Functional and genomic characterization of a novel probiotic Lactobacillus johnsonii KD1 against shrimp WSSV infection

White Spot syndrome virus (WSSV) causes rapid shrimp mortality and production loss worldwide. This study demonstrates potential use of Lactobacillus johnsonii KD1 as an anti-WSSV agent for post larva shrimp cultivation and explores some potential mechanisms behind the anti-WSSV properties. Treatment of Penaeus vannamei shrimps with L. johnsonii KD1 prior to oral challenge with WSSV-infected tissues showed a significantly reduced mortality. In addition, WSSV copy numbers were not detected and shrimp immune genes were upregulated. Genomic analysis of L. johnsonii KD1 based on Illumina and Nanopore platforms revealed a 1.87 Mb chromosome and one 15.4 Kb plasmid. Only one antimicrobial resistance gene (ermB) in the chromosome was identified. Phylogenetic analysis comparing L. johnsonii KD1 to other L. johnsonii isolates revealed that L. johnsonii KD1 is closely related to L. johnsonii GHZ10a isolated from wild pigs. Interestingly, L. johnsonii KD1 contains isolate-specific genes such as genes involved in a type I restriction-modification system and CAZymes belonging to the GT8 family. Furthermore, genes coding for probiotic survival and potential antimicrobial/anti-viral metabolites such as a homolog of the bacteriocin helveticin-J were found. Protein–protein docking modelling suggests the helveticin-J homolog may be able to block VP28–PmRab7 interactions and interrupt WSSV infection

σ(B) Activation under Environmental and Energy Stress Conditions in Listeria monocytogenes

To measure σ(B) activation in Listeria monocytogenes under environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the σ(B)-dependent opuCA and clpC genes in strains having null mutations in genes encoding regulator of sigma B proteins (rsbT and rsbV) and sigma B (sigB) and in the L. monocytogenes wild-type 10403S strain under different stress conditions. The ΔsigB, ΔrsbT, and ΔrsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and σ(B) were all required for opuCA expression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of clpC was RsbT, RsbV, and σ(B) dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or σ(B) dependent in the presence of either CCCP or salt. opuCA transcript levels did not increase in the presence of rapidly lethal stresses (i.e., pH 2.5 or 13 mM cumene hydroperoxide) despite the enhanced survival of the wild type compared with the survival of the mutant strains under these conditions. These findings highlight the importance of complementing phenotypic characterizations with gene expression studies to identify direct and indirect effects of null mutations in regulatory genes, such as sigB. Overall, our data show that while σ(B) activation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the σ(B) regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators

RsbT and RsbV Contribute to σ(B)-Dependent Survival under Environmental, Energy, and Intracellular Stress Conditions in Listeria monocytogenes

Sigma B (σ(B)) is a stress-responsive alternative sigma factor that has been identified in various gram-positive bacteria. Seven different regulators of sigma B (Rsbs) are located in the sigB operons of both Bacillus subtilis and Listeria monocytogenes. In B. subtilis, these proteins contribute to regulation of σ(B) activity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway. RsbT contributes to regulation of σ(B) activity in response to environmental stresses, while RsbV contributes to σ(B) activation under both environmental and energy stresses in B. subtilis. To probe L. monocytogenes Rsb roles in σ(B)-mediated responses to various stresses, in-frame deletions were created in rsbT and rsbV. Phenotypic characterization of the L. monocytogenes rsbT and rsbV null mutants revealed that both mutants were similar to the ΔsigB strain in their abilities to survive under environmental stress conditions (exposure to synthetic gastric fluid, pH 2.5, acidified brain heart infusion broth [BHI], or oxidative stress [13 mM cumene hydroperoxide]). Under energy stress conditions (carbon starvation in defined media, entry into stationary phase, or reduced intracellular ATP), both ΔrsbT and ΔrsbV showed survival reductions similar to that of the ΔsigB strain. These observations suggest that the pathways for Rsb-dependent regulation of σ(B) activity differ between L. monocytogenes and B. subtilis. As σ(B) also activates transcription of the L. monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of ΔprfAP1rsbT and ΔprfAP1rsbV double mutants in hemolysis and tissue culture assays. Both double mutants showed identical phenotypes to ΔprfAP1P2 and ΔprfAP1sigB double mutants, i.e., reduced hemolysis activity and reduced plaque size in mouse fibroblast cells. These findings indicate that RsbT and RsbV both contribute to σ(B) activation in L. monocytogenes during exposure to environmental and energy stresses as well as during tissue culture infection

Evidence of international transmission of mobile colistin resistant monophasic Salmonella Typhimurium ST34

Abstract S. 4,[5],12:i:-, a monophasic variant of S. enterica serovar Typhimurium, is an important multidrug resistant serovar. Strains of colistin-resistant S. 4,[5],12:i:- have been reported in several countries with patients occasionally had recent histories of travels to Southeast Asia. In the study herein, we investigated the genomes of S. 4,[5],12:i:- carrying mobile colistin resistance (mcr) gene in Thailand. Three isolates of mcr-3.1 carrying S. 4,[5],12:i:- in Thailand were sequenced by both Illumina and Oxford Nanopore platforms and we analyzed the sequences together with the whole genome sequences of other mcr-3 carrying S. 4,[5],12:i:- isolates available in the NCBI Pathogen Detection database. Three hundred sixty-nine core genome SNVs were identified from 27 isolates, compared to the S. Typhimurium LT2 reference genome. A maximum-likelihood phylogenetic tree was constructed and revealed that the samples could be divided into three clades, which correlated with the profiles of fljAB-hin deletions and plasmids. A couple of isolates from Denmark had the genetic profiles similar to Thai isolates, and were from the patients who had traveled to Thailand. Complete genome assembly of the three isolates revealed the insertion of a copy of IS26 at the same site near iroB, suggesting that the insertion was an initial step for the deletions of fljAB-hin regions, the hallmark of the 4,[5],12:i:- serovar. Six types of plasmid replicons were identified with the majority being IncA/C. The coexistence of mcr-3.1 and bla CTX-M-55 was found in both hybrid-assembled IncA/C plasmids but not in IncHI2 plasmid. This study revealed possible transmission links between colistin resistant S. 4,[5],12:i:- isolates found in Thailand and Denmark and confirmed the important role of plasmids in transferring multidrug resistance

Lysis Profiles of <i>Salmonella</i> Phages on <i>Salmonella</i> Isolates from Various Sources and Efficiency of a Phage Cocktail against <i>S.</i> Enteritidis and <i>S.</i> Typhimurium

Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are major foodborne pathogens of concern worldwide. Bacteriophage applications have gained more interest for biocontrol in foods. This study isolated 36 Salmonella phages from several animal farms in Thailand and tested them on 47 Salmonella strains from several sources, including farms, seafood processing plant and humans in Thailand and USA. Phages were classified into three major groups. The estimated phage genome size showed the range from 50 &#177; 2 to 200 &#177; 2 kb. An effective phage cocktail consisting of three phages was developed. Approximately 4 log CFU/mL of S. Enteritidis and S. Typhimurium could be reduced. These phages revealed a burst size of up to 97.7 on S. Enteritidis and 173.7 PFU/cell on S. Typhimurium. Our phage cocktail could decrease S. Enteritidis on chicken meat and sunflower sprouts by 0.66 log CFU/cm2 and 1.27 log CFU/g, respectively. S. Typhimurium on chicken meat and sunflower sprouts were decreased by 1.73 log CFU/cm2 and 1.17 log CFU/g, respectively. Overall, animal farms in Thailand provided high abundance and diversity of Salmonella phages with the lysis ability on Salmonella hosts from various environments and continents. A developed phage cocktail suggests a potential biocontrol against Salmonella in fresh foods

Data from: Alternative Sigma factors regulate overlapping as well as distinct stress response and metabolic functions in Listeria monocytogenes stationary phase cells

Please cite as: Renato H. Orsi, Soraya Chaturongakul, Haley F. Oliver, Lalit Ponnala, Ahmed Gaballa, Martin Wiedmann. (2020) Data from: Alternative Sigma factors regulate overlapping as well as distinct stress response and metabolic functions in Listeria monocytogenes stationary phase cells. [dataset] Cornell University eCommons Repository. https://doi.org/10.7298/0mjr-6c90Listeria monocytogenes has the ability to regulate and fine tune gene expression to adapt to diverse stress conditions encountered during foodborne transmission. To further understand the specific contributions of alternative Sigma factors to regulation of L. monocytogenes gene expression, deep RNA sequencing was performed on L. monocytogenes strain 10403S and five isogenic mutants (four strains bearing in-frame null mutations in three out of four alternative Sigma factor genes, ΔCHL, ΔBHL, ΔBCL and ΔBCH and one strain bearing null mutations in all four genes, ΔBCHL), grown to stationary phase. Our data showed that 184, 35, 34, and 20 genes were positively regulated by SigmaB, SigmaL, SigmaH, and SigmaC (posterior probability > 0.9 and fold change [FC] > 5.0), including 112 and 6 genes identified as directly regulated by SigmaB and SigmaH, respectively (as supported by identification of appropriate putative upstream promoter elements). SigmaB-dependent genes showed the highest FC (based on comparisons between the ΔCHL and the ΔBCHL strain) with 44 genes showing a FC > 100; only four SigmaL-dependent, and no SigmaH- or SigmaC-dependent genes showed FC >100. SigmaB also directly up-regulated four ncRNAs as well as two long 5’ untranslated regions (UTRs) that overlapped other transcription units in the opposite strand; we did not identify noncoding RNA features directly regulated by other alternative Sigma factors. While SigmaB-regulated genes identified in this study are involved in a diversity of pathways and functions, SigmaL appears to largely regulate genes involved in a few specific metabolic pathways, including positive regulation of a number of operons encoding phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs). Overall, our data show that (i) SigmaB and SigmaL directly and indirectly regulate genes involved in a number of energy metabolism-related functions, (ii) alternative Sigma factors are involved in complex regulatory networks and appear to have epistatic effects in stationary phase cells, and (iii) SigmaB regulates multiple stress response pathways while SigmaL and SigmaH positively regulate a smaller number of specific pathways

Alternative σ Factors Regulate Overlapping as Well as Distinct Stress Response and Metabolic Functions in Listeria monocytogenes under Stationary Phase Stress Condition

Listeria monocytogenes can regulate and fine-tune gene expression, to adapt to diverse stress conditions encountered during foodborne transmission. To further understand the contributions of alternative sigma (σ) factors to the regulation of L. monocytogenes gene expression, RNA-Seq was performed on L. monocytogenes strain 10403S and five isogenic mutants (four strains bearing in-frame null mutations in three out of four alternative σ factor genes, ΔCHL, ΔBHL, ΔBCL, and ΔBCH, and one strain bearing null mutations in all four genes, ΔBCHL), grown to stationary phase. Our data showed that 184, 35, 34, and 20 genes were positively regulated by σB, σL, σH, and σC (posterior probability &gt; 0.9 and Fold Change (FC) &gt; 5.0), respectively. Moreover, σB-dependent genes showed the highest FC (based on comparisons between the ΔCHL and the ΔBCHL strain), with 44 genes showing an FC &gt; 100; only four σL-dependent, and no σH- or σC-dependent genes showed FC &gt;100. While σB-regulated genes identified in this study are involved in stress-associated functions and metabolic pathways, σL appears to largely regulate genes involved in a few specific metabolic pathways, including positive regulation of operons encoding phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs). Overall, our data show that (i) σB and σL directly and indirectly regulate genes involved in several energy metabolism-related functions; (ii) alternative σ factors are involved in complex regulatory networks and appear to have epistatic effects in stationary phase cells; and (iii) σB regulates multiple stress response pathways, while σL and σH positively regulate a smaller number of specific pathways

<i>Salmonella enterica</i> multilocus sequence typing and its correlation with serotypes

<p><i>Salmonella enterica</i> is a foodborne pathogen of significant public health concern worldwide. In Thailand, <i>S. enterica</i> has also been ranked among the top five most significant bacterial agents of foodborne illnesses by the Ministry of Public Health. Conventionally, biochemical tests and antigen-antibody agglutination have been used to identify and subtype <i>S. enterica</i>, respectively. The objective of this study was to identify the serotypes of 180 <i>S. enterica</i> isolates. Multilocus sequence typing (MLST) was used to deduce the <i>S. enterica</i> serotypes based on sequence type (ST) correlation as shown in the MLST database (<a href="http://mlst.warwick.ac.uk/mlst/" target="_blank">http://mlst.warwick.ac.uk/mlst/</a>). Initially, MLST was used to confirm serotypes of 53 previously identified isolates of <i>S</i>. Enteritidis, <i>S</i>. Typhimurium, <i>S</i>. Hadar, <i>S</i>. Virchow, and <i>S</i>. Infantis isolated in Thailand. MLST and serotype correlation confirmed 52 (of 53) known isolates. MLST was performed in 127 <i>S. enterica</i> isolates of unknown serotypes from various sources. Serotypes of all 127 <i>S. enterica</i> isolates were successfully deduced based on STs. With MLST and PCR-based identification, we have shown that the majority of isolates are of monophasic <i>S</i>. Typhimurium (ST34; 43 isolates) and serotype Rissen (ST469; 37 isolates), in agreement with the top serotypes commonly found in Thailand based on the WHO National <i>Salmonella</i> and <i>Shigella</i> Center. We have also confirmed that MLST is a powerful <i>Salmonella</i> subtyping method which could be used not only as a tracking tool for an outbreak investigation at nucleotide level but also as a serotype predictor for making correlations with food safety regulations.</p

Differential Interaction between Invasive Thai Group B <i>Streptococcus</i> Sequence Type 283 and Caco-2 Cells

The emergence in Southeast Asia of invasive group B Streptococcus (GBS) infections in adults by sequence type (ST) 283 is suggested to be associated with fish consumption. Genotyping of 55 GBS clinical isolates revealed that 33/44 invasive isolates belonged to ST283/capsular polysaccharide type (CPS) III. This included 15/16 isolates recovered from younger adults aged 16–36 years. Seven ST283/CPSIII isolates from the blood, cerebrospinal fluid, or joint fluid were selected by the patient’s age at random to perform interaction studies with intestinal epithelial Caco-2 monolayers. The invasion efficiency profiles from this study classified these isolates into two groups; a higher invasion efficiency group 1 recovered from patients aged between 23 and 36 years, and a lower invasion efficiency group 2 recovered from the elderly and neonate. Intracellular survival tests revealed that only group 1 members could survive inside Caco-2 cells up to 32 h without replication. Additionally, all isolates tested were able to traverse across polarized Caco-2 monolayers. However, the timing of translocation varied among the isolates. These results indicated the potential of GBS invasion via the gastrointestinal tract and showed phenotypic variations in invasiveness, intracellular survival, and translocation efficiency between genetically closely related ST283 isolates infecting young adults and those infecting the elderly