ECTyper: in silico Escherichia coli serotype and species prediction from raw and assembled whole-genome sequence data

Escherichia coli is a priority foodborne pathogen of public health concern and phenotypic serotyping provides critical information for surveillance and outbreak detection activities. Public health and food safety laboratories are increasingly adopting whole-genome sequencing (WGS) for characterizing pathogens, but it is imperative to maintain serotype designations in order to minimize disruptions to existing public health workflows. Multiple in silico tools have been developed for predicting serotypes from WGS data, including SRST2, SerotypeFinder and EToKi EBEis, but these tools were not designed with the specific requirements of diagnostic laboratories, which include: speciation, input data flexibility (fasta/fastq), quality control information and easily interpretable results. To address these specific requirements, we developed ECTyper (https://github.com/phac-nml/ecoli_serotyping) for performing both speciation within Escherichia and Shigella , and in silico serotype prediction. We compared the serotype prediction performance of each tool on a newly sequenced panel of 185 isolates with confirmed phenotypic serotype information. We found that all tools were highly concordant, with 92–97 % for O-antigens and 98–100 % for H-antigens, and ECTyper having the highest rate of concordance. We extended the benchmarking to a large panel of 6954 publicly available E. coli genomes to assess the performance of the tools on a more diverse dataset. On the public data, there was a considerable drop in concordance, with 75–91 % for O-antigens and 62–90 % for H-antigens, and ECTyper and SerotypeFinder being the most concordant. This study highlights that in silico predictions show high concordance with phenotypic serotyping results, but there are notable differences in tool performance. ECTyper provides highly accurate and sensitive in silico serotype predictions, in addition to speciation, and is designed to be easily incorporated into bioinformatic workflows.

Isolation and sequencing of three RB49-like bacteriophages infecting O antigen-producing E. coli strains

E. coli strains 4s, F5 and F17, whose O antigens are structurally characterized and shown to effectively shield the cell surface from bacteriophage attack, were used as the hosts to isolate novel RB49-like bacteriophages. Three novel phage isolates were obtained, and their genomes were sequenced and annotated. Despite high overall identity levels of these genomic sequences, the variants of large distal tail fiber subunit, orthologous to the bacteriophage T2 long tail receptor recognition protein gp38, were unique for each phage, suggesting their role in host range determination. The annotated genomes are available via NCBI Genbank, acc. numbers MZ504876-MZ504878.

Rationalizing the design of a broad coverage Shigella vaccine based on evaluation of immunological cross-reactivity among S. flexneri serotypes

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials.

Salmonella serotype prediction using the GalaxyTrakr SeqSero2 workflow v1

Salmonella serotypes are defined by two surface structures, O antigen and two H antigens. Traditional serotype determination is performed with the Salmonella serological somatic (O) and flagellar (H) tests and paired with biochemical confirmation. More than 2,600 Salmonella serotypes have been described in the White-Kauffmann-Le Minor scheme. Molecular methods for serotype determination have been developed based on genes responsible for serotype antigens. These genes are encoded in the rfb gene cluster, fliC, and fljB. SeqSero2 is a bioinformatic pipeline that uses whole genome sequence (WGS) data from pure-culture isolates to perform in silico analysis to determine the antigenic formula, including somatic (O) antigens and both flagellar (H) antigens. This provides continuity with the well-established scheme for phenotypic Salmonella serotypes. PURPOSE: This document outlines the steps required to run SeqSero2 v1.1.1 on a collection of isolates in the GalaxyTrakr environment. This is performed by utilizing a custom workflow called “SeqSero2 v1.1.1 collection workflow” and downloading the resulting table. SCOPE: This protocol covers the following tasks: 1. set up an account in GalaxyTrakr 2. Create a new history/workspace 3. Upload data 4. Execute the SeqSero2 workflow 5. Download the results

High-Resolution Genomic Profiling of Carbapenem-Resistant Klebsiella pneumoniae Isolates: A Multicentric Retrospective Indian Study

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a threat to public health in India due to its high dissemination, mortality, and limited treatment options. Its genomic variability is reflected in the diversity of sequence types, virulence factors, and antimicrobial resistance (AMR) mechanisms. This study aims to characterize the clonal relationships and genetic mechanisms of resistance and virulence in CRKP isolates in India. Materials and Methods: We characterized 344 retrospective K. pneumoniae clinical isolates collected from 8 centers across India collected in 2013-2019. Susceptibility to antibiotics was tested with VITEK 2. Capsular types, MLST, virulence genes, AMR determinants, plasmid replicon types, and a single-nucleotide polymorphism (SNP) phylogeny were inferred from their whole genome sequences. Results: Phylogenetic analysis of the 325 Klebsiella isolates that passed QC revealed 3 groups: K. pneumoniae sensu stricto (n=307), K. quasipneumoniae (n=17), and K. varicolla (n=1). Sequencing and capsular diversity analysis of the 307 K. pneumoniae sensu stricto isolates revealed 28 sequence types, 26 K-locus types, and 11 O-locus types, with ST231, KL51, and O1V2 being predominant. blaOXA-48-like and blaNDM-1/5 were present in 73.2% and 24.4% of isolates respectively. The major plasmid replicon types associated with carbapenase genes were IncF (51.0%), and Col group (35.0%). Conclusion: Our study documents for the first time the genetic diversity of K- and O-antigens circulating in India. The results demonstrate the practical applicability of genomic surveillance and its utility in tracking the population dynamics of CRKP. It alerts us to the urgency for longitudinal surveillance of these virulent and transmissible lineages.

Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.

Ways to Reduce the Level of Contamination at the Stages of Tableted Chemical Cholera Vaccine Production

Objective of the study was an assessment of the degree of contamination of cholera chemical vaccine at the stages of preparation and determination of the ways to reduce it.Materials and methods. Liquid and lyophilized components of the cholera chemical vaccine used in the study: cholerogen-anatoxin and O-antigens of Vibrio cholerae 569B and V. cholerae M-41 strains, as well as auxiliary substances (sucrose, talc, calcium stearate, starch). Granulation was carried out in a device that works on a fluidized bed principle, GPCG 2 (GLATT, Germany). Subsequent tabletizing of the mixture was performed using MiniTabT compression machine (LUXNER, Germany). Studies were conducted on the evaluation of “microbiological purity” at the stages of manufacturing of the cholera chemical vaccine, tablets coated with an enteric coating. Positive or negative growth of microorganisms on Petri dishes with nutrient media was determined on visual inspection.Results and conclusions. The dynamics of changes in microbial contamination at certain technological stages of vaccine production has been revealed. It is shown that the solutions of antigens in the process of separation are subject to microbial contamination which is associated with the use of ammonium sulfate during precipitation and non-sterile water at the stage of dialysis. Sterility of semi-finished products has been achieved through twophase filtration of choleragen-anatoxin and sterilization of O-antigens of V. cholerae 569B and V. cholerae M-41 strains with flowing steam at (100±1) °C for 30 minutes. In order to decrease microbial contamination at the stage of granulation additional fine filters were installed in the air-supply system. Further on comparative assessment of microbial purity of vaccine batches obtained using both, direct compression and preliminary granulation, was carried out. It has been experimentally demonstrated that granulation of the components of a tablet mixture of cholera vaccine leads to a decrease in the level of bacterial contamination and improves the microbiological purity of the finished dosage form.

Detection and Diagnosis of Xylella fastidiosa by Specific Monoclonal Antibodies

Monoclonal antibodies (MAb) specific to Xylella fastidiosa were obtained through hybridoma technology using heat-treated somatic O antigens from LMG 17159strain. Ten stable hybrydoma clones secreting MAb were selected and their isotype was determined. The MAbs 2G1/PPD, IgG1 showed specificity for X. fastidiosa, detecting all the analyzed strains representing different subspecies, STs and hosts. Polyclonal antibodies (PAb) against X. fastidiosa were also produced and antiserum 17159-O/IVIA was selected for the highest titre and its excellent detection capability. MAb 2G1/PPD was tested against strain IVIA 5235 in PBS and in spiked raw extract samples from almond, olive, citrus, and other hosts and its sensitivity by DAS-ELISA was 104 CFU mL−1. The MAb also reacted with high affinity and avidity against X. fastidiosa by DASI-ELISA and Tissue print-ELISA. The diagnostic parameters of DAS-ELISA based on MAb were calculated and compared with the gold standard real-time PCR. The diagnostic specificity of MAb2G1/PPD was 100%, the diagnostic sensitivity was 88.5% compared to Harper’s real-time PCR and 89.9% compared to Francis’ real-time PCR. The agreement between the techniques was almost perfect according to the estimated Cohen’s kappa-index, even in symptomless almond trees. The developed immunological techniques represent sustainable and low-cost analysis tools, based on specific, homogeneous, and well-characterized MAbs, which can be obtained in unlimited quantities in a reproducible way and constitute a guarantee for the standardization of commercial kits. They are a valuable option within a polyphasic strategy for the detection of X. fastidiosa.

Cryo-EM structure of the full-length WzmWzt ABC transporter required for lipid-linked O antigen transport

O antigens are important cell surface polysaccharides in gram-negative bacteria where they extend core lipopolysaccharides in the extracellular leaflet of the outer membrane. O antigen structures are serotype specific and form extended cell surface barriers endowing many pathogens with survival benefits. In the ABC transporter-dependent biosynthesis pathway, O antigens are assembled on the cytosolic side of the inner membrane on a lipid anchor and reoriented to the periplasmic leaflet by the channel-forming WzmWzt ABC transporter for ligation to the core lipopolysaccharides. In many cases, this process depends on the chemical modification of the O antigen’s nonreducing terminus, sensed by WzmWzt via a carbohydrate-binding domain (CBD) that extends its nucleotide-binding domain (NBD). Here, we provide the cryo-electron microscopy structure of the full-length WzmWzt transporter fromAquifex aeolicusbound to adenosine triphosphate (ATP) and in a lipid environment, revealing a highly asymmetric transporter organization. The CBDs dimerize and associate with only one NBD. Conserved loops at the CBD dimer interface straddle a conserved peripheral NBD helix. The CBD dimer is oriented perpendicularly to the NBDs and its putative ligand-binding sites face the transporter to likely modulate ATPase activity upon O antigen binding. Further, our structure reveals a closed WzmWzt conformation in which an aromatic belt near the periplasmic channel exit seals the transporter in a resting, ATP-bound state. The sealed transmembrane channel is asymmetric, with one open and one closed cytosolic and periplasmic portal. The structure provides important insights into O antigen recruitment to and translocation by WzmWzt and related ABC transporters.