Vaccine value profile for Klebsiella pneumoniae.

Klebsiella pneumoniae causes community- and healthcare-associated infections in children and adults. Globally in 2019, an estimated 1.27 million (95% Uncertainty Interval [UI]: 0.91-1.71) and 4.95 million (95% UI: 3.62-6.57) deaths were attributed to and associated with bacterial antimicrobial resistance (AMR), respectively. K. pneumoniae was the second leading pathogen in deaths attributed to AMR resistant bacteria. Furthermore, the rise of antimicrobial resistance in both community- and hospital-acquired infections is a concern for neonates and infants who are at high risk for invasive bacterial disease. There is a limited antibiotic pipeline for new antibiotics to treat multidrug resistant infections, and vaccines targeted against K. pneumoniae are considered to be of priority by the World Health Organization. Vaccination of pregnant women against K. pneumoniae could reduce the risk of invasive K.pneumoniae disease in their young offspring. In addition, vulnerable children, adolescents and adult populations at risk of K. pneumoniae disease with underlying diseases such as immunosuppression from underlying hematologic malignancy, chemotherapy, patients undergoing abdominal and/or urinary surgical procedures, or prolonged intensive care management are also potential target groups for a K. pneumoniae vaccine. A 'Vaccine Value Profile' (VVP) for K.pneumoniae, which contemplates vaccination of pregnant women to protect their babies from birth through to at least three months of age and other high-risk populations, provides a high-level, holistic assessment of the available information to inform the potential public health, economic and societal value of a pipeline of K. pneumoniae vaccines and other preventatives and therapeutics. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public-private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the WHO. All contributors have extensive expertise on various elements of the K.pneumoniae VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing.

A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome-each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).

Polypolish: Short-read polishing of long-read bacterial genome assemblies.

Long-read-only bacterial genome assemblies usually contain residual errors, most commonly homopolymer-length errors. Short-read polishing tools can use short reads to fix these errors, but most rely on short-read alignment which is unreliable in repeat regions. Errors in such regions are therefore challenging to fix and often remain after short-read polishing. Here we introduce Polypolish, a new short-read polisher which uses all-per-read alignments to repair errors in repeat sequences that other polishers cannot. Polypolish performed well in benchmarking tests using both simulated and real reads, and it almost never introduced errors during polishing. The best results were achieved by using Polypolish in combination with other short-read polishers.

Genomic Diversity and Antimicrobial Resistance of Haemophilus Colonizing the Airways of Young Children with Cystic Fibrosis.

Respiratory infection during childhood is a key risk factor in early cystic fibrosis (CF) lung disease progression. Haemophilus influenzae and Haemophilus parainfluenzae are routinely isolated from the lungs of children with CF; however, little is known about the frequency and characteristics of Haemophilus colonization in this context. Here, we describe the detection, antimicrobial resistance (AMR), and genome sequencing of H. influenzae and H. parainfluenzae isolated from airway samples of 147 participants aged ≤12 years enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program, Melbourne, Australia. The frequency of colonization per visit was 4.6% for H. influenzae and 32.1% for H. parainfluenzae, 80.3% of participants had H. influenzae and/or H. parainfluenzae detected on at least one visit, and using genomic data, we estimate 15.6% of participants had persistent colonization with the same strain for at least two consecutive visits. Isolates were genetically diverse and AMR was common, with 52% of H. influenzae and 82% of H. parainfluenzae displaying resistance to at least one drug. The genetic basis for AMR could be identified in most cases; putative novel determinants include a new plasmid encoding blaTEM-1 (ampicillin resistance), a new inhibitor-resistant blaTEM allele (augmentin resistance), and previously unreported mutations in chromosomally carried genes (pbp3, ampicillin resistance; folA/folP, cotrimoxazole resistance; rpoB, rifampicin resistance). Acquired AMR genes were more common in H. parainfluenzae than H. influenzae (51% versus 21%, P = 0.0107) and were mostly associated with the ICEHin mobile element carrying blaTEM-1, resulting in more ampicillin resistance in H. parainfluenzae (73% versus 30%, P = 0.0004). Genomic data identified six potential instances of Haemophilus transmission between participants, of which three involved participants who shared clinic visit days. IMPORTANCE Cystic fibrosis (CF) lung disease begins during infancy, and acute respiratory infections increase the risk of early disease development and progression. Microbes involved in advanced stages of CF are well characterized, but less is known about early respiratory colonizers. We report the population dynamics and genomic determinants of AMR in two early colonizer species, namely, Haemophilus influenzae and Haemophilus parainfluenzae, collected from a pediatric CF cohort. This investigation also reveals that H. parainfluenzae has a high frequency of AMR carried on mobile elements that may act as a potential reservoir for the emergence and spread of AMR to H. influenzae, which has greater clinical significance as a respiratory pathogen in children. This study provides insight into the evolution of AMR and the colonization of H. influenzae and H. parainfluenzae in a pediatric CF cohort, which will help inform future treatment.

Population genomics of hypervirulent Klebsiella pneumoniae clonal-group 23 reveals early emergence and rapid global dissemination.

Severe liver abscess infections caused by hypervirulent clonal-group CG23 Klebsiella pneumoniae have been increasingly reported since the mid-1980s. Strains typically possess several virulence factors including an integrative, conjugative element ICEKp encoding the siderophore yersiniabactin and genotoxin colibactin. Here we investigate CG23's evolutionary history, showing several deep-branching sublineages associated with distinct ICEKp acquisitions. Over 80% of liver abscess isolates belong to sublineage CG23-I, which emerged in ~1928 following acquisition of ICEKp10 (encoding yersiniabactin and colibactin), and then disseminated globally within the human population. CG23-I's distinguishing feature is the colibactin synthesis locus, which reportedly promotes gut colonisation and metastatic infection in murine models. These data show circulation of CG23 K. pneumoniae decades before the liver abscess epidemic was first recognised, and provide a framework for future epidemiological and experimental studies of hypervirulent K. pneumoniae. To support such studies we present an open access, completely sequenced CG23-I human liver abscess isolate, SGH10.

Comparative genomics of Crohn's disease-associated adherent-invasive Escherichia coli.

OBJECTIVE: Adherent-invasive Escherichia coli (AIEC) are a leading candidate bacterial trigger for Crohn's disease (CD). The AIEC pathovar is defined by in vitro cell-line assays examining specific bacteria/cell interactions. No molecular marker exists for their identification. Our aim was to identify a molecular property common to the AIEC phenotype. DESIGN: 41 B2 phylogroup E. coli strains were isolated from 36 Australian subjects: 19 patients with IBD and 17 without. Adherence/invasion assays were conducted using the I-407 epithelial cell line and survival/replication assays using the THP-1 macrophage cell line. Cytokine secretion tumour necrosis factor ((TNF)-α, interleukin (IL) 6, IL-8 and IL-10) was measured using ELISA. The genomes were assembled and annotated, and cluster analysis performed using CD-HIT. The resulting matrices were analysed to identify genes unique/more frequent in AIEC strains compared with non-AIEC strains. Base composition differences and clustered regularly interspaced palindromic repeat (CRISPR) analyses were conducted. RESULTS: Of all B2 phylogroup strains assessed, 79% could survive and replicate in macrophages. Among them, 11/41 strains (5 CD, 2 UCs, 5 non-IBD) also adhere to and invade epithelial cells, a phenotype assigning them to the AIEC pathovar. The AIEC strains were phylogenetically heterogeneous. We did not identify a gene (or nucleic acid base composition differences) common to all, or the majority of, AIEC. Cytokine secretion and CRISPRs were not associated with the AIEC phenotype. CONCLUSIONS: Comparative genomic analysis of AIEC and non-AIEC strains did not identify a molecular property exclusive to the AIEC phenotype. We recommend a broader approach to the identification of the bacteria-host interactions that are important in the pathogenesis of Crohn's disease.