Biology subjects

Djordjevic, S. P.

Publications and source records attributed to Djordjevic, S. P..

9 recordsLinked to original sources

bioRxiv2025

Escape from antimicrobial CRISPR-Cas9 in E. coli ST131 depends on the genetic context of the target gene.

Escherichia coli (E. coli) is a common bacterium in the human gut and an important cause of intestinal and extraintestinal infections. Some E. coli sequence types (ST) are associated with high pathogenicity. The Extraintestinal Pathogenic E. coli (ExPEC) ST131 is a globally distributed multidrug-resistant human pathogen associated with urinary tract and bloodstream infections. Antibiotic-resistant infections often lead to antibiotic treatment failure, underscoring the need of developing alternative treatments. The highly selective antimicrobial potential of CRISPR-Cas9 has been demonstrated in a range of model organisms. However, the effectiveness of CRISPR-Cas9 in combating ST131-associated infections and the consequences of CRISPR-Cas9 treatment, such as the emergence of escapers, remains unclear. Here, we investigated the antimicrobial activity of CRISPR-Cas9 against ST131 and assessed the frequency and genetic basis of escape. We conjugatively delivered CRISPR-Cas9 to ST131 isolates which carried cefotaxime-resistance-encoding target gene blaCTX-M-15 in the chromosome and characterized escape subpopulations. Two main types of escapers emerged: blaCTX-M-15-positive escapers carried dysfunctional CRISPR-Cas9 systems and arose at a [~]10-5 frequency. Instead, blaCTX-M-15-negative escapers presented chromosomal deletions involving blaCTX-M-15 loss. The frequency of blaCTX-M-15 loss depended on the blaCTX-M-15 genetic context. Specifically, blaCTX-M-15-negative escapers emerged at low frequency ([~]10-5) in isolates where blaCTX-M-15 was located downstream of insertion sequence (IS) ISEcp1, while escapers emerged with high frequency ([~]10-3) in isolates where blaCTX-M-15 was flanked by IS26. This work emphasizes how the genetic context of target genes can drive the outcome of CRISPR-Cas9 tools, where the presence of IS26 may drive increased frequencies of escape. IMPORTANCEIn the past decade CRISPR-Cas9 has emerged as an efficient antimicrobial tool capable of selective elimination of targeted bacteria. Even though it has been well described that bacteria can evolve to escape targeting by CRISPR-Cas9, the mechanisms of bacterial escape and their consequences remain largely elusive. In this study, we demonstrate the antimicrobial efficacy of CRISPR-Cas9 against natural isolates of Escherichia coli ST131, a clinically relevant pathogen, and elucidate the mechanism of escape from antimicrobial activity. We identify two distinct mechanisms of escape, which involve either dysfunctional CRISPR-Cas9 activity, or loss of the target gene (blaCTX-M-15), with the latter occurring at frequencies that depend on the genetic context of the target gene. These findings provide important insights into the frequency and mechanisms of bacterial escape from CRISPR-Cas9-based antimicrobials and offer a foundation for the development of more effective treatments.

microbiology↗

bioRxiv2024

Occurrence of "under-the-radar" antibiotic resistance in anthropogenically affected produce

With global climate change, treated-wastewater (TWW) irrigation and manure amendment are becoming increasingly important in sustainable agriculture in water- and nutrient-stressed regions. Yet, these practices can potentially disseminate pathogens and antimicrobial resistance (AMR) determinants to crops, resulting in serious health risks to humans through the food chain. Previous studies demonstrated that pathogen and AMR indicators from wastewater and manure survive poorly in the environment, suggesting that ecological barriers prevent their dissemination. However, we recently found that these elements can persist below detection levels in low quality TWW-irrigated soil, and potentially proliferate under favorable conditions. This "under the radar" phenomenon was further investigated here, in TWW irrigated- and poultry litter-amended lettuce plants, using an enrichment platform that resembles gut conditions, and an analytical approach that combined molecular and cultivation-based techniques. Enrichment uncovered clinically-relevant multidrug resistant pathogen indicators and a myriad of antibiotic resistance genes in the litter amended and TWW-irrigated lettuce that were not detected by direct analyses, or in the enriched freshwater irrigated samples. Selected resistant E. coli isolates were capable of horizontally transferring plasmids carrying multiple resistance genes to a susceptible strain. Overall, our study underlines the hidden risks of under-the-radar pathogen and AMR determinants in anthropogenically affected agroenvironments, providing a novel platform to improve quantitative microbial risk assessment models in the future.

microbiology↗

bioRxiv2024

Multiple variants of IncF plasmid alleles discovered within single bacterial cells challenge previous assumptions

IncF plasmids are diverse mobile genetic elements found in bacteria from the Enterobacteriaceae family and often carry critical antibiotic and virulence gene cargo. The classification of IncF plasmids using the plasmid Multi-Locus Sequence Typing (pMLST) tool compares the sequences of IncF alleles against a database to create a plasmid sequence type (ST). Accurate identification of plasmid STs is epidemiologically useful because it enables an assessment of the crucial IncF plasmid lineages associated with pandemic and emerging enterobacterial sequence types, inferring important information about specific bacterial lineages. Our initial observations showed discrepancies in IncF allele variants reported by pMLST in a collection of 898 Escherichia coli ST131 genomes. To evaluate the limitations of the pMLST tool, we interrogated an in-house and publicly available repository of 70324 E. coli genomes of various STs and other Enterobacterales genomes (n=1247). All short-read genomes and representatives selected for long-read sequencing were used to assess allele variants and to compare the output with the real biological situation. When multiple allele variants occurred in a single bacterial genome, the python and web versions of the tool randomly selected one allele to report, leading to limited and inaccurate ST identification. Discrepancies were detected in 5804 of 72469 genomes (8.01%). Long read sequencing of 27 carefully selected genomes confirmed multiple IncF allele variants present on one plasmid, or two separate IncF plasmids present in a single bacterial cell. The pMLST tool was unable to accurately distinguish allele variants and their location on replicons using short-read nor long-read genome sequences. ImportancePlasmid sequence type is crucial for describing IncF plasmids due to their capacity to carry important antibiotic and virulence gene cargo and consequently due to their association with disease-causing enterobacterial lineages exhibiting resistance to clinically relevant antibiotics in humans and food animals. As a result, precise reporting of IncF allele variants in IncF plasmids is necessary. Comparison of the FAB formulae generated by the plasmid Multi-Locus Sequence Typing (pMLST) tool with annotated long-read genome sequences identified inconsistencies, including examples where multiple IncF allele variants were present on the same plasmid but missing in the FAB formula, or in cases where two IncF plasmids were detected in one bacterial cell and the pMLST output provided information only about one plasmid. Such inconsistencies may cloud interpretation of IncF plasmid replicon type in specific bacterial lineages or inaccurate assumptions of host strain clonality.

genomics↗

bioRxiv2023

Xanthomonas rydalmerenesis sp. nov., a novel plant bacteria isolated from Fragaria x ananassa

We describe five bacterial isolates that were isolated from Fragaria x ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, biochemical characterisation identified these isolates as members of the Xanthomonas genus. To determine their species, we conducted further analysis using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates belonged to the Xanthomonas genus but could not classify species. Genome relatedness indices and extensive phylogenetic analysis confirmed that the isolates belonged to the Xanthomonas genus and represented a new species. Based on the absence of virulence factors typically found in Xanthomonas spp. genomes, we suggest that these isolates are non-pathogenic. This conclusion was supported by a pathogenicity assay. Based on these findings, we propose the name Xanthomonas rydalmerenesis, with DAR34855 = ICMP24941 as the type strain.

genetics↗

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↗

bioRxiv2020

Post-weaning shifts in microbiome composition and metabolism revealed by over 25,000 pig gut metagenome assembled genomes

Using a previously described metagenomics dataset of 27 billion reads, we reconstructed over 50,000 metagenome-assembled genomes (MAGs) of organisms resident in the porcine gut, 46.5% of which were classified as >70% complete with a <10% contamination rate, and 24.4% were nearly complete genomes. Here we describe the generation and analysis of those MAGs using time-series samples. The gut microbial communities of piglets appear to follow a highly structured developmental program in the weeks following weaning, and this development is robust to treatments including an intramuscular antibiotic treatment and two probiotic treatments. The high resolution we obtained allowed us to identify specific taxonomic "signatures" that characterize the microbiome development immediately after weaning. Additionally, we characterized the carbohydrate repertoire of the organisms resident in the porcine gut, identifying 294 carbohydrate active enzymes. We tracked the shifts in abundance of these enzymes across time, and identified the species and higher-level taxonomic groups carrying each of these enzymes in their MAGs, raising the possibility of modifying the piglet microbiome through the tailored provision of carbohydrate substrates.

microbiology↗

bioRxiv2020

Community composition and development of the post-weaning piglet gut microbiome

BackgroundIntensive farming practices can increase exposure of animals to infectious agents against which antibiotics are used. Besides leading to antimicrobial resistance (AMR), orally administered antibiotics are well known to cause dysbiosis. To counteract dysbiotic effects, numerous studies in the past two decades sought to understand whether probiotics are a valid tool to help re-establish a healthy gut microbial community after antibiotic treatment. However, although dysbiotic effects of antibiotics are well investigated, little is known about the effects of intramuscular antibiotic treatment on the gut microbiome and a few studies attempted to study treatment effects using phylogenetic diversity analysis techniques. In this study we sought to determine the effects of two probiotic- and one intramuscularly administered antibiotic treatment on the developing gut microbiome of post-weaning piglets between their 3rd and 9th week of life. MethodsShotgun metagenomic sequences from over 800 faecal time-series samples derived from 126 piglets and 42 sows were analysed in a phylogenetic framework to characterise the developing gut microbial community composition of post-weaning piglets. We assessed the effects of intramuscular antibiotic treatment and probiotic oral treatment on the diversity of these gut microbial communities using alpha and beta diversity measures. ResultsDifferences between individual hosts such as breed, litter, and age, were found to be important contributors to variation in the community composition. Host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appeared to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life. Treatment effects of the antibiotic and probiotic treatments were found but were subtle and included a higher representation of Mollicutes associated with intramuscular antibiotic treatment, and an increase of Lactobacillus associated with probiotic treatment. DiscussionThe discovery of correlations between experimental factors and microbial community composition is more commonly addressed with OTU-based methods and rarely analysed via phylogenetic diversity measures. The latter method, although less intuitive than the former, suffers less from library size normalization biases, and it proved to be instrumental in this study for the discovery of correlations between microbiome composition and host-, and treatment factors.

microbiology↗

bioRxiv2017

Porcine commensal Escherichia coli: A reservoir for class 1 integrons associated with IS26

Porcine faecal waste is a serious environmental pollutant. Carriage of antimicrobial resistance and virulence-associated genes (VAGs) and the zoonotic potential of commensal Escherichia coli from swine is largely unknown. Furthermore, little is known about the role of commensal E. coli as contributors to the mobilisation of antimicrobial resistance genes between food animals and the environment. Here, we report whole genome sequence analysis of 141 E. coli from the faeces of healthy pigs. Most strains belonged to phylogroups A and B1 and carried i) a class 1 integron; ii) VAGs linked with extraintestinal infection in humans; iii) antimicrobial resistance genes blaTEM, aphAl, cmlA, strAB, tet(A)A, dfrA12, dfrA5, sul1, sul2, sul3; iv) IS26; and v) heavy metal resistance genes (merA, cusA, terA). Carriage of the sulphonamide resistance gene sul3 was notable in this study. The 141 strains belonged to 42 multilocus sequence types, but clonal complex 10 featured prominently. Structurally diverse class 1 integrons that were frequently associated with IS26 carried unique genetic features that were also identified in extraintestinal pathogenic E. coli (ExPEC) from humans. This study provides the first detailed genomic analysis and point of reference for commensal E. coli of porcine origin, facilitating tracking of specific lineages and the mobile resistance genes they carry.\n\nConflict of Interest StatementNone to declare.

genomics↗

bioRxiv2017

Evaluation of ddRADseq for reduced representation metagenome sequencing

Background Who is doing what is the ultimate open question in microbiome study. Shotgun metagenomics is often applied to gain knowledge of functional roles for bacteria in microbial communities, where the data can be used to predict protein encoding genes and enzymatic pathways present in the community, sometimes leading to testable hypotheses for microbial function. We describe a method and basic analysis for a metagenomic adaptation of the double digest restriction site associated DNA sequencing (ddRADseq) protocol for reduced representation metagenome profiling. This technique takes advantage of the sequence specificity of restriction endonucleases to construct an Illumina-compatible sequencing library containing DNA fragments that are between a pair of restriction sites located within close proximity. This results in a reduced sequencing library with coverage breadth that can be tuned by size selection.\n\nResultsWe assessed the performance of the metagenomic ddRADseq approach by applying the method to human stool samples and generating sequence data. We evaluate the extent to which ddRADseq data provides an unbiased reduced representation for microbiome profiling.\n\nConclusionAlthough ddRADseq does introduce some bias in taxonomic representation, the bias is likely to be small relative to DNA extraction bias. ddRADseq appears feasible and could have value as a tool for metagenome-wide association studies.

genomics↗