Application of a broad range lytic phage LPST94 for biological control of salmonella in foods

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Salmonella, one of the most common food-borne pathogens, is a significant public health and economic burden worldwide. Lytic phages are viable alternatives to conventional technologies for pathogen biocontrol in food products. In this study, 40 Salmonella phages were isolated from environmentally sourced water samples. We characterized the lytic range against Salmonella and among all isolates, phage LPST94 showed the broadest lytic spectrum and the highest lytic activity. Electron microscopy and genome sequencing indicated that LPST94 belongs to the Ackermannviridae family. Further studies showed this phage is robust, tolerating a wide range of pH (4–12) and temperature (30–60◦C) over 60 min. The efficacy of phage LPST94 as a biological control agent was evaluated in various food products (milk, apple juice, chicken breast, and lettuce) inoculated with non-typhoidal Salmonella species at different temperatures. Interestingly, the anti-Salmonella efficacy of phage LPST94 was greater at 4◦C than 25◦C, although the efficacy varied between different food models. Adding phage LPST94 to Salmonella inoculated milk decreased the Salmonella count by 3 log10 CFU/mL at 4◦C and 0.84 to 2.56 log10 CFU/mL at 25◦C using an MOI of 1000 and 10000, respectively. In apple juice, chicken breast, and lettuce, the Salmonella count was decreased by 3 log10 CFU/mL at both 4◦C and 25◦C after applying phage LPST94 at an MOI of 1000 and 10,000, within a timescale of 48 h. The findings demonstrated that phage LPST94 is a promising candidate for biological control agents against pathogenic Salmonella and has the potential to be applied across different food matrices

Bacterial Strains and Plasmids.

<p>* All <i>Y</i>. <i>pestis</i> strains contained the pMT1 and pPCP plasmids.</p><p>Bacterial Strains and Plasmids.</p

<i>Y</i>. <i>pestis crp</i>-deficient Mutant 26°C Growth Kinetics.

<p>(A) Growth curves of CO92, CO92Δ<i>crp</i>, and plasmid complemented CO92Δ<i>crp</i>: pBluescript/<i>crp</i> when grown in BCS medium supplemented with 0.2% glucose or 0.2% K-gluconate. Growth of the CO92 <i>crp</i>-deficient mutant when cultured in medium supplemented with 0.2% K-gluconate was significantly impaired relative to all other bacterial strain and media conditions (Tukey’s HSD P < 0.05). (B) Growth curves of KIM6+ and KIM6+Δ<i>crp</i> when cultured in BCS medium supplemented with either 0.2% glucose, 0.2% glycerol, or a combination of both 0.2% glucose and 0.2% glycerol. Growth of the KIM6+ <i>crp</i>-deficient mutant when cultured in medium solely supplemented with 0.2% glycerol was significantly impaired relative to all other bacterial strain and media conditions (Tukey’s HSD P < 0.05). (C) Growth kinetics of the CO92 and KIM6+ <i>crp</i>-deficient mutants and respective isogenic controls when grown in HIB medium at 26°C.</p

CsrA does not alter <i>Y</i>. <i>pestis hmsH</i>, <i>hmsP</i>, and <i>hmsT</i> Transcript Levels.

<p>Relative mRNA abundance of <i>hmsH</i>, <i>hmsP</i>, and <i>hmsT</i> among the <i>csrA</i>-deficient mutants and the respective parental strains calculated via the ΔΔC_T method in reference to the <i>gyrB</i> gene. Dashed lines reflect two-fold thresholds for alterations in relative transcript levels. No significant change in relative mRNA abundance among the deletion mutants and the respective controls were determined by one-way ANOVA comparing the average of 2 independent cDNA samples per strain, each consisting of 3 technical replicates per target.</p

<i>Y</i>. <i>pestis csrA</i>-deficient Mutant 26°C Growth Kinetics.

<p>(A) Growth of CO92, CO92Δ<i>csrA</i>, and the chromosomal restoration mutant CO92Δ<i>csrA csrA</i>’ in BCS medium supplemented with either 0.2% glucose or 0.2% K-gluconate during incubation at 26°C. (B) Growth kinetics of the CO92 and KIM6+ <i>csrA</i>-deficient mutants and respective controls when cultured in HIB medium at 26°C. No significant alteration in growth kinetics as determined by repeated measures ANOVA was calculated among the <i>csrA</i>-deficient mutants and the respective controls, regardless of media type or available carbon source.</p

<i>Y</i>. <i>pestis</i> Biofilm Production is not Influenced by Exogenous cAMP.

<p>(A) Relative biofilm production of KIM6+ and KIM6+Δ<i>crp</i> when grown in BCS medium supplemented with 0.2% glucose containing either 3 mM cAMP prepared in K-phosphate buffer or an equal volume of plain K-phosphate buffer. (B) Relative biofilm production of CO92 and CO92Δ<i>crp</i> when grown in BCS medium supplemented with 0.2% glucose containing either 3 mM cAMP prepared in K-phosphate buffer or an equal volume of plain K-phosphate buffer. Identical source inoculum per strain was utilized for each media type. Error bars reflect standard deviation from the mean derived from two independent experiments, each consisting of 6 technical replicates. No statistically significant change in crystal violet absorption as determined by one-way ANOVA was calculated for both the <i>crp</i>-deficient mutants and the respective parental controls when cultured in media containing 3mM cAMP or an equal volume of K-phosphate buffer.</p

CRP Enables Robust <i>Y</i>. <i>pestis</i> Biofilm Production.

<p>Relative biofilm production of CO92, CO92Δ<i>crp</i>, and plasmid complemented CO92Δ<i>crp</i>: pBluescript/<i>crp</i> when cultured in HIB medium or BCS medium supplemented with either 0.2% K-gluconate or 0.2% glucose. (A) Following 24 hours post-inoculation. (B) After 72 hours post-inoculation. Error bars reflect standard deviation from the mean derived from two independent experiments, each consisting of 6 technical replicates. * P-value <0.005 determined by Tukey’s HSD post-hoc analysis.</p

Relative Crystal Violet Absorption of the CO92 Glycogen-deficient Mutants.

<p>* P-value < 0.05 as determined by Tukey’s HSD post-hoc analysis comparing two independent experiments, each consisting of 6 technical replicates.</p><p>Relative Crystal Violet Absorption of the CO92 Glycogen-deficient Mutants.</p

CRP-Mediated Carbon Catabolite Regulation of <i>Yersinia pestis</i> Biofilm Formation Is Enhanced by the Carbon Storage Regulator Protein, CsrA

<div><p>The natural transmission of <i>Yersinia pestis</i> is reliant upon biofilm blockage of the flea vector. However, the environmentally-responsive adaptive regulators which facilitate <i>Y</i>. <i>pestis</i> biofilm production in accordance with the flea midgut milieu are not well understood. We seek to establish the impact of available carbon source metabolism and storage upon <i>Y</i>. <i>pestis</i> biofilm production. Our findings demonstrate that <i>Y</i>. <i>pestis</i> biofilm production is subject to carbon catabolite regulation in which the presence of glucose impairs biofilm production; whereas, the sole metabolism of alternate carbon sources promotes robust biofilm formation. This observation is facilitated by the cAMP receptor protein, CRP. In accordance with a stark growth defect, deletion of <i>crp</i> in both CO92 and KIM6+ <i>Y</i>. <i>pestis</i> strains significantly impaired biofilm production when solely utilizing alternate carbon sources. Media supplementation with cAMP, a small-molecule activator of CRP, did not significantly alter <i>Y</i>. <i>pestis</i> biofilm production. Furthermore, CRP did not alter mRNA abundance of previously-characterized <i>hms</i> biofilm synthesis and regulation factors. Therefore, our findings indicate CRP does not confer a direct stimulatory effect, but may indirectly promote <i>Y</i>. <i>pestis</i> biofilm production by facilitating the alternate carbon source expression profile. Additionally, we assessed the impact of the carbon storage regulator protein, CsrA, upon <i>Y</i>. <i>pestis</i> biofilm production. Contrary to what has been described for <i>E</i>. <i>coli</i>, <i>Y</i>. <i>pestis</i> biofilm formation was found to be enhanced by CsrA. Regardless of media composition and available carbon source, deletion of <i>csrA</i> significantly impaired <i>Y</i>. <i>pestis</i> biofilm production. CsrA was found to promote <i>Y</i>. <i>pestis</i> biofilm production independent of glycogen regulation. Loss of <i>csrA</i> did not significantly alter relative <i>hmsH</i>, <i>hmsP</i>, or <i>hmsT</i> mRNA abundance. However, deletion of <i>hmsP</i> in the <i>csrA</i>-deficient mutant enabled excessive biofilm production, suggesting CsrA enables potent <i>Y</i>. <i>pestis</i> biofilm production through cyclic diguanylate regulation.</p></div

CsrA is a Positive Regulator of <i>Y</i>. <i>pestis</i> Biofilm Production.

<p>Relative biofilm production of the <i>csrA</i>-deficient mutants after 24 hours inoculation of BCS medium supplemented with 0.2% K-gluconate, HIB medium, or BCS medium supplemented with 0.2% glucose. (A) CO92, CO92Δ<i>csrA</i>, and the chromosomal restoration mutant CO92Δ<i>csrA csrA’</i>. (B) KIM6+, KIM6+Δ<i>csrA</i>, and the chromosomal restoration mutant KIM6+Δ<i>csrA csrA’</i>. Results reflect the average of two independently derived <i>csrA</i>-deficient mutants (CO92 clones 5a and 3b, and KIM6+ clones 2:14 and 4:12). Two biological replicates were assessed per mutant, each consisting of 6 technical replicates. Error bars reflect standard deviation from the mean among biological replicates. * P-value < 0.005 as determined by Tukey’s HSD post-hoc analysis. (C) Impaired Congo red assimilation of the <i>Y</i>. <i>pestis csrA</i>-deficient mutants. Phenotypic evaluation of Congo red binding of the CO92 and KIM6+ <i>csrA</i>-deficient mutants, chromosomal <i>csrA</i> restoration mutants, parental isogenic controls, and pigmentation locus negative (biofilm-deficient) mutants after 48 hours post-inoculation of Congo red plates supplemented with 0.2% K-gluconate.</p