Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria

Abstract

Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti-S. enterica antibody response.

A simple, quantitative assay for the detection of viable but non-culturable (VBNC) bacteria

“…The Molecular Probes Bac Light stains are compatible across numerous bacterial species such as Agrobacterium tumefaciens , Clostridium perfringens , Klebsiella pneumoniae , Pseudomonas aeruginosa , Salmonella oranienburg , Staphylococcus aureus and Streptococcus pyogenes ( Hu et al, 2017 ; Lindbäck et al, 2010 ; Park and Kim, 2018 ; Robertson et al, 2019 ; Molecular Probes, 2004 ). The Biotium stains have been used with E. coli, Leptothrix , Lactobacillus fermentum, Lactobacillus reuteri , Pleurotus osreatus , and Salmonella enterica , ( Adamski and Pietr, 2019 ; Bhave et al, 2015 ; Duplantis et al, 2015 ; Furutani et al, 2011 ; Hernandez et al, 2012 ; Kim et al, 2016 ; Rodes et al, 2013 ; Biotium, 2020 ).…”

Enzymes from Marine Polar Regions and Their Biotechnological Applications

“…The authors applied the same approach to Salmonella enterica and the Gram-positive Mycobacterium [62]. The TS S. enterica strains were shown to be safe in research or diagnostic laboratories but were still capable of stimulating a protective immune response [63]. The TS strain of M. tuberculosis could be a safe research or diagnostic strain that is incapable of causing serious disease in humans while being identical to wild-type M. tuberculosis except for the TS phenotype [62].…”

Use of essential gene, encoding prophobilinogen deaminase from extreme psychrophilic Colwellia sp. C1, to generate temperature-sensitive strain of Francisella novicida

“…This type of genome engineering often renders the pathogen temperature sensitive and unable to revert to growth in the normal mesophilic range at a detectable level (Duplantis et al . , ). The biological basis of this approach is the highly conserved requirement for functional activity of about 300 bacterial essential proteins, most of which are enzymes (Baba et al .…”