Clonal Clusters Research Articles

Profound reduction of HIV-1 reservoir cells over three decades of antiretroviral therapy started in early infancy.

HIV-1 reservoir cells persist indefinitely during suppressive antiretroviral therapy (ART) in individuals who acquire infection in adulthood, but little is known about the longitudinal evolution of viral reservoir cells during long-term ART started during early infancy. We studied two fraternal twins who acquired HIV-1 perinatally, started ART at week 10 after birth and remained on ART for 28 years. We observed that the frequency of genome intact proviruses, determined by single-genome near full-length proviral sequencing, declined by approximately 4,000- to 13,000-fold during this period, indicating enhanced decay rates of intact proviruses even after adjusting for dilution effects from somatic growth. Despite analyzing more than one billion PBMC after 28 years of ART in each participant, no intact proviruses were detected in one participant, and one intact provirus was isolated in the other. The longitudinal decline of defective proviruses in the two participants was more similar to proviral decay kinetics reported in individuals who started ART during adulthood; moreover, clonal sequence clusters were readily detectable for defective proviruses but not for intact proviruses after 28 years of ART in the two twins. Together, these data suggest decreased long-term stability and increased immunological vulnerability of intact proviruses during long-term ART started in early infancy.

Genomic epidemiology of carbapenem-resistant Enterobacterales at a New York City hospital over a 10-year period reveals complex plasmid-clone dynamics and evidence for frequent horizontal transfer of bla KPC.

Transmission of carbapenem-resistant Enterobacterales (CRE) in hospitals has been shown to occur through complex, multifarious networks driven by both clonal spread and horizontal transfer mediated by plasmids and other mobile genetic elements. We performed nanopore long-read sequencing on CRE isolates from a large urban hospital system to determine the overall contribution of plasmids to CRE transmission and identify specific plasmids implicated in the spread of bla KPC (the Klebsiella pneumoniae carbapenemase [KPC] gene). Six hundred and five CRE isolates collected between 2009 and 2018 first underwent Illumina sequencing for genome-wide genotyping; 435 bla KPC-positive isolates were then successfully nanopore sequenced to generate hybrid assemblies including circularized bla KPC-harboring plasmids. Phylogenetic analysis and Mash clustering were used to define putative clonal and plasmid transmission clusters, respectively. Overall, CRE isolates belonged to 96 multilocus sequence types (STs) encoding bla KPC on 447 plasmids which formed 54 plasmid clusters. We found evidence for clonal transmission in 66% of CRE isolates, over half of which belonged to four clades comprising K. pneumoniae ST258. Plasmid-mediated acquisition of bla KPC occurred in 23%-27% of isolates. While most plasmid clusters were small, several plasmids were identified in multiple different species and STs, including a highly promiscuous IncN plasmid and an IncF plasmid putatively spreading bla KPC from ST258 to other clones. Overall, this points to both the continued dominance of K. pneumoniae ST258 and the dissemination of bla KPC across clones and species by diverse plasmid backbones. These findings support integrating long-read sequencing into genomic surveillance approaches to detect the hitherto silent spread of carbapenem resistance driven by mobile plasmids.

Survey of oomycetes and pathogenicity of Phytophthora cinnamomi associated with root rot disease of western white pine (Pinus monticola)

Western white pine (Pinus monticola) with genetic tolerance to white pine blister rust disease caused by Cronartium ribicola constitutes an invaluable resource for the mitigation of this disease as well as the restoration of the species and its utilization in managed forests. In the last several years, typical root-rot symptoms and sudden tree death have been observed in some western white pine plantations located in British Columbia (BC) in Canada, and a few Phytophthora species were found to be associated with symptomatic trees. Our objectives were to survey one of the plantations located in south-west BC on Vancouver Island to confirm the presence of these Phytophthora species and other oomycetes and test their pathogenicity on western white pine by completing Koch’s postulates. We used direct plating of root samples on PARP(H)-CMA media and by baiting for oomycetes in soil and root samples. Ten oomycetes, including four Phytophthora species (Phytophthora cinnamomi, P. cactorum-like, P. cryptogea and P. pseudocryptogea) were isolated from roots and soil samples of diseased Pi. monticola trees. The Phytophthora species were identified using ITS, β-tubulin and TEF1 gene regions. Koch’s postulates were validated with controlled inoculations of 1-year old seedlings; mortality occurred as early as 15 days after inoculation with P. cinnamomi when the seedlings were maintained in warm temperature conditions (25°C). Molecular typing of two P. cinnamomi cultures using Oxford Nanopore MinIONTM sequencing indicated that they belong to one of the two main clonal clusters of mating type A2 that have been recognized as the main drivers of the global P. cinnamomi epidemic.

Phenotypic and molecular characterization of Streptococcus pneumoniae serotype 3 isolates from blood and respiratory samples in Canada: CANWARD 2007-21.

Lower respiratory infections and invasive disease caused by Streptococcus pneumoniae serotype 3 remain major clinical challenges around the world, despite widespread availability of updated vaccines. As part of CANWARD, antimicrobial susceptibility testing and serotyping were performed on all S. pneumoniae isolates from 2007 to 2021. A subset of 226/264 (85.6%) serotype 3 isolates were selected for WGS to determine sequence type (ST)/clonal cluster (CC) and correspondence of antimicrobial resistance determinants (erm, mefAE, tetM, cat, folA, folP) with resistance phenotype. Of the 3,039 S. pneumoniae isolates obtained from 2007 to 2021, 8.7% (n = 264) were serotype 3, with 64.0% of respiratory origin and 36.0% from blood. Of 226 sequenced serotype 3 isolates, 184 (81.4%) were ST180 (GPSC12). The proportion of ST8561 (single locus variant of ST180) increased from 7.2% to 16.6% during the study period. An increasing proportion of serotype 3 isolates had phenotypic resistance (P = 0.0007) and genetic resistance determinants (P = 0.004), comparing 2017-21 to 2007-11, largely due to a recently expanded ST180 clade with cat, tetM and mef determinants. S. pneumoniae serotype 3 from GPSC12 continues to dominate throughout Canada, with an increase in the proportion of ST8561. The proportion of serotype 3 isolates that are phenotypically resistant and with genetic resistance determinants is increasing over time, reflecting a global increase in GPSC12 genotypes with known resistance determinants. Phylogenomic characterization of isolates collected over time and from around the world may facilitate improved treatment and enhanced prevention strategies, including new vaccines with activity against S. pneumoniae serotype 3.

Population genomics uncovers global distribution, antimicrobial resistance, and virulence genes of the opportunistic pathogen Klebsiella aerogenes

Klebsiella aerogenes is an understudied and clinically important pathogen. We therefore investigate its population structure by genome analysis aligned with metadata. We sequence 130 non-duplicated K. aerogenes clinical isolates and identify two inter-patient transmission events. We then retrieve all publicly available K. aerogenes genomes (n= 1,026, accessed by January 1, 2023) and analyze them with our 130 genomes. We develop a core-genome multi-locus sequence-typing scheme. We find that K. aerogenes is a species complex comprising four phylogroups undergoing evolutionary divergence, likely forming three species. We delineate remarkable clonal diversity and identify three worldwide-distributed carbapenemase-encoding clonal clusters, representing high-risk lineages. We uncover that K. aerogenes has an open genome equippedby a large arsenal of antimicrobial resistance genes. We identify two genetic regions specific for K. aerogenes, encoding a type VI secretion system and flagella/chemotaxis for motility, respectively, both contributing to the virulence. These results provide much-needed insights into the population structure and pan-genomes of K. aerogenes.

Comparative Genomic Analysis of Prophages in Human Vaginal Isolates of Streptococcus agalactiae.

Prophages, viral genomes integrated into bacterial genomes, are known to enhance bacterial colonization, adaptation, and ecological fitness, providing a better chance for pathogenic bacteria to disseminate and cause infection. Streptococcus agalactiae (Group B Streptococcus or GBS) is a common bacterium found colonizing the genitourinary tract of humans. However, GBS-colonized pregnant women are at risk of passing the organism to the neonate, where it can cause severe infections. GBS typically encode one or more prophages in their genomes, yet their role in pathogen fitness and virulence has not yet been described. Sequencing and bioinformatic analysis of the genomic content of GBS human isolates identified 42 complete prophages present in their genomes. Comparative genomic analyses of the prophage sequences revealed that the prophages could be classified into five distinct clusters based on their genomic content, indicating significant diversity in their genetic makeup. Prophage diversity was also identified across GBS capsule serotypes, sequence types (STs), and clonal clusters (CCs). Comprehensive genomic annotation revealed that all GBS strains encode paratox, a protein that prevents the uptake of DNA in Streptococcus, either on the chromosome, on the prophage, or both, and each prophage genome has at least one toxin-antitoxin system.

Clonal transmission of blaIMP-4-carrying ST196 Klebsiella pneumoniae isolates mediated by the IncN plasmid in China

ObjectivesTo investigate the clinical and molecular epidemiological characteristics of blaIMP-4-carrying Klebsiella pneumoniae in a tertiary hospital in China. MethodsTen carbapenem-resistant K. pneumoniae (CRKP) isolates carrying the blaIMP-4 gene were collected. Molecular characteristics were analysed using whole-genome sequencing. Plasmid conjugation experiments were used to analyse conjugation of the plasmids. We compared and analysed K. pneumoniae-carrying blaIMP-4 genomic datasets obtained from the National Center for Biotechnology Information (NCBI) with the strains in this study. ResultsAll 10 CRKP isolates carrying blaIMP-4 were collected from 10 adult patients in the respiratory intensive care unit. These strains were only sensitive to polymyxins and tigecycline due to them simultaneously carrying multiple resistance genes, namely blaOKP-A-5, fosA, oqxA, and oqxB. Notably, R29 harboured two carbapenemase genes (blaNDM-1 and blaIMP-4). These strains had similar drug-resistant phenotypes and genes, all belonging to sequence type (ST)196. Additionally, the patients had experienced spatiotemporal intersection during hospitalization, suggesting that these strains underwent clonal transmission, but they belonged to different clonal clusters from the blaIMP-4-positive K. pneumoniae currently published in the NCBI. Among the 10 strains, blaIMP-4 was located on the IncN plasmid, and six strains had successfully transferred the plasmid to the recipient strain EC600 through plasmid conjugation. ConclusionsThe blaIMP-4-positive ST196 CRKP isolate showed clonal distribution in the respiratory intensive care unit, which was mediated by the IncN plasmid. Consequently, there should be increased monitoring of carbapenem-resistant strains in clinical settings to prevent and control its transmission.

Abstract 6097: The role of B-cell receptor repertoire in lung squamous premalignant lesions

Abstract Objective: B cells and plasma cells (PCs) play a vital role in the pathogenesis of lung cancer and provide prognostic predictive value. However, the role of B cells in the bronchial premalignant lesions (PMLs), the precursor of lung squamous cell carcinoma, is poorly understood. Prior work has shown that progression of proliferative subtype, a PML molecular subtype enriched with bronchial dysplasia, to a higher histology grade is associated with downregulation of antigen processing and presentation. In this study, we seek to understand the role of B cells, PCs, and the B-cell receptor (BCR) repertoire in progression of the bronchial PML. Methods: We performed bulk targeted BCR sequencing on 69 endobronchial biopsies obtained from 29 subjects at high-risk for developing lung cancer. The Immcantation pipeline were applied to obtain the V(D)J germline segment assignment using the human reference from IMGT, clonal cluster assignment, and mutational load quantification for each BCR sequences by pooling all the BCR sequences from the same subjects. The clones with frequency less than 10-4 were filtered out. We then performed the repertoire analysis, such as clonality diversity, isotype switching frequency, and somatic hypermutation rate (SHM) rate at sample levels and investigated the association with transcriptional signature derived from bulk RNA-seq and the clinical progression of PMLs. Results: The B cell and PC transcriptional signatures and B cell chemoattractants gene expression are associated with progression in proliferative subtype PMLs. The number of BCR clones, the class switch from IgG3 to IgG1, and the mutational rates of total and CDR3 regions were positively correlated with the B cells and PCs transcriptional signatures. Among the 31 PMLs of proliferative subtype, regressive lesions showed higher proportions of IgG heavy chain usage, higher SHM rate in CDR3 and total BCR regions, and higher frequency of class switch from IgD/M to IgG and from IgG3 to IgG1. Biopsies within the same patient had a higher proportion of shared BCR clones when they were sampled at the same timepoint than when they were sampled from the same anatomic location at different timepoints. The preserved clones of BCRs at the same anatomic location over different times had higher clonality, more class switch from IgD/M to IgG or IgA and from IgG3 to IgG1, and increased SHM rates than the non-preserved clones. Conclusions: These results suggest B and plasma cells exert anti-tumor effects that prevent bronchial PMLs from progressing to higher grade lesions by switching to IgG1 BCRs and increasing SHM rates. Citation Format: Darren J. Chiu, Carter Merenstein, Marc Lenburg, Sarah Mazzilli, Jennifer Beane. The role of B-cell receptor repertoire in lung squamous premalignant lesions [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 6097.

Connexin 43 Prevents Radiation-Induced Intestinal Damage via the Ca2+-Dependent PI3K/Akt Signaling Pathway.

Radiation-induced intestinal damage (RIID) is a common side effect of radiotherapy in patients with abdominopelvic malignancies. Gap junctions are special structures consisting of connexins (Cxs). This study aimed to investigate the expression and role of connexins in RIID and underlying mechanism. In this study, a calcein-AM fluorescence probe was used to detect changes in gap junctional intercellular communication in intestinal epithelial IEC-6 cells. Our results show that gap junctional intercellular communication of IEC-6 cells was reduced at 6, 12, 24, and 48 h after irradiation, with the most pronounced effect at 24 h. Western blotting and immunofluorescence results showed that the expression of Cx43, but not other connexins, was reduced in irradiated intestinal epithelial cells. Silencing of Cx43 reduced gap junctional intercellular communication between irradiated intestinal epithelial cells with increased ROS and intracellular Ca2+ levels. Furthermore, knockdown of Cx43 reduced the number of clonal clusters, decreased cell proliferation with increased cytotoxicity and apoptosis. Western blotting results showed that silencing of Cx43 resulted in changed γ-H2AX and PI3K/AKT pathway proteins in irradiated intestinal epithelial cells. Administration of the PI3K/AKT pathway inhibitor LY294002 inhibited the radioprotective effects in Cx43-overexpressing intestinal epithelial cells. Our study demonstrated that Cx43 expression is decreased by ionizing radiation, which facilitates the radioprotection of intestinal epithelial cells.

Multilocus sequence typing schemes for the emerging swine pathogen Mycoplasma hyosynoviae

Mycoplasma (M.) hyosynoviae is a commensal of the upper respiratory tract in swine, which has the potential to spread systemically, usually resulting in arthritis in fattening pigs and gilts. To date, very little is known about the epidemiology of M. hyosynoviae, mainly due to a lack of suitable typing methods. Therefore, this study aimed to develop both a conventional multi locus sequence typing (MLST) and a core genome (cg) MLST scheme. The development of the cgMLST was based on whole genome sequences of 64 strains isolated from pigs and wild boars during routine diagnostics as well as nine publicly available genomes. A cgMLST scheme containing 390 target genes was established using the Ridom© SeqSphere+ software. Using this scheme as a foundation, seven housekeeping genes were selected for conventional MLST based on their capability to reflect genome wide relatedness and subsequently, all 73 strains were typed by applying both methods. Core genome MLST results revealed a high diversity of the studied strain population and less than 100 allele differences between epidemiologically unrelated strains were only detected for four isolates from the US. On the other hand, seven clonal clusters (≤ 12 allele differences) comprising 20 isolates were identified. Comparison of the two typing methods resulted in highly congruent phylogenetic trees and an Adjusted Rand Coefficient of 0.893, while cgMLST showed marginally higher resolution when comparing closely related isolates, indicated by a slightly higher Simpson’s ID (0.992) than conventional MLST (Simpson’s ID = 0.990). Overall, both methods seem well suited for epidemiological analyses for scientific as well as diagnostic purposes. While MLST is faster and cheaper, cgMLST can be used to further differentiate closely related isolates.

Inside Mycobacterium bovis SB0120 spoligotype circulating in Italy: analysis of the most frequent genotypes by whole genome sequencing.

Bovine tuberculosis (bTB) is a chronic inflammatory disease primarily caused by Mycobacterium bovis. The infection affects domestic animals and wildlife, posing a zoonotic risk to humans. To understand the dynamics of transmission and genetic diversity in Italy's M. bovis population, we conducted whole-genome sequencing (WGS) analysis on two prevalent genotypes, belonging to Spoligotype SB0120, identified in different geographical and temporal contexts. By comparing these genomes with international M. bovis isolates, we identified a distinct clade within the lineage La1.2, encompassing the Italian SB0120 isolates, indicating a genomic segregation of Italian M. bovis from other European isolates. Within Italy, a significant level of genetic variability emerged across regions, while isolates within epidemiologically linked outbreaks exhibited minimal genetic diversity. Additionally, isolates derived from cattle and wild boars within a tuberculosis hotspot in Central Italy and from cattle and black pigs in Sicily formed unified clonal clusters. This indicates the presence of persistent strains circulating in the examined regions. The genetic diversity within herds was limited, as specific clones endured over time within certain herds. This research enhances our comprehension of the epidemiology and transmission patterns of bTB in Italy, thereby aiding the development of precise control strategies and disease management. Using WGS and implementing standardized protocols and databases will be pivotal in combating bTB and promoting One-Health approaches to address this noteworthy public health concern.

Staphylococcus epidermidis joint isolates: Whole-genome sequencing demonstrates evidence of hospital transmission and common antimicrobial resistance.

We investigated genetic, epidemiologic, and environmental factors contributing to positive Staphylococcus epidermidis joint cultures. Retrospective cohort study with whole-genome sequencing (WGS). We identified S. epidermidis isolates from hip or knee cultures in patients with 1 or more prior corresponding intra-articular procedure at our hospital. WGS and single-nucleotide polymorphism-based clonality analyses were performed, including species identification, in silico multilocus sequence typing (MLST), phylogenomic analysis, and genotypic assessment of the prevalence of specific antibiotic resistance and virulence genes. Epidemiologic review was performed to compare cluster and noncluster cases. In total, 60 phenotypically distinct S. epidermidis isolates were identified. After removal of duplicates and impure samples, 48 isolates were used for the phylogenomic analysis, and 45 (93.7%) isolates were included in the clonality analysis. Notably, 5 S. epidermidis strains (10.4%) showed phenotypic susceptibility to oxacillin yet harbored mecA, and 3 (6.2%) strains showed phenotypic resistance despite not having mecA. Smr was found in all isolates, and mupA positivity was not observed. We also identified 6 clonal clusters from the clonality analysis, which accounted for 14 (31.1%) of the 45 S. epidermidis isolates. Our epidemiologic investigation revealed ties to common aspirations or operative procedures, although no specific common source was identified. Most S. epidermidis isolates from clinical joint samples are diverse in origin, but we identified an important subset of 31.1% that belonged to subclinical healthcare-associated clusters. Clusters appeared to resolve spontaneously over time, suggesting the benefit of routine hospital infection control and disinfection practices.

Age-Specific Induction of Mutant p53 Drives Clonal Hematopoiesis in Adult Mice Leading to Acute Myeloid Leukemia

Background: Acute Myeloid Leukemia (AML) is a devastating hematological malignancy commonly observed in the elderly population. Mutations in TP53 can give rise to clonal hematopoiesis (CH), a condition preceding leukemia, where mutated hematopoietic stem cells (HSCs) proliferate without developing overt malignancy. The mechanisms underlying progression of TP53 mutant CH to AML and lymphoma remain incompletely understood. Through the creation of a traceable conditional Trp53-mutant CH model and complementary genetic models, we demonstrate that the age of HSCs is critical in determining whether the transformation leads to myeloid or lymphoid malignancy. Methods: To induce p53 mutation in early hematopoietic stem cells, we generated two cohorts of mice, one with Vav-Cre-mediated recombination of a conditional Trp53 allele ( Vav-Cre; Trp53 fl/wmR172H) and another with recombination of homozygous conditional floxed alleles ( Vav-Cre; Trp53 fl/fl), allowing us to compare the effects of p53 mutation versus complete loss of p53 in hematopoietic stem cells. To investigate the impact of Trp53 mutation on adult HSCs and their clonal evolution into myeloid malignancies, we developed an inducible mouse model driven by Mx1-cre. The inducible conditional mouse model allowed us to induce a somatic Trp53 mutation in HSCs of mice at 4 months of age, which is equivalent to young adult humans, and track the recombined cells using the mTmG allele ( Fig. 1A). Results: Mice with Trp53 mutations and Trp53 deletion introduced early in life developed lymphoma with complete penetrance, indicating that early-age mutation or deletion of Trp53 is more likely to lead to lymphoma development. However, when a somatic Trp53 mutation was induced in adult HSCs ( Mx1-Cre;Trp53 wmR172H/fl;mTmG mice), it resulted in myeloid-biased hematopoiesis leading to AML. Fluorescence microscopy analysis of the bone marrow (BM) clearly showed the presence of clonal clusters of GFP+ cells ( Fig. 1B). These GFP+ cells formed distinct colonies ( Fig. 1C) and were detectable in the peripheral blood ( Fig. 1D). The population of Trp53 mutant (GFP+) cells increased over time ( Fig. 1E). Flow cytometry analysis of peripheral blood after three months revealed an increased myeloid population defined as CD11b+ ( Fig. 1F). While the p53 wild-type group showed relatively stable population levels over time, the p53 mutant population displayed a significant expansion with increasing duration following pIpC injection. CyTOF analysis using a panel of 31 antibodies showed a slight increase in the myeloid lineage in p53 mutant and null mice compared to p53 wild-type, but no significant expansion of a specific stem/progenitor cell population in p53 mutant GFP+ cells was observed. After a one-year follow-up period, the Mx1-Cre;Trp53 wmR172H/fl;mTmG mice developed various myeloid malignancies (11 out of 25 mice), including AML, acute erythroid leukemia (AEL), and myeloid sarcoma ( Fig 1G). Peripheral blood smears analysis revealed the presence of large blast cells expressing GFP ( Fig. 1H). Characterizing the AML revealed a significant expansion of myeloid cells not only in the BM but also in the liver and spleen, indicating widespread dissemination of the disease ( Fig. 1I). Immunofluorescence analysis of the BM confirmed the accumulation of the mutant p53 in GFP+ cells ( Fig. 1J). Among the mice, 8 exhibited AML, 2 had AEL, and 2 presented a mixed disease phenotype consisting of both AML and AEL in the Mx1-Cre;Trp53 wmR172H/fl;mTmG cohort. The AML cells were identified as CD11b+Ly6G+CD3- ( Fig. 1K), while the AEL cells were characterized as CD34+CD71+ ( Fig. 1L). Conclusion: This data demonstrates the importance of age in the development of p53 mutant myeloid malignancies, and that p53 mutations in adult HSCs lead to the development of myeloid, but not lymphoid malignancies, highlighting the different mechanisms underlying the development of these types of malignancies, and suggest potential preventative or therapeutic strategies for TP53-mutant AML.

Deciphering the evolution of the temporal and geographic distribution of French Mycobacterium bovis genotypes using a high throughput SNP-targeted amplicon sequencing method.

Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex, is a highly clonal pathogen. However, several lineages of M. bovis have been described worldwide and nine different clusters were identified in France. Targeted amplicon sequencing using next-generation sequencing technology of eighty-eight phylogenetically informative single nucleotide polymorphisms (SNPs) were used to infer the phylogenetic relationship of 630 strains of the National Reference Laboratory isolated between 1979 and 2018 from various animal species. This study allowed classifying 618 different genotypic profiles (combination of a spoligotype and 8 loci-MIRU-VNTR profiles) into the nine previously identified clusters. A global analysis of the entire collection of the National Reference Laboratory has made it possible to represent the evolution of clonal complexes and clusters in time and space for better assessing epidemiological changes of bovine tuberculosis in France.

Clonal aggregation of fluconazole-resistant Candida tropicalis isolated from sterile body fluid specimens from patients in Hefei, China.

Candida tropicalis, a human conditionally pathogenic yeast, is distributed globally, especially in Asia-Pacific. The increasing morbidity and azole resistance of C. tropicalis have made clinical treatment difficult. The correlation between clonality and antifungal susceptibility of clinical C. tropicalis isolates has been reported. To study the putative correlation in C. tropicalis isolated from normally sterile body fluid specimens and explore the distinct clonal complex (CC) in Hefei, 256 clinical C. tropicalis isolates were collected from four teaching hospitals during 2016-2019, of which 30 were fluconazole-resistant (FR). Genetic profiles of 63 isolates, including 30 FR isolates and 33 fluconazole-susceptible (FS) isolates, were characterized using multilocus sequence typing (MLST). Phylogenetic analysis of the data was conducted using UPGMA (unweighted pair group method with arithmetic averages) and the minimum spanning tree algorithm. MLST clonal complexes (CCs) were analyzed using the goeBURST package. Among 35 differentiated diploid sequence types (DSTs), 16 DSTs and 1 genotype were identified as novel. A total of 35 DSTs were assigned to five major CCs based on goeBURST analysis. CC1 (containing DST376, 505, 507, 1221, 1222, 1223, 1226, and 1229) accounted for 86.7% (26/30) of the FR isolates. However, the genetic relationships among the FS isolates were relatively decentralized. The local FR CC1 belongs to a large fluconazole non-susceptible CC8 in global isolates, of which the putative founder genotype was DST225. The putative correlation between MLST types and antifungal susceptibility of clinical C. tropicalis isolates in Hefei showed that DSTs are closely related to FR clones.

Outbreak of OXA-232-producing carbapenem-resistant Klebsiella pneumoniae ST15 in a Chinese teaching hospital: a molecular epidemiological study.

The incidence of OXA-232-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) has been on the rise in China over the past five years, potentially leading to nosocomial epidemics. This study investigates the first outbreak of CRKP in the Second Affiliated Hospital of Jiaxing University. Between February 2021 and March 2022, 21 clinical isolates of OXA-232-producing CRKP were recovered from 16 patients in the Second Affiliated Hospital of Jiaxing University. We conducted antimicrobial susceptibility tests, whole genome sequencing, and bioinformatics to determine the drug resistance profile of these clinical isolates. Whole-genome sequencing revealed that all 21 OXA-232-producing CRKP strains belonged to the sequence type 15 (ST15) and shared similar resistance, virulence genes, and plasmid types, suggesting clonal transmission between the environment and patients. Integrated genomic and epidemiological analysis traced the outbreak to two clonal transmission clusters, cluster 1 and cluster 2, including 14 and 2 patients. It was speculated that the CRKP transmission mainly occurred in the ICU, followed by brain surgery, neurosurgery, and rehabilitation department. Phylogenetic analysis indicated that the earliest outbreak might have started at least a year before the admission of the index patient, and these strains were closely related to those previously isolated from two major adjacent cities, Shanghai and Hangzhou. Comparative genomics showed that the IncFII-type and IncHI1B-type plasmids of cluster 2 had homologous recombination at the insertion sequence sites compared with the same type of plasmids in cluster 1, resulting in the insertion of 4 new drug resistance genes, including TEM-1, APH(6)-Id, APH(3'')-Ib and sul2. Our study observed the clonal spread of ST15 OXA-232-producing between patients and the hospital environment. The integration of genomic and epidemiological data offers valuable insights and facilitate the control of nosocomial transmission.

Group B streptococci (GBS) strains evading molecular diagnostics showed novel chromosomal deletions encompassing the CAMP-factor (cfb) encoding gene.

A highly conserved fragment adjacent to the cfb gene encoding the CAMP factor is the target of PCR-based molecular diagnostic systems for the identification of S. agalactiae (group B streptococci (GBS)). Six PCR-negative, culture-positive GBS strains were whole genome sequenced to assess why they escaped molecular diagnostics. GBS strains did not constitute a clonal cluster and presented variably sized chromosomal deletions (from 7 to 33 kb) which always included the cfb gene, a finding never described before. GBS strains that escape molecular diagnostics are considered rare; however, they can cause false-negative results using molecular diagnostics alone, affecting medical decisions.

Multicentre genetic diversity study of carbapenem-resistant Enterobacterales: predominance of untypeable pUVA-like blaKPC bearing plasmids.

Carbapenem-resistant Enterobacterales (CRE) are an urgent public health threat. A better understanding of the molecular epidemiology and transmission dynamics of CRE is necessary to limit their dissemination within healthcare settings. We sought to investigate the mechanisms of resistance and spread of CRE within multiple hospitals in Maryland. From 2016 to 2018, all CRE were collected from any specimen source from The Johns Hopkins Medical Institutions. The isolates were further characterized using both phenotypic and genotypic approaches, including short- and/or long-read WGS. From 2016 to 2018, 302 of 40 908 (0.7%) unique Enterobacterales isolates were identified as CRE. Of CRE, 142 (47%) were carbapenemase-producing CRE with KPC (80.3%) predominating among various genera. Significant genetic diversity was identified among all CRE with high-risk clones serving as major drivers of clonal clusters. Further, we found the predominance of pUVA-like plasmids, with a subset harbouring resistance genes to environmental cleaning agents, involved in intergenus dissemination of blaKPC genes. Our findings provide valuable data to understand the transmission dynamics of all CRE within the greater Maryland region. These data can help guide targeted interventions to limit CRE transmission in healthcare facilities.

Genomic analysis of the population structure of Paenibacillus larvae in New Zealand.

New Zealand is a remote country in the South Pacific Ocean. The isolation and relatively late arrival of humans into New Zealand has meant there is a recorded history of the introduction of domestic species. Honey bees (Apis mellifera) were introduced to New Zealand in 1839, and the disease American foulbrood was subsequently found in the 1870s. Paenibacillus larvae, the causative agent of American foulbrood, has been genome sequenced in other countries. We sequenced the genomes of P. larvae obtained from 164 New Zealand apiaries where American foulbrood was identified in symptomatic hives during visual inspection. Multi-locus sequencing typing (MLST) revealed the dominant sequence type to be ST18, with this clonal cluster accounting for 90.2% of isolates. Only two other sequence types (with variants) were identified, ST5 and ST23. ST23 was only observed in the Otago area, whereas ST5 was limited to two geographically non-contiguous areas. The sequence types are all from the enterobacterial repetitive intergenic consensus I (ERIC I) genogroup. The ST18 and ST5 from New Zealand and international P. larvae all clustered by sequence type. Based on core genome MLST and SNP analysis, localized regional clusters were observed within New Zealand, but some closely related genomes were also geographically dispersed, presumably due to hive movements by beekeepers.

Large Multicountry Outbreak of Invasive Listeriosis by a Listeria monocytogenes ST394 Clone Linked to Smoked Rainbow Trout, 2020 to 2021.

Whole-genome sequencing (WGS) has revolutionized surveillance of infectious diseases. Disease outbreaks can now be detected with high precision, and correct attribution of infection sources has been improved. Listeriosis, caused by the bacterium Listeria monocytogenes, is a foodborne disease with a high case fatality rate and a large proportion of outbreak-related cases. Timely recognition of listeriosis outbreaks and precise allocation of food sources are important to prevent further infections and to promote public health. We report the WGS-based identification of a large multinational listeriosis outbreak with 55 cases that affected Germany, Austria, Denmark, and Switzerland during 2020 and 2021. Clinical isolates formed a highly clonal cluster (called Ny9) based on core genome multilocus sequence typing (cgMLST). Routine and ad hoc investigations of food samples identified L. monocytogenes isolates from smoked rainbow trout filets from a Danish producer grouping with the Ny9 cluster. Patient interviews confirmed consumption of rainbow trout as the most likely infection source. The Ny9 cluster was caused by a MLST sequence type (ST) ST394 clone belonging to molecular serogroup IIa, forming a distinct clade within molecular serogroup IIa strains. Analysis of the Ny9 genome revealed clpY, dgcB, and recQ inactivating mutations, but phenotypic characterization of several virulence-associated traits of a representative Ny9 isolate showed that the outbreak strain had the same pathogenic potential as other serogroup IIa strains. Our report demonstrates that international food trade can cause multicountry outbreaks that necessitate cross-border outbreak collaboration. It also corroborates the relevance of ready-to-eat smoked fish products as causes for listeriosis. IMPORTANCE Listeriosis is a severe infectious disease in humans and characterized by an exceptionally high case fatality rate. The disease is transmitted through consumption of food contaminated by the bacterium Listeria monocytogenes. Outbreaks of listeriosis often occur but can be recognized and stopped through implementation of whole-genome sequencing-based pathogen surveillance systems. We here describe the detection and management of a large listeriosis outbreak in Germany and three neighboring countries. This outbreak was caused by rainbow trout filet, which was contaminated by a L. monocytogenes clone belonging to sequence type ST394. This work further expands our knowledge on the genetic diversity and transmission routes of an important foodborne pathogen.