Biology subjects

Kenyon, J. J.

Publications and source records attributed to Kenyon, J. J..

7 recordsLinked to original sources

bioRxiv2026

Diversity at the Acinetobacter baumannii K locus: towards a comprehensive in silico database for prediction of capsular polysaccharide types

Acinetobacter baumannii is one of the most critical bacterial pathogens requiring novel approaches for infection control and treatment to curb the spread of highly resistant isolates. Epidemiological surveillance, and the design and application of many non-antibiotic interventions, require rapid and accurate prediction of diverse capsular polysaccharide (CPS) types from whole genome sequence data. The internationally adopted CPS typing system relies on the in-silico detection of CPS biosynthesis genes both in and outside the K locus (KL), utilising a reference sequence database that is compatible with the bioinformatics tool, Kaptive. In this study, 168 novel loci were added to the database following an extensive survey of publicly available A. baumannii genomes and non-redundant sequence entries, bringing the total number of reference sequences to 409 KL and 10 extra-locus genes. All novel loci conformed to the characteristic K locus configuration described previously, with conserved core genes for CPS biosynthesis flanking a central and highly variable region that includes type-specific structural genes. Across the 409 KL, there were 1000 protein clusters. Annotations for 309 novel clusters were curated and validated using a variety of sequence-and structure-based approaches. Most proteins (n=781) were encoded by genes found in [≤]4 KL, consistent with extensive structural diversity of CPS types in A. baumannii as reported previously. However, gene distribution analysis predicted shared structural features, with several KL predicting the same CPS type or K unit. Validation of the updated database against 46,185 publicly available genomes revealed that 32 K loci were present in 87.1% of sequenced isolates, with an overrepresentation of genomes with KL2, KL3, KL18, KL9 or KL22. IMPACT STATEMENTCapsular polysaccharide (CPS) typing is embedded in in-silico genomic approaches utilised for characterisation and epidemiological surveillance of Acinetobacter baumannii isolates. As new information regarding CPS diversity and structures becomes available, continual updates to the integrated typing database are essential to ensure accuracy and endurance as a valuable scientific resource. The rapid expansion of genome data released into public databases, particularly from previously underrepresented geographies and isolation sources in recent years, provided an opportunity to capture further diversity in CPS encoding regions to enhance the utility of the typing database. A total of 168 novel K loci were identified and added to the database, and an analysis of gene distribution across all 409 loci predicted shared CPS features. This work is expected to enhance KL typing and prediction of CPS structural features for studies involving diagnostics, surveillance and therapeutics applications. DATA AVAILABILITYAll genome sequences used in this study are publicly available with details and accession numbers listed in Supplementary Table 1. The updated database with 409 KL and 10 extra-locus reference sequences is freely available at https://github.com/johannajkenyon/Abaumannii_surface_polysaccharide_loci.

microbiology↗

bioRxiv2024

Characterisation of the carbapenem-resistant Acinetobacter baumannii clinical reference isolate BAL062 (CC2:KL58:OCL1): resistance properties and capsular polysaccharide structure

The carbapenem resistant Acinetobacter baumannii isolate BAL062 is a clinical reference isolate used in several recent experimental studies. It is from a ventilator associated pneumonia (VAP) patient in an intensive care unit at the Hospital for Tropical Diseases (HTD), Ho Chi Minh City, Vietnam in 2009. Here, BAL062 was found to belong to the B sub-lineage of global clone 2 (GC2) isolates in the previously reported outbreak (2008 and 2012) of carbapenem-resistant VAP A. baumannii at the HTD. While related sub-lineage B outbreak isolates were extensively antibiotic resistant and carry GC2-associated genomic resistance islands, AbGRI1, AbGRI2 and AbGRI3, BAL062 has lost AbGRI3 and three aminoglycoside resistance genes, armA, aacA4 and aphA1, leading to amikacin and kanamycin susceptibility. The location of Tn2008VAR found in the chromosome of this sub-lineage was also corrected. Like many of the outbreak isolates, BAL062 carries the KL58 gene cluster at the capsular polysaccharide (CPS) synthesis locus and an annotation key is provided. As information about K type is important for development of novel CPS-targeting therapies, the BAL062 K58-type CPS structure was established using NMR spectroscopy. It is most closely related to K2 and K93, sharing similar configurations and linkages between K units and contains the rare higher monosaccharide, 5,7-diacetamido-3,5,7,9-tetradeoxy-O_SCPLOWDC_SCPLOW-glycero-O_SCPLOWLC_SCPLOW-manno-non-2-ulosonic acid (5,7-di-N-acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac), the 8-epimer of Pse5Ac7Ac (5,7-di-N-acetylpseudaminic acid). Inspection of publicly available A. baumannii genomes revealed a wide distribution of the KL58 locus in geographically diverse isolates belonging to several sequence types that were recovered over two decades from clinical, animal, and environmental sources. IMPORTANCEMany published experimental studies aimed at developing a clearer understanding of the pathogenicity of carbapenem resistant Acinetobacter baumannii strains currently causing treatment failure due to extensive antibiotic resistance are undertaken using historic, laboratory adapted isolates. However, it is ideal if not imperative that recent clinical isolates are used in such studies. The clinical reference isolate characterized here belongs to the dominant A. baumannii GC2 clone causing extensively resistant infections, and has been used in various recent studies. Correlation of resistance profiles and resistance gene data is key to identifying genes available for gene knockout and complementation analyses, and we have mapped the antibiotic resistance genes to find candidates. Novel therapies, such as bacteriophage or monoclonal antibody therapies, currently under investigation as alternatives or adjuncts to antibiotic treatment to combat difficult-to-treat CRAb infections often exhibit specificity for specific structural epitopes of the capsular polysaccharide (CPS), the outer-most polysaccharide layer. Here, we have solved the structure of the CPS type found in BAL062 and other extensively resistant isolates. As consistent gene naming and annotation are important for locus identification and interpretation of experimental studies, we also have correlated automatic annotations to the standard gene names.

microbiology↗

bioRxiv2023

Genomic Analysis of Diverse Environmental Acinetobacter Isolates Identifies Plasmids, Antibiotic Resistance Genes, and Capsular Polysaccharides Shared with Clinical Strains

Acinetobacter baumannii, an important pathogen known for its widespread antibiotic resistance, has been the focus of extensive research within its genus, primarily involving clinical isolates. Consequently, data on environmental A. baumannii and other Acinetobacter species remain limited. Here, we utilised Illumina and Nanopore sequencing to analyse the genomes of ten Acinetobacter isolates representing six different species sourced from aquatic environments in South Australia. All ten isolates were phylogenetically distinct compared to clinical and other non-clinical Acinetobacter strains, often tens of thousands of SNPs from their nearest neighbours. Despite the genetic divergence, we identified pdif modules (sections of mobilised DNA) carrying clinically important antimicrobial resistance genes in species other than A. baumannii, including carbapenemase oxa58, tetracycline resistance gene tet(39) and macrolide resistance genes msr(E)-mph(E). All of these pdif modules were located on plasmids with high sequence homology to those circulating in globally distributed A. baumannii ST1 and ST2 clones. The environmental A. baumannii isolate characterised here (SAAb472; ST350) did not possess any native plasmids; however, it could capture two clinically important plasmids (pRAY and pACICU2) with high transfer frequencies. Furthermore, A. baumannii SAAb472 possessed virulence genes and a capsular polysaccharide type analogous to clinical strains. Our findings highlight the potential for environmental Acinetobacter species to acquire and disseminate clinically important antimicrobial resistance genes, underscoring the need for further research into the ecology and evolution of this important genus. IMPORTANCEAntimicrobial resistance (AMR) is a global threat to human, animal, and environmental health. Studying AMR in environmental bacteria is crucial to understand the emergence and dissemination of resistance genes and pathogens, and to identify potential reservoirs and transmission routes. This study provides novel insights into the genomic diversity and AMR potential of environmental Acinetobacter species. By comparing the genomes of aquatic Acinetobacter isolates with clinical and non-clinical strains, we revealed that they are highly divergent yet carry pdif modules that encode resistance to antibiotics commonly used in clinical settings. We also demonstrated that an environmental A. baumannii isolate can acquire clinically relevant plasmids and carries virulence factors similar to those of hospital-associated strains. These findings suggest that environmental Acinetobacter species may serve as reservoirs and vectors of clinically important genes. Consequently, further research is warranted to comprehensively understand the ecology and evolution of this genus.

microbiology↗

bioRxiv2023

Identification of further variation at the lipooligosaccharide outer core locus in Acinetobacter baumannii genomes and extension of the OCL reference sequence database for Kaptive

The outer core locus (OCL) that includes genes for the synthesis of the variable outer core region of the lipooligosaccharide (LOS) is one of the key epidemiological markers used for tracing the spread of Acinetobacter baumannii, a bacterial pathogen of global concern. In this study, we screened 12476 publicly available A. baumannii genome assemblies for novel OCL sequences, detecting six new OCL types that were designated OCL17-OCL22. These were compiled with previously characterised OCL to create an updated version of the A. baumannii OCL reference database, providing a total of 22 OCL reference sequences for use with the bioinformatics tool, Kaptive. Use of this database against the 12476 downloaded assemblies found OCL1 to be the most common locus, present in 73.6% of sequenced genomes assigned by Kaptive with a match confidence score of Good or above. OCL1 was most common amongst isolates belonging to sequence types, ST1, ST2, ST3 and ST78, all of which are over-represented clonal lineages associated with extensive antibiotic resistance. The highest level of diversity in OCL types was found in ST2, with eight different OCL identified. The updated OCL reference database is available for download from GitHub (https://github.com/katholt/Kaptive; under version v 2.0.5), and has been integrated for use on Kaptive-Web (https://kaptive-web.erc.monash.edu/) and PathogenWatch (https://pathogen.watch/), enhancing current methods for A. baumannii strain identification, classification and surveillance. IMPACT STATEMENTIn the absence of effective treatment options for multi-drug resistant Acinetobacter baumannii, the highest-ranking critical priority bacterial pathogen of global concern, national and global surveillance is necessary to detect, track and subsequently curb the spread of isolates that resist current therapies. Several epidemiological markers are used to characterise A. baumannii strains by detecting genetic differences in specific regions of the genome. One of these is the chromosomal OC locus (OCL) responsible for the synthesis of the outer core (OC) component of the lipooligosaccharide (LOS). Here, we provide an update to the international A. baumannii OCL reference sequence database, extending the number of known OCL types to assist with clinical surveillance of important strains or clonal lineages. Data summaryO_LIThe updated A. baumannii OCL reference sequence database including 22 annotated OCL sequences is available for download under Kaptive v. 2.0.5 at https://github.com/katholt/Kaptive. C_LIO_LIGenome assemblies or GenBank records used as representative reference sequences are listed in Table 1 and acknowledged in each record in the database. C_LI O_TBL View this table: org.highwire.dtl.DTLVardef@1d32ff1org.highwire.dtl.DTLVardef@1f0af8borg.highwire.dtl.DTLVardef@94f185org.highwire.dtl.DTLVardef@7c8b7aorg.highwire.dtl.DTLVardef@a93255_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONInformation on A. baumannii OCL reference sequences used to populate the updated OCL database for Kaptive v 2.0.5 C_TABLECAPTION C_TBL

microbiology↗

bioRxiv2022

An update to the database for Acinetobacter baumannii capsular polysaccharide locus typing extends the extensive and diverse repertoire of genes found at and outside the K locus

2.Several novel non-antibiotic therapeutics for the critical priority bacterial pathogen, Acinetobacter baumannii, rely on specificity to the cell-surface capsular polysaccharide (CPS). Hence, prediction of CPS type deduced from genes in whole genome sequence data underpins the development and application of these therapies. In this study, we provide a comprehensive update to the A. baumannii K locus reference sequence database for CPS typing (available in Kaptive v. 2.0.1) to include 145 new KL, providing a total of 237 KL reference sequences. The database was also reconfigured for compatibility with the updated Kaptive v. 2.0.0 code that enables prediction of K type from special logic parameters defined by detected combinations of KL and additional genes outside the K locus. Validation of the database against 8994 publicly available A. baumannii genome assemblies from NCBI databases identified the specific KL in 73.45% of genomes with perfect, very high or high confidence. Poor sequence quality or the presence of insertion sequences were the main reasons for lower confidence levels. Overall, 17 KL were overrepresented in available genomes, with KL2 the most common followed by the related KL3 and KL22. Substantial variation in gene content of the central portion of the K locus, that usually includes genes specific to the CPS type, included 34 distinct groups of genes for synthesis of various complex sugars and >400 genes for forming linkages between sugars or adding non-sugar substituents. A repertoire of 681 gene types were found across the 237 KL, with 88.4% found in <5% of KL. 3. Significance as a BioResource to the communityNew therapies that target the bacterial polysaccharide capsule (CPS) show promise as effective tools to curb the high mortality rates associated with extensively resistant A. baumannii; one of the worlds most troublesome Gram-negative pathogens. As important information about the CPS structure produced by an isolate can be extracted from Whole Genome Sequences (WGS), simple bioinformatic tools and definitive sequence databases are needed to facilitate robust prediction of CPS type from WGS data. Here, we provide a comprehensive update to the international CPS sequence typing database for A. baumannii, increasing the utility of this resource for prediction of CPS type from WGS to assist with clinical surveillance, and/or the design and application of CPS-targeted therapies. This study is expected to further inform epidemiological tracking efforts, as well as the design of therapeutics targeting the CPS, enhancing global efforts to identify, trace and treat infections caused by this pathogen. 4. Data summaryO_LIThe updated A. baumannii KL reference sequence database including 241 fully annotated gene clusters is available for download under Kaptive v. 2.0.1 at https://github.com/katholt/Kaptive. C_LIO_LIGenome assemblies, short read data, or GenBank records used as representative reference sequence for each K locus are listed in Supplementary Table S1, and are referenced within each entry in the A. baumannii KL reference sequence database. C_LI The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2022

What are the reference strains of Acinetobacter baumannii referring to?

We assembled the whole genome sequence (WGS) of a collection of 43 non-redundant modern clinical isolates and four broadly used reference strains of Acinetobacter baumannii. Comparison of these isolates and their WGS confirmed the high heterogeneity in capsule loci, sequence types, the presence of virulence and antibiotic resistance genes. However, a significant portion of clinical isolates strongly differ when compared to several reference strains in the light of colony morphology, cellular density, capsule production, natural transformability and in vivo virulence. These genetic and phenotypic differences between current circulating strains of A. baumannii and established reference strains could hamper the study of A. baumannii as an entity. The broadly used reference strains led to the current state of the art of the A. baumannii field, however, we propose that established reference strains in the A. baumannii field should be carefully used, because of the high genetic and phenotypic heterogeneities. In this study, we generated a collection of high-quality nucleotide sequences of 43 modern clinical isolates with the corresponding multi-level phenotypic characterizations. Beside the contribution of novel fundamental observations generated in this study, the phenotypic and genetic data, along with the bacterial strains themselves, will be further accessible using the first open access online platform called "Acinetobase". Therefore, a rational choice of modern strains will be possible to select the ones that suit the needs of specific biological questions.

microbiology↗

bioRxiv2019

Identification of Acinetobacter baumannii loci for capsular polysaccharide (KL) and lipooligosaccharide outer core (OCL) synthesis in genome assemblies using curated reference databases compatible with Kaptive

Multiply antibiotic resistant Acinetobacter baumannii infections are a global public health concern and accurate tracking of the spread of specific lineages is needed. Variation in the composition and structure of capsular polysaccharide (CPS), a critical determinant of virulence and phage susceptibility, makes it an attractive epidemiological marker. The outer core (OC) of lipooligosaccharide also exhibits variation. To take better advantage of the untapped information available in whole genome sequences, we have created a curated reference database of the 92 publicly available gene clusters at the locus encoding proteins responsible for biosynthesis and export of CPS (K locus), and a second database for the 12 gene clusters at the locus for outer core biosynthesis (OC locus). Each entry has been assigned a unique KL or OCL number, and is fully annotated using a simple, transparent and standardised nomenclature. These databases are compatible with Kaptive, a tool for in silico typing of bacterial surface polysaccharide loci, and their utility was validated using a) >630 assembled A. baumannii draft genomes for which the KL and OCL regions had been previously typed manually, and b) 3386 A. baumannii genome assemblies downloaded from NCBI. Among the previously typed genomes, Kaptive was able to confidently assign KL and OCL types with 100% accuracy. Among the genomes retrieved from NCBI, Kaptive detected known KL and OCL in 87% and 90% of genomes, respectively indicating that the majority of common KL and OCL types are captured within the databases; 13 KL were not detected in any public genome assembly. The failure to assign a KL or OCL type may indicate incomplete or poor-quality genomes. However, further novel variants may remain to be documented. Combining outputs with multi-locus sequence typing (Institut Pasteur scheme) revealed multiple KL and OCL types in collections of a single sequence type (ST) representing each of the two predominant globally-distributed clones, ST1 of GC1 and ST2 of GC2, and in collections of other clones comprising >20 isolates each (ST10, ST25, and ST140), indicating extensive within-clone replacement of these loci. The databases are available at https://github.com/katholt/Kaptive and will be updated as further locus types become available. Data Summary1. Databases including fully annotated gene cluster sequences for A. baumannii K loci and OC loci are available for download at https://github.com/katholt/Kaptive 2. The Kaptive software, which can be used to screen new genomes against the K and O locus database is available at https://github.com/katholt/Kaptive (command-line code) and http://kaptive.holtlab.net/ (interactive web service). 3. Details of the Kaptive search results validating in silico serotyping of K and O loci using our approach are provided as supplementary files, Dataset 1 (92 KL reference sequences and 12 OCL reference sequences), Dataset 2 (642 genomes assembled from reads available in NCBI SRA) and Dataset 3 (3415 genome assemblies downloaded from NCBI GenBank). Impact statementThe ability to identify and track closely related isolates is key to understanding, and ultimately controlling, the spread of multiply antibiotic resistant A. baumannii causing difficult to treat infections, which are an urgent public health threat. Extensive variation in the KL and OCL gene clusters responsible for biosynthesis of capsule and the outer core of lipooligosaccharide, respectively, are potentially highly informative epidemiological markers. However, clear, well-documented identification of each variant and simple-to-use tools and procedures are needed to reliably identify them in genome sequence data. Here, we present curated databases compatible with the available web-based and command-line Kaptive tool to make KL and OCL typing readily accessible to assist epidemiological surveillance of this species. As many bacteriophage recognise specific properties of the capsule and attach to it, capsule typing is also important in assessing the potential of specific phage for therapy on a case by case basis.

microbiology↗