Biology subjects

Al-Khanaq, H.

Publications and source records attributed to Al-Khanaq, H..

5 recordsLinked to original sources

bioRxiv2024

Reversible excision of the wzy locus in Salmonella Typhimurium may aid recovery following phage predation

Bacteriophage (phage) are promising novel antimicrobials but a key challenge to their effective implementation is the rapid emergence of phage resistance. An improved understanding of phage-host interactions is therefore needed. The Anderson phage typing scheme differentiates closely related strains of Salmonella enterica serovar Typhimurium (S. Typhimurium) based on sensitivity to a panel of phage preparations. Switches in phage type are indicative of changes in phage sensitivity and inform on the dynamics of phage interaction with their host bacteria. We investigated the molecular basis of switches between the relatively phage sensitive S. Typhimurium DT8 and phage resistant DT30 strains that are present in the same phylogenetic clade. DT30 strains emerged from DT8 strains predominantly by deletion of a genomic region affecting the wzy locus encoding an O-antigen polymerase. The deletion site was flanked by two perfect direct repeats designated attL and attR. During broth culture in the presence of a typing phage that used O-antigen as primary receptor the {Delta}wzy genotype increased in frequency compared with culture in the absence of phage and removal of attL prevented deletion of the wzy locus. Co-culture of S. Typhimurium DT8 with a strain lacking wzy resulted in reversion of the latter to wild type. We propose a model in which reversible deletion of the wzy locus enables recovery of S. Typhimurium DT8 following predation by phage that use O-antigen as their primary receptor. This was consistent with ancestral state reconstruction of DT8 and DT30 phylogeny that supported a model of reversible transition from DT8 to DT30 in natural populations. ImportanceS. Typhimurium is a major pathogen of livestock that adversely affects productivity and animal welfare and poses a risk of foodborne disease in the human population. Antibiotics are used to control Salmonella infections in livestock that contributes to the antimicrobial resistance global emergency. Viruses of bacteria (phage) are one alternative to antibiotics to control Salmonella in the food chain but their successful implementation as antimicrobials is restricted by the rapid emergence of resistance to phage. A better understanding of the outcome of phage-bacteria interactions is needed to optimise the design and implementation of phage-based antimicrobials. This study identifies a genetic mechanism that confers resistance to phage that use O-antigen as a receptor on the surface of Salmonella. The mechanism is likely to impart a fitness cost on the bacterium but importantly the mechanism has the potential to be revert to a fully fit state once phage predation ceases. A model for how the mechanism may contribute to survival and recovery following phage predation is proposed.

microbiology↗

bioRxiv2023

Investigating how Salmonella colonise alfalfa using a whole genome screen.

Enteropathogenic bacteria including Salmonella regularly cause outbreaks of infection from fresh produce posing a significant public health threat. Salmonellas ability to persist on fresh produce for extended periods is partly attributed to its capacity to form biofilms, which poses a challenge to food decontamination and facilitates persistence in the food chain. Preventing biofilm formation on food products and in food processing environments is crucial for reducing the incidence of foodborne diseases. Understanding the mechanisms of colonisation and establishment on fresh produce will inform the development of decontamination approaches. We used Transposon-directed Insertion site sequencing (TraDIS-Xpress) to investigate the mechanisms employed by Salmonella enterica serovar Typhimurium to colonise and establish itself on fresh produce at critical timepoints following infection. We established an alfalfa infection model and compared the findings to those obtained from glass surfaces. Our research revealed dynamic changes in the pathways associated with biofilm formation over time, with distinct plant-specific and glass-specific mechanisms for biofilm formation, alongside the identification of shared genes playing pivotal roles in both contexts. Notably, we observed variations in the significance of factors such as flagella biosynthesis, lipopolysaccharide (LPS) production, and stringent response regulation in biofilm development on plant versus glass surfaces. Understanding the genetic underpinnings of biofilm formation on both biotic and abiotic surfaces offers valuable insights that can inform the development of targeted antibacterial therapeutics, ultimately enhancing food safety throughout the food processing chain. FundingThe authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC); ERH, JAA, HAK, MAW and ET were supported by the BBSRC Institute Strategic Programme Microbes and Food Safety BB/X011011/1 and its constituent project BBS/E/F/000PR13635. NV was supported by the Food Safety Research Network grant BB/X002985/1 awarded to ET. Data availabilityNucleotide sequence data supporting the analysis in this study has been deposited in ArrayExpress under the accession number E-MTAB-13495. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2023

Collateral sensitivity increases the efficacy of a rationally designed bacteriophage combination to control Salmonella enterica

The ability of virulent bacteriophages to lyse bacteria influences bacterial evolution, fitness, and population structure. Knowledge of both host susceptibility and resistance factors is crucial for the successful application of bacteriophages as biological control agents in clinical therapy, food processing and agriculture. In this study, we isolated twelve bacteriophages termed SPLA phage which infect the foodborne pathogen Salmonella enterica. To determine phage host range, a diverse collection of Enterobacteriaceae and Salmonella enterica were used and genes involved in infection by six SPLA phages were identified using Salmonella Typhimurium strain ST4/74. Candidate host receptors included lipopolysaccharide, cellulose and BtuB. Lipopolysaccharide was identified as susceptibility factor for phage SPLA1a and mutations in LPS biosynthesis genes spontaneously emerged during culture with S. Typhimurium. Conversely, LPS was a resistance factor for phage SPLA5b that suggested that emergence of LPS mutations in culture with SPLA1a may represent a case of collateral sensitivity to SPLA5b. We show that combination therapy with SPLA1a and SPLA5b was more successful in limiting the emergence of phage resistance compared to monotherapy. Identification of host susceptibility and resistance genes and understanding infection dynamics is critical step in rationale design of phage cocktails against specific bacterial pathogens.

microbiology↗

bioRxiv2023

A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses

Bacteriophages (phages) within the Przondovirus genus are T7-like podoviruses belonging to the Studiervirinae subfamily, within the Autographiviridae family and have a highly conserved genome organisation. The genome size of these phages ranges from 37 kb to 42 kb, encode 50-60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA - a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics. IMPACT STATEMENTThe current workflows employed for phage genome assembly are often error-prone and can lead to many incomplete phage genomes being deposited within databases. This can create challenges when performing comparative genomics, and may also lead to incorrect taxonomic assignment. To overcome these challenges we proposed HYPPA, a workflow that can produce complete and high-quality phage genomes without the need for laborious lab-based validation. DATA SUMMARYPhage raw reads are available from the National Centre for Biotechnology Information Sequence Read Archive (NCBI-SRA) under the BioProject number PRJNA914245. Phage annotated genomes have been deposited at GenBank under the accessions OQ579023-OQ579032 (Table 1). Bacterial WGS data for clinical preterm infant samples have been deposited at GenBank under BioProject accession PRJNA471164 (Table S1). Bacterial raw reads for food samples are available from NCBI-SRA with individual accessions (SAMN33593347-SAMN33593351), and can be found under the BioProject number PRJNA941224 (Table S1). Strain-specific details for bacteria and publicly-available phages used in these analyses, along with accessions for the latter can be found in Table S1 and Table S6, respectively. The CL1-CL8 clinical Klebsiella strains (Table S1) were under a Materials Transfer Agreement, for which sequencing data and strain information is not available. O_TBL View this table: org.highwire.dtl.DTLVardef@8cae53org.highwire.dtl.DTLVardef@8e512org.highwire.dtl.DTLVardef@1f71a67org.highwire.dtl.DTLVardef@1e6947dorg.highwire.dtl.DTLVardef@11ebc58_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONPrzondoviruses within the collection to date and data relating to the closest database relative. C_TABLECAPTION C_TBL

microbiology↗

bioRxiv2022

Strain and serovar variants of Salmonella enterica exhibit diverse tolerance to food chain-related stress

Non-Typhoidal Salmonella (NTS) continues to be a leading cause of foodborne illness worldwide. Food manufacturers implement hurdle technology by combining more than one approach to control food safety and quality, including preservatives such as organic acids, refrigeration, and heating. We assessed the variation in survival in stresses of genotypically diverse isolates of Salmonella enterica to identify genotypes with potential elevated risk to sub-optimal processing or cooking. Sub-lethal heat treatment, survival in desiccated conditions and growth in the presence of NaCl or organic acids were investigated. S. Gallinarum strain 287/91 was most sensitive to all stress conditions. While none of the strains replicated in a food matrix at 4{degrees}C, S. Infantis strain S1326/28 retained the greatest viability, and six strains exhibited a significantly reduced viability. A S. Kedougou strain exhibited the greatest resistance to incubation at 60{degrees}C in a food matrix that was significantly greater than S. Typhimurium U288, S Heidelberg, S. Kentucky, S. Schwarzengrund and S. Gallinarum strains. Two isolates of monophasic S. Typhimurium, S04698-09 and B54 Col9 exhibited the greatest tolerance to desiccation that was significantly more than for the S. Kentucky and S. Typhimurium U288 strains. In general, the presence of 12mM acetic acid or 14mM citric acid resulted in a similar pattern of decreased growth in broth, but this was not observed for S. Enteritidis, and S. Typhimurium strains ST4/74 and U288 S01960-05. Acetic acid had a moderately greater effect on growth despite the lower concentration tested. A similar pattern of decreased growth was observed in the presence of 6% NaCl, with the notable exception that S. Typhimurium strain U288 S01960-05 exhibited enhanced growth in elevated NaCl concentrations. An understanding of the molecular basis of phenotypic variation in response to stress has the potential to improve process validation during food challenge tests, improve processing, and result in more reliable risk assessments in the food industry.

microbiology↗