism in dual ESCC/HPC carcinogenesis.

Clonal relationship of extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT lymphoma) involving multiple organ systems with review of the literature

Clonal relationship of extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT lymphoma) involving multiple organ systems with review of the literature

Prevalence and Population Diversity of Listeria monocytogenes Isolated from Dairy Cattle Farms in the Cantabria Region of Spain.

Simple SummaryThe origin and prevalence of Listeria monocytogenes was studied in dairy cattle farms in order to examine its diversity and determine its possible persistence in manure. The utilization of manure for agricultural purposes is common in many countries. While properly treated and managed manure is an effective and safe fertilizer, foodborne illness outbreaks can occur, as many of the most prominent foodborne pathogens are carried by healthy livestock. It is, therefore, necessary to study the origin and persistence of zoonotic agents in general and of L. monocytogenes in particular, in order to avoid recirculation in farms and reduce risk for human populations.Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. Here we show the prevalence and transmission of L. monocytogenes in dairy farms in the Cantabria region, on the northern coast of Spain. A total of 424 samples was collected from 14 dairy farms (5 organic and 9 conventional) and 211 L. monocytogenes isolates were recovered following conventional microbiological methods. There were no statistically significant differences in antimicrobial resistance ratios between organic and conventional farms. A clonal relationship among the isolates was assessed by pulsed field gel electrophoresis (PFGE) analysis and 64 different pulsotypes were obtained. Most isolates (89%, n = 187) were classified as PCR serogroup IVb by using a multiplex PCR assay. In this case, 45 isolates of PCR serogroup IVb were whole genome-sequenced to perform a further analysis at genomic level. In silico MLST analysis showed the presence of 12 sequence types (ST), of which ST1, ST54 and ST666 were the most common. Our data indicate that the environment of cattle farms retains a high incidence of L. monocytogenes, including subtypes involved in human listeriosis reports and outbreaks. This pathogen is shed in the feces and could easily colonize dairy products, as a result of fecal contamination. Effective herd and manure management are needed in order to prevent possible outbreaks.

Molecular epidemiology, virulence and antimicrobial resistance of Bulgarian methicillin resistant Staphylococcus aureus isolates.

Severe infections of virulent methicillin-resistant Staphylococcus aureus (MRSA) are a serious health problem. The present study aimed to investigate clonal spread, virulence and antimicrobial resistance rates of Bulgarian MRSA isolates in 2016-2020. Molecular identification and mecA gene detection were performed with PCR. Clonal relatedness was evaluated by RAPD PCR and MLST. MRSA epidemiology, virulence and resistance patterns were investigated by PCR. All 27 isolates were identified as S. aureus and were mecA positive, and all were susceptible to linezolid, tigecycline and vancomycin. The toxin genes hlg (in 92.6% of isolates), seb (77.8%), sei (77.8%), seh (59.3%), sej (55.6%), and seg (48.1%), were frequently found among the isolates. Epidemiological typing by RAPD identified 4 clones (16 isolates) and 11 were with a unique profile. MLST analysis of the same MRSA isolates showed five MLST clonal complexes and 11 ST types, including CC5 (33.3%) (ST5, ST221, ST4776), CC8 (22.2%) (ST8, ST239, ST72), CC15 (ST582), CC22 (14.8%) (ST217, ST5417), CC30 (ST30) CC398 (ST398), and CC59 (ST59). The isolates from CC5 showed higher virulence potential and almost all were macrolide resistant (ermB or ermC positive). CC8 isolates showed higher level of resistance. To the best of our knowledge, this study is the first describing the clonal spreading of Bulgarian MRSA and the association with their virulence and resistance determinants. Monitoring of MRSA epidemiology, resistance and virulence profile can lead to better prevention and faster therapeutic choice in cases of severe infections.

Detection of different colistin resistance mechanisms among multidrug resistant Klebsiella pneumoniae isolates in Bulgaria.

The more frequent usage of colistin resulted in an increase of colistin resistance due to lipopolysaccharide modifications. The aim of this study was to reveal the prevalence and mechanisms of colistin resistance among multidrug-resistant Klebsiella pneumoniae isolates collected in Bulgaria. One hundred multidrug resistant K. pneumoniae isolates were collected in a period between 2017 and 2018. Among them, 29 colistin resistant and 8 heteroresistant isolates were observed and further investigated. Clonal relatedness was detected by RAPD and MLST. Сarbapenemases, two component system phoQ/phoP, pmrA/B, and mgrB were investigated by PCR amplification and Sanger sequencing. Among 37 colistin nonsusceptible isolates, we detected 25 NDM-1 producers. The isolates belonged mainly to ST11 (80%), and also to ST147, ST35, ST340, ST219 (1-2 members per clone). Nine colistin resistant isolates showed changes in mgrB. IS903B-like elements truncated mgrB in five isolates. In two isolates, premature stopcodon (Q30stopcodon) was observed and another two isolates did not amplify mgrB, possibly due to bigger deletion or insertion. No isolates showed phoQ/phoP and pmrA/B mutations except for pmrB (four isolates had R256G). All isolates with IS903B insertions belonged to ST11 clone. The mgrB alterations play major role in colistin resistance in K. pneumoniae isolates studied in the current work. We report truncation of mgrB by IS903 like element in colistin resistant NDM-1 producing K. pneumoniae ST11 clone in Bulgaria.