Clonal Relationship Research Articles

Clonal relation between Salmonella enterica subspecies enterica serovar Dublin strains of bovine and food origin in Germany.

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) is a host-adapted serovar causing enteritis and/or systemic diseases in cattle. As the serovar is not host-restricted, it may cause infections in other animals, including humans with severe illness and higher mortality rates than other non-typhoidal serovars. As human infections are mainly caused by contaminated milk, milk products and beef, information on the genetic relationship of S. Dublin strains from cattle and food should be evaluated. Whole-genome sequencing (WGS) of 144 S. Dublin strains from cattle and 30 strains from food origin was performed. Multilocus sequence typing (MLST) revealed mostly sequence type ST-10 from both, cattle and food isolates. In total, 14 of 30 strains from food origin were clonally related to at least one strain from cattle, as detected by core-genome single nucleotide polymorphisms typing as well as core-genome MLST. The remaining 16 foodborne strains fit into the genome structure of S. Dublin in Germany without outliers. WGS proved to be a powerful tool not only to gain information on the epidemiology of Salmonella strains but also to detect clonal relations between organisms isolated from different stages of production. This study has shown a high genetic correlation between S. Dublin strains from cattle and food and, therefore, the potential to cause human infections. S. Dublin strains of both origins share an almost identical set of virulence factors, emphasizing their potential to cause severe clinical manifestations in animals, but also in humans and thus the need for effective control of S. Dublin in a farm-to-fork strategy.

IncHI1 plasmids are epidemic vectors that mediate transmission of tet(X4) in Escherichia coli isolated from China.

This study aimed to investigate the genetic factors promoting widespread Q6 dissemination of tet(X4) between Escherichia coli and to characterize the genetic contexts of tet(X4). We isolated E. coli from feces, water, soil and flies collected across a large-scale chicken farm in China in 2020. Antimicrobial susceptibility testing and PFGE typing were used to identify tigecycline resistance and assess clonal relationships among isolates. Plasmids present and genome sequences were analyzed by conjugation, S1 pulsed-field gel electrophoresis (PFGE), plasmid stability testing and whole-genome sequencing. A total of 204 tigecycline-resistant E. coli were isolated from 662 samples. Of these, we identified 165 tet(X4)-carrying E. coli and these strains exhibited a high degree of multidrug resistance. Based on the geographical location distribution of the sampled areas, number of samples in each area and isolation rate of tigecycline-resistant strains and tet(X4)-carrying isolates, 72 tet(X4)-positive isolates were selected for further investigation. Tigecycline resistance was shown to be mobile in 72 isolates and three types of tet(X4)-carrying plasmids were identified, they were IncHI1 (n = 67), IncX1 (n = 3) and pO111-like/IncFIA(HI1) (n = 2). The pO111-like/IncFIA(HI1) is a novel plasmid capable of transferring tet(X4). The transfer efficiency of IncHI1 plasmids was extremely high in most cases and IncHI1 plasmids were stable when transferred into common recipient strains. The genetic structures flanked by IS1, IS26 and ISCR2 containing tet(X4) were complex and varied in different plasmids. The widespread dissemination of tigecycline-resistant E. coli is a major threat to public health. This data suggests careful use of tetracycline on farms is important to limit spread of resistance to tigecycline. Multiple mobile elements carrying tet(X4) are in circulation with IncHI1 plasmids the dominant vector in this setting.

Wastewater treatment plants, an "escape gate" for ESCAPE pathogens.

Antibiotics are an essential tool of modern medicine, contributing to significantly decreasing mortality and morbidity rates from infectious diseases. However, persistent misuse of these drugs has accelerated the evolution of antibiotic resistance, negatively impacting clinical practice. The environment contributes to both the evolution and transmission of resistance. From all anthropically polluted aquatic environments, wastewater treatment plants (WWTPs) are probably the main reservoirs of resistant pathogens. They should be regarded as critical control points for preventing or reducing the release of antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic-resistance genes (ARGs) into the natural environment. This review focuses on the fate of the pathogens Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae spp. (ESCAPE) in WWTPs. All ESCAPE pathogen species, including high-risk clones and resistance determinants to last-resort antibiotics such as carbapenems, colistin, and multi-drug resistance platforms, were detected in wastewater. The whole genome sequencing studies demonstrate the clonal relationships and dissemination of Gram-negative ESCAPE species into the wastewater via hospital effluents and the enrichment of virulence and resistance determinants of S. aureus and enterococci in WWTPs. Therefore, the efficiency of different wastewater treatment processes regarding the removal of clinically relevant ARB species and ARGs, as well as the influence of water quality factors on their performance, should be explored and monitored, along with the development of more effective treatments and appropriate indicators (ESCAPE bacteria and/or ARGs). This knowledge will allow the development of quality standards for point sources and effluents to consolidate the WWTP barrier role against the environmental and public health AR threats.

Whole-exome sequencing reveals the metastatic potential of hepatocellular carcinoma from the perspective of tumor and circulating tumor DNA.

Relapse of hepatocellular carcinoma (HCC) due to vascular invasion is common, but the genomic mechanisms remain unclear, and molecular determinants of high-risk relapse cases are lacking. We aimed to reveal the evolutionary trajectory of microvascular invasion (MVI) and develop a predictive signature for relapse in HCC. Whole-exome sequencing was performed on tumor and peritumor tissues, portal vein tumor thrombus (PVTT), and circulating tumor DNA (ctDNA) to compare the genomic profiles between 5 HCC patients with MVI and 5 patients without MVI. We conducted an integrated analysis of exome and transcriptome to develop and validate a prognostic signature in two public cohorts and one cohort from Zhongshan Hospital, Fudan University. Shared genomic landscapes and identical clonal origins among tumor, PVTT, and ctDNA were observed in MVI ( +) HCC, suggesting that genomic changes favoring metastasis occur at the primary tumor stage and are inherited in metastatic lesions and ctDNA. There was no clonal relatedness between the primary tumor and ctDNA in MVI ( - ) HCC. HCC had dynamic mutation alterations during MVI and exhibited genetic heterogeneity between primary and metastatic tumors, which can be comprehensively reflected by ctDNA. A relapse-related gene signature named RGSHCC was developed based on the significantly mutated genes associated with MVI and shown to be a robust classifier of HCC relapse. We characterized the genomic alterations during HCC vascular invasion and revealed a previously undescribed evolution pattern of ctDNA in HCC. A novel multiomics-based signature was developed to identify high-risk relapse populations.

Worldwide transmission of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae: an analysis of publicly available genomes.

ST11-KL64 is an internationally distributed lineage of carbapenem-resistant Klebsiella pneumoniae and is the most common type in China. The international and interprovincial (in China) transmission of ST11-KL64 CRKP remains to be elucidated. We used both static clusters defined based on a fixed cutoff of ≤21 pairwise single-nucleotide polymorphisms and dynamic groups defined by modeling the likelihood to be linked by a transmission threshold to investigate the transmission of ST11-KL64 strains based on genome sequences mining. We analyzed all publicly available genomes (n = 730) of ST11-KL64 strains, almost all of which had known carbapenemase genes with KPC-2 being dominant. We identified 4 clusters of international transmission and 14 clusters of interprovincial transmission across China of ST11-KL64 strains. We found that dynamic grouping could provide further resolution for determining clonal relatedness in addition to the widely adopted static clustering and therefore increases the confidence for inferring transmission.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious challenge for clinical management and is prone to spread in and between healthcare settings. ST11-KL64 is the dominant CRKP type in China with a worldwide distribution. Here, we used two different methods, the widely used clustering based on a fixed single-nucleotide polymorphism (SNP) cutoff and the recently developed grouping by modeling transmission likelihood, to mine all 730 publicly available ST11-KL64 genomes. We identified international transmission of several strains and interprovincial transmission in China of a few, which warrants further investigations to uncover the mechanisms for their spread. We found that static clustering based on ≤21 fixed SNPs is sensitive to detect transmission and dynamic grouping has higher resolutions to provide complementary information. We suggest the use of the two methods in combination for analyzing transmission of bacterial strains. Our findings highlight the need of coordinated actions at both international and interprovincial levels for tackling multi-drug resistant organisms.

Using Genomics To Investigate an Outbreak of Vancomycin-Resistant Enterococcus faecium ST78 at a Large Tertiary Hospital in Queensland.

To investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) sequence type 78 (ST78) in a large tertiary Australian hospital. A collection of 63 VREfm ST78 isolates, identified during a routine genomic surveillance program, were subjected to genomic epidemiological analysis based on whole-genome sequencing (WGS) data. The population structure was reconstructed using phylogenetic analysis, and a collection of publicly available VREfm ST78 genomes were used to provide global context. Core genome single nucleotide polymorphism (SNP) distances and available clinical metadata were used to characterize outbreak clusters and reconstruct transmission events. In silico genotyping confirmed that all study isolates were vanB-type VREfm carrying virulence characteristics of the hospital-associated E. faecium. Phylogenetic analysis identified two distinct phylogenetic clades, only one of which was responsible for a hospital outbreak. Four outbreak subtypes could be defined with examples of recent transmissions. Inference on transmission trees suggested complex transmission routes with unknown environmental reservoirs mediating the outbreak. WGS-based cluster analysis with publicly available genomes identified closely related Australian ST78 and ST203 isolates, highlighting the capacity for WGS to resolve complex clonal relationships between the VREfm lineages. Whole genome-based analysis has provided a high-resolution description of an outbreak of vanB-type VREfm ST78 in a Queensland hospital. Combined routine genomic surveillance and epidemiological analysis have facilitated better understanding of the local epidemiology of this endemic strain, providing valuable insight for better targeted control of VREfm. IMPORTANCE Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of health care-associated infections (HAIs) globally. In Australia, the spread of hospital-adapted VREfm is largely driven by a single clonal group (clonal complex [CC]), CC17, to which the lineage ST78 belongs. While implementing a genomic surveillance program in Queensland, we observed increased incidence of ST78 colonizations and infections among patients. Here, we demonstrate the use of real-time genomic surveillance as a tool to support and enhance infection control (IC) practices. Our results show that real-time whole-genome sequencing (WGS) can efficiently disrupt outbreaks by identifying transmission routes that in turn can be targeted using resource-limited interventions. Additionally, we demonstrate that by placing local outbreaks in a global context, high-risk clones can be identified and targeted prior to them becoming established within clinical environments. Finally, the persistence of these organism within the hospital highlights the need for routine genomic surveillance as a management tool to control VRE transmission.

Brote nosocomial causado por Serratia marcescens en una unidad de cuidados intensivos neonatal de un hospital regional. Análisis y propuestas de mejora

ObjectivesSerratia marcescens may cause nosocomial outbreaks in neonatal intensive care units. We describe an outbreak of Serratia marcescens in a neonatal intensive care unit and propose additional prevention and control recommendations. MethodsBetween March 2019 and January 2020, samples were taken from patients in the neonatal intensive care unit (rectal, pharyngeal, axillary and other locations) and from 15 taps and their sinks. Control measures were implemented including thorough cleaning of incubators, health education to staff and neonates’ relatives, and use of single-dose containers. PFGE was performed in 19 isolates from patients and in 5 environmental samples. ResultsFrom the first case in March 2019 to the detection of the outbreak, a month elapsed. Finally, 20 patients were infected and 5 colonized. Eighty percent of infected neonates had conjunctivitis, 25% bacteremia, 15% pneumonia, 5% wound infection, and 5% urinary tract infection. Six neonates had 2 foci of infection. Among the 19 isolates studied, 18 presented the same pulsotype and only one of the isolates from the sinkhole showed a clonal relationship with those of the outbreak. Initial measures established were ineffective to control the outbreak and were implemented with exhaustive cleaning, use of individual eye drops, environmental sampling and changing sinks. ConclusionThis outbreak presented a high number of neonates affected due to its late detection and torpid evolution. The microorganisms isolated from the neonates were related to an environmental isolate. Additional prevention and control measures are proposed, including routine weekly microbiological sampling.

Clonal relatedness of carbapenem-resistant Acinetobacter baumannii: high prevalence of ST136pas in a burn center

BackgroundCarbapenem-resistant Acinetobacter baumannii (CRAB) is a global health crisis. This study aimed to determine the clonal relatedness of antibiotic-resistant A. baumannii isolates in hospitalized patients who suffered from burn wound infection.MethodsOne hundred and six A. baumannii isolates from 562 patients with burn wound infections, were identified and examined for antimicrobial susceptibility. Detection and characterization of carbapenem-hydrolyzing class D OXA-type beta-lactamases (CHDLs) were performed by PCR assays. The clonal relatedness of A. baumannii isolates was determined by multilocus sequence typing (MLST) according to the Pasteur scheme, dual-sequence typing of blaOXA−51-like and ampC genes, and RAPD-PCR method.ResultsAll isolates were carbapenem-resistant while susceptible to colistin, minocycline, doxycycline, and ampicillin-sulbactam. The intrinsic blaOXA−51-like was detected in all isolates, and blaOXA−23-like was identified in 92.5% of isolates. However, blaOXA−143-like and blaOXA−58-like genes were not detected among isolates. Four distinct blaOXA−51-like alleles were determined as follows: blaOXA−317 (67.0%), blaOXA−90 (9.4%), blaOXA−69 (17.0%), and blaOXA−64 (6.6%) and four ampC (blaADC) allele types including ampC-25 (6.6%), ampC-39 (9.4%), ampC-1 (17.0%), and blaADC−88 (67.0%) were identified. MLST (Pasteur scheme) analysis revealed four ST types including ST136 (singleton), ST1 (CC1), ST25 (CC25), and ST78 (singleton) in 71, 18, 7, and 10 of A. baumannii strains, respectively. Five RAPD clusters including A (1.9%), B (26.4%), C (57.5%), D (7.5%), and E (1.9%) were characterized and 5 (4.7%) strains were found to be singletons.ConclusionThe present study demonstrated that there was a high prevalence of blaOXA−23-like producing CRAB in the clinical setting. The majority of isolates belonged to ST136 (singleton). However, blaOXA−23-like producing multi-drug resistant international clones including ST1, and emerging lineages (e.g. ST25 and ST78) were also identified. Interestingly, in this study ST2 was not detected.

Microbiological Characterization and Clinical Facets of Elizabethkingia Bloodstream Infections in a Tertiary Care Hospital of Eastern India.

Elizabethkingia is an emerging non-fermenting Gram-negative bacillus (NFGNB) causing bloodstream infections (BSI) associated with high mortality. It demonstrates a unique antimicrobial profile in showing susceptibility to antimicrobials effective against Gram-positive bacteria. This study was undertaken to determine the overall frequency of Elizabethkingia BSI, associated risk factors, microbiological susceptibility, and clonal relationship of Elizabethkingia isolates using Enterobacterial Repetitive Intergenic ConsensusPolymerase Chain Reaction (ERIC-PCR). Elizabethkingia isolates obtained from the blood culture of admitted patients (August 2020-December 2021) were identified by the VITEK 2 system and subjected to an antimicrobial susceptibility test by standard procedures. Demographics, co-morbidities, risk factors for survival, and outcome were summarized and analyzed by Chi-square test, Kaplan-Meier curve, and Cox regression. Clonal relatedness between Elizabethkingia isolates was analyzed using ERIC‑PCR fingerprinting with the "PAST: Paleontological statistics software package". Of 13,747 blood samples received during the study period, 13.59% were culture positive, and 14.60% were NFGNBs. The frequency of Elizabethkingia spp. among all NFGNBs in BSI was 29.30%, and the overall prevalence in BSI was 4.21%. In patients with Elizabethkingia BSI, Foley's catheter was present in 81.25% of the cases. 100% susceptibility was observed to linezolid, followed by vancomycin (98.75%) and chloramphenicol (89.5%). The 30-day mortality rate in the patients of Elizabethkingia BSI was 26.25%. The Presence of COVID-19, pneumonia, diabetes mellitus (DM), mechanical ventilation (MV), and prior antibiotics were significantly different (p<0.05) between the survival and death groups. ERIC-PCR profile dendrogram of Elizabethkingia isolates showed ten major clusters indicating high genetic diversity. Elizabethkingia was responsible for one-third of NFGNB BSI in a single-center study, with approximately 26% of 30-day all-cause mortality. Most isolates were susceptible to linezolid, vancomycin, and chloramphenicol. COVID-19 was the most significant risk factor associated with mortality. ERIC-PCR of Elizabethkingia isolates exhibited high genetic diversity.

Carbapenemase-producing Klebsiella pneumoniae in community settings: a cross-sectional study in Belgrade, Serbia.

Aim: The types of carbapenemases and clonal relatedness among community isolates of carbapenemase-producing Klebsiella pneumoniae in Belgrade, Serbia, were determined. Materials & methods: During the period 2016-2020, K. pneumoniae community isolates were screened for carbapenemases, and carbapenemase production was confirmed by multiplex PCR. Clonality was determined based on genetic profiles obtained by enterobacterial repetitive intergenic consensus PCR. Results: Carbapenemase genes were detected in 114 of 4800 isolates (2.4%). The most frequent gene was blaOXA-48-like. Most isolates (70.5%) were grouped in ten clusters. Cluster 11 contained 16.4% of all blaOXA-48-like-positive isolates, and all blaKPC-positive isolates were grouped in one cluster. Conclusion: Laboratory-based detection and surveillance are highly recommended in order to control the spread of resistance in community settings.

Abstract IA11: Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions

Abstract Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia. Citation Format: Laura D. Wood. Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA11.

Listeria monocytogenes clones circulating in the natural environment of the Czech Republic and Slovakia

<i>Listeria monocytogenes</i> is not only a pathogen causing a serious food-borne disease in humans but can also occur as a saprophyte in the natural environment. This study aimed to evaluate the occurrence of <i>L.</i> <i>monocytogenes</i> obtained from the natural environment of the Czech Republic (hereinafter Czechia) and Slovakia in 2016–2018 and to compare the clonal relationship of strains circulating in the environment with the strains originating from the food chain and humans. Altogether, 217 samples of mud, surface water, vegetation and soil were collected in 61 locations. Samples were processed according to the modified EN ISO 11290-1 standard. The obtained <i>L.</i> <i>monocytogenes</i> isolates were characterised using serotyping, macrorestriction analysis, followed by pulsed-field gel electrophoresis, whole genome sequencing (WGS), and antimicrobial susceptibility testing. <i>L. monocytogenes</i> were detected in 8.8% of the examined samples and were isolated in 15 locations, mainly from the mud from the banks of the surface water sources. Altogether, 25 <i>L. monocytogenes</i> strains were obtained from 19 positive samples. Serotypes 1/2a, 4b, and 1/2b were detected among the strains. Twenty combined <i>Asc</i>I<i>/Apa</i>I pulsotypes were obtained by macrorestriction analysis. Altogether, 12 sequence types (STs) were detected using Multi-locus sequence typing (MLST) by WGS, with ST451 being the most frequent. The core genome MLST analysis revealed a heterogeneous population of environmental strains. No phenotype resistance was detected by antimicrobial susceptibility testing. Screening of antimicrobial-resistance genes using the platform ResFinder revealed the genes <i>fosX</i> in 24 isolates and <i>bla</i>TEM-116 in one isolate. The occurrence of <i>L.</i> <i>monocytogenes</i> in various samples from natural environments within wide altitude range during different seasons of the year may highlight this bacterium's remarkable adaptability and exceptional tolerance to external factors. Serotype distribution of the strains circulating in the natural environment of Czechia and Slovakia seems to reflect distribution in the human population more than in the food chain.

A Nationwide Genomic Study of Clinical Klebsiella pneumoniae Carrying blaOXA-232 and rmtF in China.

OXA-232 carbapenemase is becoming a threat in China due to its high prevalence, mortality, and limited treatment options. However, little information is available on the impact of OXA-232-producing Klebsiella pneumoniae in China. This study aims to characterize the clonal relationships, the genetic mechanisms of resistance, and the virulence of OXA-232-producing K. pneumoniae isolates in China. We collected 81 OXA-232-producing K. pneumoniae clinical isolates from 2017 to 2021. Antimicrobial susceptibility testing was performed using the broth microdilution method. Capsular types, multilocus sequence types, virulence genes, antimicrobial resistance (AMR) determinants, plasmid replicon types, and single-nucleotide polymorphism (SNP) phylogeny were inferred from whole-genome sequences. OXA-232-producing K. pneumoniae strains were resistant to most antimicrobial agents. These isolates showed partial differences in susceptibility to carbapenems: all strains were resistant to ertapenem, while the resistance rates to imipenem and meropenem were 67.9% and 97.5%, respectively. Sequencing and capsular diversity analysis of the 81 K. pneumoniae isolates revealed 3 sequence types (ST15, ST231, and one novel ST [ST-V]), 2 K-locus types (KL112 and KL51), and 2 O-locus types (O2V1 and O2V2). The predominant plasmid replicon types associated with the OXA-232 and rmtF genes were ColKP3 (100%) and IncFIB-like (100%). Our study summarized the genetic characteristics of OXA-232-producing K. pneumoniae circulating in China. The results demonstrate the practical applicability of genomic surveillance and its utility in providing methods to prevent transmission. It alerts us to the urgent need for longitudinal surveillance of these transmissible lineages. IMPORTANCE In recent years, the detection rate of carbapenem-resistant K. pneumoniae has increased and represents a major threat to clinical anti-infective therapy. Compared with KPC-type carbapenemases and NDM-type metallo-β-lactamases, OXA-48 family carbapenemases are another important resistance mechanism mediating bacterial resistance to carbapenems. In this study, we investigated the molecular characteristics of OXA-232 carbapenemase-producing K. pneumoniae isolated from several hospitals to clarify the epidemiological dissemination characteristics of such drug-resistant strains in China.

Clostridioides difficile infections were predominantly driven by fluoroquinolone-resistant Clostridioides difficile ribotypes 176 and 001 in Slovakia in 2018–2019

To investigate the epidemiology of Clostridioides difficile infection (CDI) in Slovakian hospitals after the emergence of ribotype 176 (027-like) in 2016. Between 2018 and 2019, European Centre for Disease Control and Prevention CDI surveillance protocol v2.3 was applied to 14 hospitals, with additional data collected on recent antimicrobial use and the characterization of C. difficile isolates. The mean hospital incidence of CDI was 4.1 cases per 10,000 patient bed-days. One hundred and five (27.6%) in-hospital deaths were reported among the 381 cases. Antimicrobial treatment within the previous 4 weeks was recorded in 90.5% (333/368) of cases. Ribotype (RT)176 was detected in 50% (n=185/370, 14 hospitals) and RT001 was detected in 34.6% (n=128/370,13/14 hospitals) of cases with RT data. Overall, 86% (n=318/370) of isolates were resistant to moxifloxacin by Thr82Ile in GyrA (99.7%). Multi-locus variable tandem repeat analysis showed clonal relatedness of predominant RTs within and between hospitals. Seven of 14 sequenced RT176 isolates and five of 13 RT001 isolates showed between zero and three allelic differences by whole-genome multi-locus sequence typing. The majority of sequenced isolates (24/27) carried the erm(B) gene and 16/27 also carried the aac(6')-aph(2'') gene with the corresponding antimicrobial susceptibility phenotypes. Nine RT176 strains carried the cfr(E)gene and one RT001 strain carried the cfr(C) gene, but without linezolid resistance. The newly-predominant RT176 and endemic RT001 are driving the epidemiology of CDI in Slovakia. In addition to fluoroquinolones, the use of macrolide-lincosamide-streptogramin B antibiotics can represent another driving force for the spread of these epidemic lineages. In C. difficile, linezolid resistance should be confirmed phenotypically in strains with detected cfr gene(s).

Characterisation of OXA-48-like carbapenemases in Escherichia coli and Klebsiella pneumoniae from North India.

The oxacillinase-48 (OXA-48)-like carbapenemases are class D β-lactamases and increasingly reported in Enterobacterial species. The detection of these carbapenemases is challenging and little information is available on the epidemiology and plasmid characteristics of OXA-48-like carbapenemase producers. We detected the presence of OXA-48-like carbapenemases in 500 clinical isolates of Escherichia coli and Klebsiella pneumoniae, followed by detection of other carbapenemases, extended spectrum β-lactamases (ESBLs) and 16S rRNA methyltransferases in OXA-48 producers. Clonal relatedness was studied using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Finally, plasmid characterisation was performed through conjugation experiment, S1-PFGE and Southern hybridisation. Around 40% of E. coli and K. pneumoniae isolates harboured OXA-48-like β-lactamases. Two OXA-48 allele variants, OXA-232 and OXA-181 were detected in our study. OXA-48 producers co-harbored diverse drug-resistant genes belonging to other classes of carbapenemases, ESBLs and 16S rRNA methyltransferases. OXA-48-like carbapenemase producers exhibited high clonal diversity. Bla OXA-48 carrying plasmids were conjugative, untypable and their size was ~ 45kb and ~ 104.5kb in E. coli and K. pneumoniae respectively. In conclusion, OXA-48-like carbapenemases have emerged as major cause of carbapenem resistance in Enterobacteriaceae and probably still being underreported. Strict surveillance and adequate detection methods are needed to prevent the dissemination of OXA-48-like carbapenemases.

Progression and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma and B-cell prolymphocytic leukemia: Report from the 2021 SH/EAHP Workshop.

Session 3 of the 2021 Workshop of the Society for Hematopathology/European Association for Haematopathology examined progression and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and B-cell prolymphocytic leukemia (B-PLL). Thirty-one cases were reviewed by the panel. Additional studies such as immunohistochemistry and molecular genetic testing, including whole-exome sequencing and expression profiling, were performed in select cases. Session 3 included 27 CLL/SLL cases and miscellaneous associated proliferations, 3 cases of B-PLL, and 1 case of small B-cell lymphoma. The criteria for -accelerated CLL/SLL are established for lymph nodes, but extranodal disease can be diagnostically challenging. Richter transformation (RT) is a broad term and includes true transformation from original CLL/SLL clone(s) and clonally unrelated neoplasms. The morphologic, immunophenotypic, and genetic spectrum is diverse with classical and highly unusual examples. T-cell proliferations can also be encountered in CLL/SLL. B-cell prolymphocytic leukemia is a rare, diagnostically challenging disease due to its overlaps with other lymphoid neoplasms. The workshop highlighted complexity of progression and transformation in CLL/SLL and B-PLL, as well as diagnostic caveats accompanying heterogeneous presentations of RT and other manifestations of disease progression. Molecular genetic studies are pivotal for diagnosis and determination of clonal relationship, and to predict response to treatment and identify resistance to targeted therapy.

Molecular Epidemiology of Haemophilus influenzae Strains with Reduced Susceptibility and Genetic Profiles of Resistance in the Postvaccination Period.

Haemophilus influenzae serotype b has been the main cause of invasive infections in children, during the prevaccination period. More than 20 years after the introduction of the conjugate vaccine against Hib, HiNT has emerged as the cause of localized infections in children and adults. The main objective of this work is to evaluate the susceptibility and resistance mechanisms of H. influenzae strains from carriers and describe the molecular epidemiology and their clonal relationships by multilocus sequence typing (MLST). Sixty-nine strains from clinical cases and asymptomatic carriers from 2009 to 2019 were analyzed, confirmed as H. influenzae, and serotyped by polymerase chain reaction. The susceptibility to antibiotics was evaluated by E-test strips. Genotyping was performed by MLST. HiNT was the most frequent in all age groups. Resistance to ampicillin, sulfamethoxazole+trimethoprim, and amoxicillin+clavulanic acid was detected, with the production of β-lactamase being the main resistance mechanism. Among 21 HiNT strains with complete allelic MLST profiles, 19 new sequence types were described, reinforcing the already reported heterogeneity of nontypeable strains, and only one clonal complex (cc-1355) was observed. Our results show a high percentage of colonization regardless of age, increased antimicrobial resistance, and high genetic diversity, along with an increased number of cases caused by HiNT strains. These findings reinforce the need for continuous surveillance for HiNT strains as it has been reported worldwide after the introduction of the Hib conjugate vaccine.

Abstract 1045: RePhyNER: Overcoming limitations of access to matched germline for serial liquid biopsy applications

Abstract Background: Comparison of a blood or tissue sample with a matched germline control is a key step for accurate detection of somatic mutations. This is particularly important for tracking minimal residual disease (MRD), since alterations erroneously considered to be tumor derived may lead to false detection. Use of blood leukocytes as a germline control is critical not only for appropriate censoring of constitutional allelic variants, but also for addressing those associated with clonal hematopoiesis (CH). However, such matched germline is not always available, or may be suboptimal when contaminated with tumor cells. We hypothesized that accurate tumor genotyping might be feasible without a matched germline, using a dedicated algorithmic framework relying on clonal relationships of alleles of interest (i.e., phylons). Methods: Here, we introduce RePhyNER (Recursive Phylon Nomination, Enumeration, and Recovery), an algorithm for addressing this challenge in serial liquid biopsies. RePhyNER relies on the assumption that when comparing two specimens from the same individual (e.g., before and after therapy), the genomic variants of interests (e.g., tumor somatic mutations) behave differently in their allelic levels when compared to variants needing to be censored (e.g., germline constitutional alleles or CH). Using the change in mean allelic level of all variants in ≥2 samples, RePhyNER relies on a recursive approach to test each candidate somatic variant’s corresponding change gradient against this distribution using count-based statistics. Results: We used simulations to assess the importance of multiple parameters including fraction of contaminating variants or alleles and mean allelic fold-change. These simulations showed substantially superior performance of a recursive approach and revealed that for ≥2-fold changes in the mean VAF in a pair of samples, RePhyNER accurately removed &amp;gt;99% of contaminating alleles while preserving ~100% of true variants. We then applied RePhyNER to 108 plasma cell-free DNA (cfDNA) samples from 47 patients with classic Hodgkin Lymphoma (cHL) profiled by PhasED-Seq and compared MRD-detection performance with or without matched germline. RePhyNER substantially improved specificity by ~30% (68% vs 99%), and Precision by ~50% (46% vs 96%), while only modestly reducing sensitivity (100% vs 91%). We next applied PhasED-Seq to a cohort of cHL patients (n=65) without matched germline information. MRD positivity within the first 2 cycles of therapy was associated with inferior outcome when using RePhyNER (P&amp;lt;0.05 vs ns), correctly reclassifying 26% of detected patients to undetected. Conclusions: RePhyNER enables germline-free and accurate genotyping for MRD detection. Notably, RePhyNER obviates the need for additional germline profiling overcoming key limitations described above and avoids the additional associated costs of sequencing. Citation Format: Mohammad Shahrokh Esfahani, Stefan Alig, Emily Hamilton, Joseph Schroers-Martin, Brian Sworder, Jan Boegeholz, Mari Olsen, Chih Long Liu, David Kurtz, Maximilian Diehn, Ash Alizadeh. RePhyNER: Overcoming limitations of access to matched germline for serial liquid biopsy applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1045.

Towards the molecular era of discriminating multiple lung cancers.

In the era of histopathology-based diagnosis, the discrimination between multiple lung cancers (MLCs) poses significant uncertainties and has thus become a clinical dilemma. However, recent significant advances and increased application of molecular technologies in clonal relatedness assessment have led to more precision in distinguishing between multiple primary lung cancers (MPLCs) and intrapulmonary metastasis (IPMs). This review summarizes recent advances in the molecular identification of MLCs and compares various methods based on somatic mutations, chromosome alterations, microRNAs, and tumor microenvironment markers. The paper also discusses current challenges at the forefront of genomics-based discrimination, including the selection of detection technology, application of next-generation sequencing, and intratumoral heterogeneity (ITH). In summary, this paper highlights an entrance into the primary stage of molecule-based diagnostics.

The human bone marrow plasma cell compartment in rheumatoid arthritis - Clonal relationships and anti-citrulline autoantibody producing cells

A majority of circulating IgG is produced by plasma cells residing in the bone marrow (BM). Long-lived BM plasma cells constitute our humoral immune memory and are essential for infection-specific immunity. They may also provide a reservoir of potentially pathogenic autoantibodies, including rheumatoid arthritis (RA)-associated anti-citrullinated protein autoantibodies (ACPA). Here we investigated paired human BM plasma cell and peripheral blood (PB) B-cell repertoires in seropositive RA, four ACPA+ RA patients and one ACPA− using two different single-cell approaches, flow cytometry sorting, and transcriptomics, followed by recombinant antibody generation. Immunoglobulin (Ig) analysis of >900 paired heavy-light chains from BM plasma cells identified by either surface CD138 expression or transcriptome profiles (including gene expression of MZB1, JCHAIN and XBP1) demonstrated differences in IgG/A repertoires and N-linked glycosylation between patients. For three patients, we identified clonotypes shared between BM plasma cells and PB memory B cells. Notably, four individuals displayed plasma cells with identical heavy chains but different light chains, which may indicate receptor revision or clonal convergence. ACPA-producing BM plasma cells were identified in two ACPA+ patients. Three of 44 recombinantly expressed monoclonal antibodies from ACPA+ RA BM plasma cells were CCP2+, specifically binding to citrullinated peptides. Out of these, two clones reacted with citrullinated histone-4 and activated neutrophils. In conclusion, single-cell investigation of B-cell repertoires in RA bone marrow provided new understanding of human plasma cells clonal relationships and demonstrated pathogenically relevant disease-associated autoantibody expression in long-lived plasma cells.