Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens.

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing Escherichia coli and Listeria monocytogenes by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning.

Mycobacterium abscessus is one of the most common and pathogenic nontuberculous mycobacteria (NTM) isolated in clinical laboratories. It consists of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Due to their different antibiotic susceptibility pattern, a rapid and accurate identification method is necessary for their differentiation. Although matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has proven useful for NTM identification, the differentiation of M. abscessus subspecies is challenging. In this study, a collection of 325 clinical isolates of M. abscessus was used for MALDI-TOF MS analysis and for the development of machine learning predictive models based on MALDI-TOF MS protein spectra. Overall, using a random forest model with several confidence criteria (samples by triplicate and similarity values >60%), a total of 96.5% of isolates were correctly identified at the subspecies level. Moreover, an improved model with Spanish isolates was able to identify 88.9% of strains collected in other countries. In addition, differences in culture media, colony morphology, and geographic origin of the strains were evaluated, showing that the latter had an impact on the protein spectra. Finally, after studying all protein peaks previously reported for this species, two novel peaks with potential for subspecies differentiation were found. Therefore, machine learning methodology has proven to be a promising approach for rapid and accurate identification of subspecies of M. abscessus using MALDI-TOF MS.

Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification.

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

Genomic epidemiology of persistently circulating MDR Shigella sonnei strains associated with men who have sex with men (MSM) in Belgium (2013-19).

OBJECTIVES: Shigella sonnei resistant to first-line antibiotics azithromycin and ciprofloxacin are on the rise globally. The aim of this study was to describe the epidemiology of MDR S. sonnei in Belgium and to identify origins and circulating clusters through WGS. METHODS: We undertook demographic, temporal and geographical analysis of 930 S. sonnei isolates submitted to the Belgian National Reference Centre for Salmonella and Shigella between 2017 and 2019. Phylogenetic analysis of WGS data, genotyping and identification of genetic markers of antimicrobial resistance was performed on 372 Belgian isolates submitted between 2013 and 2019. RESULTS: S. sonnei was identified in 75% (930/1253) of Belgian Shigella isolates submitted between 2017 and 2019. Overall, 7% (69/930) of isolates were resistant to ciprofloxacin alone, 6% (57/930) showed reduced susceptibility to azithromycin alone, and 24% (223/930) exhibited both. Men were at higher risk of carrying a double resistant S. sonnei strain, compared with women (risk ratio = 8.6, 95% CI = 5.4-13.9). Phylogenetic analysis revealed four independent Belgian clusters of persistently circulating MDR strains, associated with men who have sex with men (MSM) and of the same genotypes as previously described international MSM-related clades. Belgian isolates carried various incompatibility (Inc)-type plasmids, the SpA plasmid and ESBL genes. CONCLUSIONS: In Belgium, S. sonnei isolates from men are much more likely to be resistant to important first-line antibiotics than isolates from women. Multiple co-circulating MDR S. sonnei clusters of different genotypes were identified in the MSM community. Further studies on risk groups are needed for targeted prevention, improved clinical and public health management and antimicrobial stewardship in Belgium.

Coverage of the national surveillance system for human Salmonella infections, Belgium, 2016-2020.

INTRODUCTION: The surveillance of human salmonellosis in Belgium is dependent on the referral of human Salmonella isolates to the National Reference Center (NRC). Knowledge of current diagnostic practices and the coverage of the national Salmonella surveillance system are important to correctly interpret surveillance data and trends over time, to estimate the true burden of salmonellosis in Belgium, and to evaluate the appropriateness of implementing whole-genome sequencing (WGS) at this central level. METHODS: The coverage of the NRC was defined as the proportion of all diagnosed human Salmonella cases in Belgium reported to the NRC and was assessed for 2019 via a survey among all licensed Belgian medical laboratories in 2019, and for 2016-2020 via a capture-recapture study using the Sentinel Network of Laboratories (SNL) as the external source. In addition, the survey was used to assess the impact of the implementation of culture-independent diagnostic tests (CIDTs) at the level of peripheral laboratory sites, as a potential threat to national public health surveillance programs. RESULTS: The coverage of the NRC surveillance system was estimated to be 83% and 85%, based on the results of the survey and on the two-source capture-recapture study, respectively. Further, the results of the survey indicated a limited use of CIDTs by peripheral laboratories in 2019. CONCLUSION: Given the high coverage and the limited impact of CIDTs on the referral of isolates, we may conclude that the NRC can confidently monitor the epidemiological situation and identify outbreaks throughout the country. These findings may guide the decision to implement WGS at the level of the NRC and may improve estimates of the true burden of salmonellosis in Belgium.

A molecular assay for rapidly distinguishing the AviPro SALMONELLA VAC T vaccine strain from wild-type field isolates.

Rapid differentiation of the AviPro Salmonella VAC T strain from wild-type Salmonella ser. Typhimurium isolates is essential for the monitoring of veterinary isolates and targeted control actions. The distinction between the two strain types is routinely made by phenotypic antimicrobial resistance testing, but this sometime leads to ambiguous results with major economic implications. In this study, we used whole-genome sequencing to identify conserved and specific mutations in resistance and virulence genes which enable to distinguish field and vaccine strains. Based on this information, we developed and validated (n = 199) a Luminex-based assay targeting seven specific single-nucleotide polymorphisms. This molecular test is able to distinguish both Salmonella ser. Typhimurium types with 100% sensitivity and specificity within one working day.

Outbreak of Central American born Shigella sonnei in two youth camps in Belgium in the summer of 2019.

In 2019, an outbreak of Shigella sonnei occurred during two youth camps in Belgium. The clustering of isolates from both camps was confirmed by next-generation sequencing, as well as a secondary infection of a technician. The outbreak strain clustered with internationally isolated strains from patients with recent travel history to Central America. This report exemplifies enhanced surveillance and international collaboration between public health institutes by enabling to link local outbreaks to region-specific sublineages circulating abroad.

Transcriptional profiling of a laboratory and clinical Mycobacterium tuberculosis strain suggests respiratory poisoning upon exposure to delamanid.

Tuberculosis (TB) is the most deadly infectious disease worldwide. To reduce TB incidence and counter the spread of multidrug resistant TB, the discovery and characterization of new drugs is essential. In this study, the transcriptional response of two Mycobacterium tuberculosis strains to a pressure of the recently approved delamanid is investigated. Total RNA sequencing revealed that the response to this bicyclic nitroimidazole shows many similarities with pretomanid, an anti-tuberculous drug from the same class. Although delamanid is found to inhibit cell wall synthesis, the expression of genes involved in this process were only mildly affected. In contrast, a clear parallel was found with components that affect aerobic respiration. This demonstrates that, besides the inhibition of cell wall synthesis, respiratory poisoning plays a fundamental role in the bactericidal effect of delamanid. Remarkably, the most highly induced genes comprise poorly characterized genes for which functional characterization might hint to the target molecule(s) of delamanid and its exact mode(s) of action.

Analyzing Phage-Host Protein-Protein Interactions Using Strep-tag® II Purifications.

After injecting their genome into the bacterial host cell, bacteriophages need to convert the host metabolism toward efficient phage production. For this, specific proteins have evolved which interact with key host proteins to inhibit, activate or redirect the function of these proteins. Since 70% of the currently annotated phage genes are hypothetical proteins of unknown function, the identification and characterization of these phage proteins involved in host-phage protein-protein interactions remains challenging. Here, we describe a method to identify phage proteins involved in host-phage protein-protein interactions using a combination of affinity purifications and mass spectrometry analyses. A bacterial strain is engineered in which a bacterial target protein is fused to a Strep-tag® II at the C-terminal end. This strain is infected with a specific bacteriophage, followed by an affinity purification of the tagged protein which allows the copurification of all bacterial and phage specific interacting proteins. After SDS-PAGE analysis and an in-gel trypsin digestion, the purified interacting proteins are identified by mass spectrometry analysis. The identification of phage proteins involved in interactions provides first hints toward the elucidation of the biological function of these proteins.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria.

Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

Viral interference of the bacterial RNA metabolism machinery.

In a recent publication, we reported a unique interaction between a protein encoded by the giant myovirus phiKZ and the Pseudomonas aeruginosa RNA degradosome. Crystallography, site-directed mutagenesis and interactomics approaches revealed this 'degradosome interacting protein' or Dip, to adopt an 'open-claw' dimeric structure that presents acidic patches on its outer surface which hijack 2 conserved RNA binding sites on the scaffold domain of the RNase E component of the RNA degradosome. This interaction prevents substrate RNAs from being bound and degraded by the RNA degradosome during the virus infection cycle. In this commentary, we provide a perspective into the biological role of Dip, its structural analysis and its mysterious evolutionary origin, and we suggest some therapeutic and biotechnological applications of this distinctive viral protein.

Structural elucidation of a novel mechanism for the bacteriophage-based inhibition of the RNA degradosome.

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing. The 2.2 Šcrystal structure reveals that this novel homo-dimeric protein has no identifiable structural homologues. Our biochemical data indicate that acidic patches on the convex outer surface bind RNase E. Through the activity of Dip, фKZ has evolved a unique mechanism to down regulate a key metabolic process of its host to allow accumulation of viral RNA in infected cells.

Experimental evidence for proteins constituting virion components and particle morphogenesis of bacteriophage ZF40.

Bacteriophage ZF40 is the only currently available, temperate Myoviridae phage infecting the potato pathogen Pectobacterium carotovorum subsp. carotovorum. Despite its unusual tail morphology, its major tail sheath and tube proteins remained uncharacterized after the initial genome annotation. Using ESI tandem mass-spectrometry, 24 structural proteins of the ZF40 virion were identified, with a sequence coverage ranging between 15.8% and 87.8%. The putative function of 16 proteins could be elucidated based on secondary structure analysis and conservative domain searches. The experimental annotation of 35% of the encoded gene products within the structural region of the genome represents a complete view of the virion structure, which can serve as the basis for future structural analysis as a model phage.

Systematic identification of hypothetical bacteriophage proteins targeting key protein complexes of Pseudomonas aeruginosa.

Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).

Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa.

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analysed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labelling and pull-down experiments. Yeast two-hybrid analysis gaveclues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets.

Development of giant bacteriophage ϕKZ is independent of the host transcription apparatus.

UNLABELLED: Pseudomonas aeruginosa bacteriophage ϕKZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ∼ 280-kb ϕKZ genome to single-nucleotide resolution, we combine 369 ϕKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the ϕKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to ϕKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the ϕKZ genome is performed by the consecutive action of two ϕKZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCE: The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage ϕKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial ß and ß'-like RNAP subunits.

Listeria phage A511, a model for the contractile tail machineries of SPO1-related bacteriophages.

Recognition of the bacterial host and attachment to its surface are two critical steps in phage infection. Here we report the identification of Gp108 as the host receptor-binding protein of the broad host-range, virulent Listeria phage A511. The ligands for Gp108 were found to be N-acetylglucosamine and rhamnose substituents of the wall teichoic acids of the bacterial cell wall. Transmission electron microscopy and immunogold-labelling allowed us to create a model of the A511 baseplate in which Gp108 forms emanating short tail fibres. Data obtained for related phages, such as Staphylococcus phages ISP and Twort, demonstrate the evolutionary conservation of baseplate components and receptor-binding proteins within the Spounavirinae subfamily, and contractile tail machineries in general. Our data reveal key elements in the infection process of large phages infecting Gram-positive bacteria and generate insights into the complex adsorption process of phage A511 to its bacterial host.

Genome and proteome analysis of 7-7-1, a flagellotropic phage infecting Agrobacterium sp H13-3.

BACKGROUND: The flagellotropic phage 7-7-1 infects motile cells of Agrobacterium sp H13-3 by attaching to and traveling along the rotating flagellar filament to the secondary receptor at the base, where it injects its DNA into the host cell. Here we describe the complete genomic sequence of 69,391 base pairs of this unusual bacteriophage. METHODS: The sequence of the 7-7-1 genome was determined by pyro(454)sequencing to a coverage of 378-fold. It was annotated using MyRAST and a variety of internet resources. The structural proteome was analyzed by SDS-PAGE coupled electrospray ionization-tandem mass spectrometry (MS/MS). RESULTS: Sequence annotation and a structural proteome analysis revealed 127 open reading frames, 84 of which are unique. In six cases 7-7-1 proteins showed sequence similarity to proteins from the virulent Burkholderia myovirus BcepB1A. Unique features of the 7-7-1 genome are the physical separation of the genes encoding the small (orf100) and large (orf112) subunits of the DNA packaging complex and the apparent lack of a holin-lysin cassette. Proteomic analysis revealed the presence of 24 structural proteins, five of which were identified as baseplate (orf7), putative tail fibre (orf102), portal (orf113), major capsid (orf115) and tail sheath (orf126) proteins. In the latter case, the N-terminus was removed during capsid maturation, probably by a putative prohead protease (orf114).