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1. Cell-free synthesis of the Salmonella specific broad host range bacteriophage, felixO1

   https://doi.org/10.1016/j.mimet.2024.106920  

   McFarlane, John A; Garenne, David; Noireaux, Vincent; Bowden, Steven D (May 2024, Journal of Microbiological Methods)

   NA (Ed.)

   Phage-based biocontrol of foodborne Salmonella is limited by the requisite use of Salmonella to propagate the phages. This limitation can be circumvented by producing Salmonella phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the Salmonella phage felixO1 using an E. coli-based CFE system. 

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2. Isothermal Amplification and Lateral Flow Nucleic Acid Test for the Detection of Shiga Toxin-Producing Bacteria for Food Monitoring

   https://doi.org/10.3390/chemosensors10060210  

   Petrucci, Sabrina; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K. (June 2022, Chemosensors)

   Foodborne bacteria have persisted as a significant threat to public health and to the food and agriculture industry. Due to the widespread impact of these pathogens, there has been a push for the development of strategies that can rapidly detect foodborne bacteria on-site. Shiga toxin-producing E. coli strains (such as E. coli O157:H7, E. coli O121, and E. coli O26) from contaminated food have been a major concern. They carry genes stx1 and/or stx2 that produce two toxins, Shiga toxin 1 and Shiga toxin 2, which are virulent proteins. In this work, we demonstrate the development of a rapid test based on an isothermal recombinase polymerase amplification reaction for two Shiga toxin genes in a single reaction. Results of the amplification reaction are visualized simultaneously for both Shiga toxins on a single lateral flow paper strip. This strategy targets the DNA encoding Shiga toxin 1 and 2, allowing for broad detection of any Shiga toxin-producing bacterial species. From sample to answer, this method can achieve results in approximately 35 min with a detection limit of 10 CFU/mL. This strategy is sensitive and selective, detecting only Shiga toxin-producing bacteria. There was no interference observed from non-pathogenic or pathogenic non-Shiga toxin-producing bacteria. A detection limit of 10 CFU/mL for Shiga toxin-producing E. coli was also obtained in a food matrix. This strategy is advantageous as it allows for timely identification of Shiga toxin-related contamination for quick initial food contamination assessments. 

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3. Molecular epidemiology and pathogenomics of extended-spectrum beta-lactamase producing- Escherichia coli and - Klebsiella pneumoniae isolates from bulk tank milk in Tennessee, USA

   https://doi.org/10.3389/fmicb.2023.1283165  

   Gelalcha, Benti D; Mohammed, Ruwaa I; Gelgie, Aga E; Kerro_Dego, Oudessa (November 2023, Frontiers in Microbiology)

   IntroductionThe rise in extended-spectrum beta-lactamase (ESBL)-producingEnterobacteriaceaein dairy cattle farms poses a risk to human health as they can spread to humans through the food chain, including raw milk. This study was designed to determine the status, antimicrobial resistance, and pathogenic potential of ESBL-producing -E. coliand -Klebsiellaspp. isolates from bulk tank milk (BTM). MethodsThirty-three BTM samples were collected from 17 dairy farms and screened for ESBL-E. coliand -Klebsiellaspp. on CHROMagar ESBL plates. All isolates were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). ResultsTen presumptive ESBL-producing bacteria, eightE. coli, and twoK. pneumoniaewere isolated. The prevalence of ESBL-E. coliand -K. pneumoniaein BTM was 21.2% and 6.1%, respectively. ESBL-E. coliwere detected in 41.2% of the study farms. Seven of the ESBL-E. coliisolates were multidrug resistant (MDR). The two ESBL-producingK. pneumoniaeisolates were resistant to ceftriaxone. Seven ESBL-E. colistrains carry thebla_CTX-Mgene, and five of them co-harboredbla_TEM-1. ESBL-E. colico-harboredbla_CTX-Mwith other resistance genes, includingqnrB19,tet(A),aadA1,aph(3’’)-Ib,aph(6)-Id),floR,sul2, and chromosomal mutations (gyrA, gyrB, parC, parE, and pmrB). MostE. coliresistance genes were associated with mobile genetic elements, mainly plasmids. Six sequence types (STs) ofE. coliwere detected. All ESBL-E. coliwere predicted to be pathogenic to humans. Four STs (three ST10 and ST69) were high-risk clones ofE. coli. Up to 40 virulence markers were detected in allE. coliisolates. One of theK. pneumoniaewas ST867; the other was novel strain.K. pneumoniaeisolates carried three types of beta-lactamase genes (bla_CTX-M,bla_TEM-1andbla_SHV). The novelK. pneumoniaeST also carried a novel IncFII(K) plasmid ST. ConclusionDetection of high-risk clones of MDR ESBL-E. coliand ESBL-K. pneumoniaein BTM indicates that raw milk could be a reservoir of potentially zoonotic ESBL-E. coliand -K. pneumoniae. 

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4. Phage defense and genome editing using novel retrons sourced from isolated environmental bacteria

   https://doi.org/10.1101/2025.01.29.635429  

   Nakamura, Kazuo L; Zhang, Karen; Mestre, Mario R; Rojas-Montero, Matías; Shipman, Seth L (January 2025, bioRxiv)

   ABSTRACT Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase, a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase, noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we present six of these novel retrons, each isolated from a different host bacterium. We characterize the full operon of these retrons and test their ability to defend against a panel ofE. coliphages. For two of these retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing inE. coli, demonstrating their potential use in a biotechnological application. 

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5. A simple BLASTn-based approach generates novel insights into the regulation and biological function of type I toxin-antitoxins

   https://doi.org/10.1128/msystems.01204-23  

   Shore, Selene_F H; Ptacek, Michael; Steen, Andrew D; Fozo, Elizabeth M (July 2024, mSystems)

   Svensson, Sarah L (Ed.)

   Bacterial chromosomal type I toxin-antitoxin systems consist of a small protein, typically under 60 amino acids, and a small RNA (sRNA) that represses toxin translation. These gene pairs have gained attention over the last decade for their contribution to antibiotic persistence and phage tolerance in bacteria. However, biological functions for many remain elusive as gene deletions often fail to produce an observable phenotype. For many pairs, it is still unknown when the toxin and/or antitoxin gene are natively expressed within the bacterium. We examined sequence conservation of three type I toxin-antitoxin systems,tisB/istR-1, shoB/ohsC, and zor/orz, in over 2,000Escherichia colistrains, including pathogenic and commensal isolates. Using our custom database, we found that these gene pairs are widespread acrossE. coliand have expression potential via BLASTn. We identified an alternative, dominant sequence variant of TisB and confirmed that it is toxic upon overproduction. Additionally, analyses revealed a highly conserved sequence in thezorOmRNA untranslated region that is required for full toxicity. We further noted that over 30% ofE. coligenomes contain anorzantitoxin gene only and confirmed its expression in a representative strain: the first confirmed report of a type I antitoxin without its cognate toxin. Our results add to our understanding of these systems, and our methodology is applicable for other type I loci to identify critical regulatory and functional features.IMPORTANCEChromosomal type I toxin-antitoxins are a class of genes that have gained increasing attention over the last decade for their roles in antibiotic persistence which may contribute to therapeutic failures. However, the control of many of these genes and when they function have remained elusive. We demonstrate that a simple genetic conservation-based approach utilizing free, publicly available data yields known and novel insights into the regulation and function of three chromosomal type I toxin-antitoxins inEscherichia coli. This study also provides a framework for how this approach could be applied to other genes of interest. 

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