Nanopore and Illumina sequencing reveal different viral populations from human gut samples.

The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments, including the human intestinal microbiome. However, bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics. Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples. Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina is preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples.

Induced Burkholderia prophages detected from the hemoculture: a biomarker for Burkholderia pseudomallei infection.

Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the environment but also in the human body. The use of phages for the diagnosis of melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is emerging as a promising novel approach, but our understanding of conditions under which Burkholderia prophages can be induced remains limited. Here, we first demonstrated the isolation of Burkholderia phages from the hemocultures of melioidosis patients. The B. pseudomallei-positive hemoculture bottles were filtered to remove bacteria, and then phages were isolated and purified by spot and double agar overlay plaque assays. Forty blood samples (hemoculture-confirmed melioidosis) were tested, and phages were found in 30% of the samples. Transmission electron microscopy and genome analysis of the isolated phages, vB_HM387 and vB_HM795, showed that both phages are Myoviruses. These two phages were stable at a pH of 5-7 and temperatures of 25-37°C, suggesting their ability to survive in human blood. The genome sizes of vB_HM387 and vB_HM795 are 36.3 and 44.0 kb, respectively. A phylogenetic analysis indicated that vB_HM387 has homologs, but vB_HM795 is a novel Myovirus, suggesting the heterogeneity of Burkholderia phages in melioidosis patients. The key finding that Burkholderia phages could be isolated from the blood of melioidosis patients highlights the potential application of phage-based assays by detecting phages in blood as a pathogen-derived biomarker of infection.

A One Health Perspective on Salmonella enterica Serovar Infantis, an Emerging Human Multidrug-Resistant Pathogen.

Salmonella enterica serovar Infantis presents an ever-increasing threat to public health because of its spread throughout many countries and association with high levels of antimicrobial resistance (AMR). We analyzed whole-genome sequences of 5,284 Salmonella Infantis strains from 74 countries, isolated during 1989-2020 from a wide variety of human, animal, and food sources, to compare genetic phylogeny, AMR determinants, and plasmid presence. The global Salmonella Infantis population structure diverged into 3 clusters: a North American cluster, a European cluster, and a global cluster. The levels of AMR varied by Salmonella Infantis cluster and by isolation source; 73% of poultry isolates were multidrug resistant, compared with 35% of human isolates. This finding correlated with the presence of the pESI megaplasmid; 71% of poultry isolates contained pESI, compared with 32% of human isolates. This study provides key information for public health teams engaged in reducing the spread of this pathogen.

Differential temporal release and lipoprotein loading in B. thetaiotaomicron bacterial extracellular vesicles.

Bacterial extracellular vesicles (BEVs) contribute to stress responses, quorum sensing, biofilm formation and interspecies and interkingdom communication. However, the factors that regulate their release and heterogeneity are not well understood. We set out to investigate these factors in the common gut commensal Bacteroides thetaiotaomicron by studying BEV release throughout their growth cycle. Utilising a range of methods, we demonstrate that vesicles released at different stages of growth have significantly different composition, with early vesicles enriched in specifically released outer membrane vesicles (OMVs) containing a larger proportion of lipoproteins, while late phase BEVs primarily contain lytic vesicles with enrichment of cytoplasmic proteins. Furthermore, we demonstrate that lipoproteins containing a negatively charged signal peptide are preferentially incorporated in OMVs. We use this observation to predict all Bacteroides thetaiotaomicron OMV enriched lipoproteins and analyse their function. Overall, our findings highlight the need to understand media composition and BEV release dynamics prior to functional characterisation and define the theoretical functional capacity of Bacteroides thetaiotaomicron OMVs.

Genomic characterization of colistin-resistant Klebsiella pneumoniae isolated from intensive care unit patients in Egypt.

BACKGROUND: Egypt has witnessed elevated incidence rates of multidrug-resistant Klebsiella pneumoniae infections in intensive care units (ICUs). The treatment of these infections is becoming more challenging whilst colistin-carbapenem-resistant K. pneumoniae is upsurging. Due to the insufficiently available data on the genomic features of colistin-resistant K. pneumoniae in Egypt, it was important to fill in the gap and explore the genomic characteristics, as well as the antimicrobial resistance, the virulence determinants, and the molecular mechanisms of colistin resistance in such a lethal pathogen. METHODS: Seventeen colistin-resistant clinical K. pneumoniae isolates were collected from ICUs in Alexandria, Egypt in a 6-month period in 2020. Colistin resistance was phenotypically detected by modified rapid polymyxin Nordmann/Poirel and broth microdilution techniques. The isolates susceptibility to 20 antimicrobials was determined using Kirby-Bauer disk diffusion method. Whole genome sequencing and bioinformatic analysis were employed for exploring the virulome, resistome, and the genetic basis of colistin resistance mechanisms. RESULTS: Out of the tested K. pneumoniae isolates, 82.35% were extensively drug-resistant and 17.65% were multidrug-resistant. Promising susceptibility levels towards tigecycline (88.24%) and doxycycline (52.94%) were detected. Population structure analysis revealed seven sequence types (ST) and K-types: ST383-K30, ST147-K64, ST17-K25, ST111-K63, ST11-K15, ST14-K2, and ST525-K45. Virulome analysis revealed yersiniabactin, aerobactin, and salmochelin siderophore systems in ˃ 50% of the population. Hypervirulence biomarkers, iucA (52.94%) and rmpA/A2 (5.88%) were detected. Extended-spectrum ß-lactamase- and carbapenemase-producers accounted for 94.12% of the population, with blaCTX-M-15, blaNDM-5, and blaOXA-48 reaching 64.71%, 82.35%, and 82.35%, respectively. Chromosomal alterations in mgrB (82.35%) were the most prevailing colistin resistance-associated genetic change followed by deleterious mutations in ArnT (23.53%, L54H and G164S), PmrA (11.76%, G53V and D86E), PmrB (11.76%, T89P and T134P), PmrC (11.76%, S257L), PhoQ (5.88%, L322Q and Q435H), and ArnB (5.88%, G47D) along with the acquisition of mcr-1.1 by a single isolate of ST525. CONCLUSIONS: In this study, we present the genotypic colistin resistance mechanisms in K. pneumoniae isolated in Egypt. More effective antibiotic stewardship protocols must be implemented by Egyptian health authorities to restrain this hazard and safeguard the future utility of colistin. This is the first characterization of a complete sequence of mcr-1.1-bearing IncHI2/IncHI2A plasmid recovered from K. pneumoniae clinical isolate belonging to the emerging high-risk clone ST525.

Application of TraDIS to define the core essential genome of Campylobacter jejuni and Campylobacter coli.

Campylobacter species are the major cause of bacterial gastroenteritis. As there is no effective vaccine, combined with the rapid increase in antimicrobial resistant strains, there is a need to identify new targets for intervention. Essential genes are those that are necessary for growth and/or survival, making these attractive targets. In this study, comprehensive transposon mutant libraries were created in six C. jejuni strains, four C. coli strains and one C. lari and C. hyointestinalis strain, allowing for those genes that cannot tolerate a transposon insertion being called as essential. Comparison of essential gene lists using core genome analysis can highlight those genes which are common across multiple strains and/or species. Comparison of C. jejuni and C. coli, the two species that cause the most disease, identified 316 essential genes. Genes of interest highlighted members of the purine pathway being essential for C. jejuni whilst also finding that a functional potassium uptake system is essential. Protein-protein interaction networks using these essential gene lists also highlighted proteins in the purine pathway being major 'hub' proteins which have a large number of interactors across the network. When adding in two more species (C. lari and C. hyointestinalis) the essential gene list reduces to 261. Within these 261 essential genes, there are many genes that have been found to be essential in other bacteria. These include htrB and PEB4, which have previously been found as core virulence genes across Campylobacter species in other studies. There were 21 genes which have no known function with eight of these being associated with the membrane. These surface-associated essential genes may provide attractive targets. The essential gene lists presented will help to prioritise targets for the development of novel therapeutic and preventative interventions.

Genomic Analysis of Carbapenem-Resistant Comamonas in Water Matrices: Implications for Public Health and Wastewater Treatments.

Comamonas spp. are Gram-negative bacteria that catabolize a wide range of organic and inorganic substrates. Comamonas spp. are abundant in aquatic and soil environments, including wastewater, and can cause opportunistic infections in humans. Because of their potential in wastewater bioaugmentation and bioremediation strategies, the identification of Comamonas species harboring genes encoding carbapenemases and other clinically important antibiotic resistance genes warrant further investigation. Here, we present an analysis of 39 whole-genome sequences comprising three Comamonas species from aquatic environments in South Australia that were recovered on media supplemented with carbapenems. The analysis includes a detailed description of 33 Comamonas denitrificans isolates, some of which carried chromosomally acquired blaGES-5, blaOXA, and aminoglycoside resistance (aadA) genes located on putative genomic islands (GIs). All blaGES-5- and blaOXA-containing GIs appear to be unique to this Australian collection of C. denitrificans. Notably, most open reading frames (ORFs) within the GIs, including all antimicrobial resistance (AMR) genes, had adjacent attC sites, indicating that these ORFs are mobile gene cassettes. One C. denitrificans isolate carried an IncP-1 plasmid with genes involved in xenobiotic degradation and response to oxidative stress. Our assessment of the sequences highlights the very distant nature of C. denitrificans to the other Comamonas species and its apparent disposition to acquire antimicrobial resistance genes on putative genomic islands. IMPORTANCE Antimicrobial resistance (AMR) poses a global public health threat, and the increase in resistance to "last-resort drugs," such as carbapenems, is alarming. Wastewater has been flagged as a hot spot for AMR evolution. Comamonas spp. are among the most common bacteria in wastewater and play a role in its bioaugmentation. While the ability of Comamonas species to catabolize a wide range of organic and inorganic substrates is well documented, some species are also opportunistic pathogens. However, data regarding AMR in Comamonas spp. are limited. Here, through the genomic analyses of 39 carbapenem-resistant Comamonas isolates, we make several key observations, including the identification of a subset of C. denitrificans isolates that harbored genomic islands encoding carbapenemase blaGES-5 or extended-spectrum ß-lactamase blaOXA alleles. Given the importance of Comamonas species in potential wastewater bioaugmentation and bioremediation strategies, as well as their status as emerging pathogens, the acquisition of critically important antibiotic resistance genes on genomic islands warrants future monitoring.

Genomic analysis of Elizabethkingia species from aquatic environments: Evidence for potential clinical transmission.

Elizabethkingia species are ubiquitous in aquatic environments, colonize water systems in healthcare settings and are emerging opportunistic pathogens with reports surfacing in 25 countries across six continents. Elizabethkingia infections are challenging to treat, and case fatality rates are high. Chromosomal bla B , bla GOB and bla CME genes encoding carbapenemases and cephalosporinases are unique to Elizabethkingia spp. and reports of concomitant resistance to aminoglycosides, fluoroquinolones and sulfamethoxazole-trimethoprim are known. Here, we characterized whole-genome sequences of 94 Elizabethkingia isolates carrying multiple wide-spectrum metallo-ß-lactamase (bla B and bla GOB) and extended-spectrum serine­ß-lactamase (bla CME) genes from Australian aquatic environments and performed comparative phylogenomic analyses against national clinical and international strains. qPCR was performed to quantify the levels of Elizabethkingia species in the source environments. Antibiotic MIC testing revealed significant resistance to carbapenems and cephalosporins but susceptibility to fluoroquinolones, tetracyclines and trimethoprim-sulfamethoxazole. Phylogenetics show that three environmental E. anophelis isolates are closely related to E. anophelis from Australian clinical isolates (∼36 SNPs), and a new species, E. umeracha sp. novel, was discovered. Genomic signatures provide insight into potentially shared origins and a capacity to transfer mobile genetic elements with both national and international isolates.

CoronaHiT: high-throughput sequencing of SARS-CoV-2 genomes.

We present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.