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Lytic bacteriophages are expected as effective tools to control infectious bacteria in human and pathogenic or spoilage bacteria in foods. Leaderless bacteriocins (LLBs) are simple bacteriocins produced by Gram-positive bacteria. LLBs do not possess an N-terminal leader peptide in the precursor, which means that they are active immediately after translation. In this study, we constructed a novel antimicrobial agent, an LLB-producing phage (LLB-phage), by genetic engineering to introduce the LLB structural gene into the lytic phage genome. To this end, lnqQ (structure gene of an LLB, lacticin Q) and trxA , an essential gene for T7 phage genome replication, were integrated in tandem into T7 phage genome using homologous recombination in Escherichia coli host strain. The recombinant lnqQ -T7 phage was isolated by a screening method using Δ trxA host strain. lnqQ -T7 phage formed a clear halo in agar plates containing both E. coli and lacticin Q-susceptible Bacillus coagulans, indicating that lnqQ -T7 phage could produce a significant amount of lacticin Q. Lacticin Q production did not exert a significant effect on the lytic cycle of T7 phage. In fact, the production of lacticin Q enhanced T7 phage lytic activity and helped to prevent the emergence of bacterial populations resistant against this phage. These results serve as a proof of principle for LLB-phages. There are different types of LLBs and phages, meaning that in the future, it may be possible to produce any number of LLB-phages which can be designed to efficiently control different types of bacterial contamination in different settings. IMPORTANCE We demonstrated that we could combine LLB and phage to construct promising novel antimicrobial agents, LLB-phage. The first LLB-phage, lnqQ -T7 phage, can control the growth of both the Gram-negative host strain and neighboring Gram-positive bacteria while preventing the emergence of phage resistance in the host strain. There are several different types of LLBs and phages, suggesting that we may be able to design a battery of LLB-phages by selecting novel combinations of LLBs and phages. These constructs could be tailored to control various bacterial contaminations and infectious diseases.

YafQ is an endoribonuclease toxin that degrades target gene transcripts such as that of tnaA , a gene encoding tryptophanase to synthesize indole from tryptophan. DinJ is the cognate antitoxin of YafQ, and the YafQ-DinJ system was reported to regulate persister formation by controlling indole production in Escherichia coli In this study, we investigated the role of YafQ-DinJ, indole production, and persister population in bacterial heat tolerance. yafQ (Δ yafQ ), dinJ (Δ dinJ ), and tnaA (Δ tnaA ) single-gene knockout mutants showed approximately 10-fold higher heat tolerance than wild-type (WT) E. coli BW25113. Persister fractions of all mutants were slightly larger than that of the WT. Interestingly, these persister cells showed an approximately 100-fold higher heat tolerance than normal cells, but there was no difference among the persister cells of all mutants and the WT in terms of heat tolerance. Indole and its derivatives promoted a drastic reduction of bacterial heat tolerance by just 10 min of pretreatment, which is not sufficient to affect persister formation before heat treatment. Surprisingly, indole and its derivatives also reduced the heat tolerance of persister cells. Among the tested derivatives, 5-iodoindole exhibited the strongest effect on both normal and persister cells. IMPORTANCE Our study demonstrated that a small persister population exhibits significantly higher heat tolerance than normal cells and that this small fraction contributes to the heat tolerance of the total bacterial population. This study also demonstrated that indole, known to inhibit persister formation, and its derivatives are very promising candidates to reduce the heat tolerance of not only normal bacterial cells but also persister cells., (Copyright © 2020 American Society for Microbiology.)

Aims The study was to identify the genes involved in phage resistance and to develop an effective biocontrol method to improve the lytic activity of phages against foodborne pathogens. Methods and results A total of 3,909 single gene-deletion mutants of Escherichia coli BW25113 from the Keio collection were individually screened for genes involved in phage resistance. Phage S127BCL3 isolated from chicken liver, infecting both E. coli BW25113 and O157: H7, was characterized and used for screening. The 10 gene-deletion mutants showed increased susceptibility to phage S127BCL3. Among them, priA gene-deletion mutant strain showed significant susceptibility to the phages S127BCL3 and T7. Furthermore, we investigated the substances that have been reported to inhibit the function of primosomal protein A (PriA) and were used to confirm increased phage susceptibility in E. coli BW25113 (Parent strain) and O157: H7. Conclusion PriA inhibitors at a low concentration showed combined effects with phage against E. coli O157: H7 and delayed the regrowth rate of phage-resistant cells. [ABSTRACT FROM AUTHOR]

Bacterial food poisoning cases due to Salmonella and E. coli O157:H7 have been linked with the consumption of a variety of food products, threatening public health around the world. This study describes the combined effects of a phage cocktail (STG2, SEG5, and PS5), EDTA, nisin, and polylysine against the bacterial cocktail consisting of S. typhimurium, S. enteritidis, and E. coli O157:H7. Overall, phage cocktail (alone or in combination with nisin or/and polylysine) not only showed great antibacterial effects against bacterial cocktail at different temperatures (4 °C, 24 °C, and 37 °C), but also totally inhibited the emergence of phage resistance during the incubation period. These results suggest that the combination of phages with nisin or/and polylysine has great potential to simultaneously control S. typhimurium, S. enteritidis, and E. coli O157:H7. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of public health concern and frequently isolated from raw beef products. Bacteriophage-based methods have been increasingly exploited to control bacterial contamination in meats. Here, we describe the isolation, characterization, and application of a lytic phage PE37 for the simultaneous bio-control of STEC O157:H7 and ESBLEC. Phage PE37, isolated from the bovine intestine, was morphologically characterized as a member of the Myoviridae family, with a broad host range and great stability under various stress conditions. Sequencing analysis revealed that the genomic DNA of phage PE37 contains genes that contribute to virion structure, replication, assembly, and host lysis. PE37 significantly reduced the viable counts of STEC O157:H7 by 4.9 and 2.6 log CFU/mL in broth after 6 h of incubation at 25 and 8 °C, respectively. Application of phage PE37 to raw beef artificially contaminated with STEC O157:H7 resulted in significant reductions in the viable counts by 2.3 and 0.9 log CFU/piece after 24 h of storage at 25 and 8 °C, respectively. Treatment of raw beef contaminated with a bacterial cocktail of STEC O157:H7 and ESBLEC with PE37 also significantly decreased the viable counts of the bacterial mixture by 1.4 and 1.0 log CFU/piece after 24 h of incubation at 25 and 8 °C, respectively. These findings suggest that bacteriophage PE37 may be a potential bio-agent for controlling STEC O157:H7 and ESBLEC contamination in raw beef. [ABSTRACT FROM AUTHOR]

Detection of foodborne pathogens at very low levels is still a challenge. A custom-built multichannel surface plasmon resonance (SPR) biosensor and simultaneous enrichment broth (SEB) were used to develop a simultaneous detection method for 3 important foodborne pathogens, Escherichia coli O157:H7 (O157:H7), Salmonella enteritidis, and Listeria monocytogenes, at a very low level. These 3 foodborne pathogens at a very low level (14, 6, and 28 CFU/25 g (mL) for O157:H7, S. enteritidis, and L. monocytogenes, respectively) were inoculated in SEB and incubated at 37 ˚C for 24 h. Sample prepared from the simultaneous enrichment culture was analyzed using the multichannel SPR biosensor and sensor chip immobilized with polyclonal antibodies specific to each of the target pathogens. O157:H7, S. enteritidis, and L. monocytogenes in chicken were detected simultaneously at an inoculum dose of 14, 6, and 28 CFU/25 g, respectively. Our method using a custom-built multichannel SPR biosensor and enrichment in SEB is expected as a rapid and simultaneous detection method for low levels of O157:H7, S. enteritidis, and L. monocytogenes in food. Practical Application Our method is expected as a rapid and simultaneous detection method for pathogens at very low levels. It has great potential for safety control of food and microbiological detection applications. [ABSTRACT FROM AUTHOR]

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Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers. [ABSTRACT FROM AUTHOR]

Extended Spectrum Beta-Lactamase-producing Escherichia coli (ESBLEC) were isolated from 12/13 (92.3%) raw chicken meat samples by using selective culture and PCR. Of the 27 ESBLEC analyzed, 33.3% (9/27) of isolates were positive for ESBL of CTX-M group 1 followed by TEM (22.2%), SHV (22.2%), CTX-M group 2 (11.1%), and CTX-M group 9 (11.1%). None of ESBLEC tested were positive for stx 1 , stx 2 , eae , ehxA , saa , and subAB genes of Shiga toxin-producing E. coli . Among 22 isolated phages, phages PBL66-CL1 and PBL116-CS6 infected 21 (77.7%) and 20 (74%) of 27 ESBLEC isolates examined, respectively. The remaining phages lysed less than 50% of the hosts tested. Compared to non-treatment, the treatment of isolate EBL116 in the broth medium with phage cocktail of PBL66-CL1 and PBL116-CS6 at 25 °C and 5 °C after 6 h significantly reduced bacterial viable counts by 5.06 and 1.33 log CFU/mL, respectively. When the treatment was performed on raw chicken meat samples, viable counts of EBL116 were also decreased by 2.02 and 1.67 log CFU/4 cm 2 meat piece after 6 h at 25 °C and 5 °C, respectively. This study demonstrates a high prevalence of ESBLEC in raw chicken meat and possible use of lytic phages as bactericide for controlling ESBLEC. [ABSTRACT FROM AUTHOR]

Abstract: Shiga toxin-producing Escherichia coli (STEC) can cause severe illnesses in humans such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, we carried out genotypic analysis of the Shiga toxin (stx) gene in 120 clinical isolates of STEC and enterohemorrhagic E. coli (EHEC) from patients in a southern district of Japan. We identified 88 stx1 + and 103 stx2 + strains. We further identified 12 stx1 + and stx2 + isolates expressing little or no Shiga toxin 1 (Stx1) and/or 2 (Stx2) by reversed passive latex agglutination (RPLA) and Vero cell toxicity assays. Among them, 1 strain could not produce Stx1, 8 could not produce Stx2, and 3 strains could produce neither. Two of the latter three strains were of the non-O157 serotype. Most of the Stx RPLA-negative strains belonged to the stx1 /stx2 subtype (11/12, [91.7%]). Our quantitative reverse transcription PCR analysis indicated that the stx genes were not effectively transcribed in the RPLA-negative strains. This is the first report of the isolation of stx-positive strains showing Stx-negative phenotype from stx1 -bearing strains and non-O157 strains. [ABSTRACT FROM AUTHOR]

This study describes the characterization of bacteriophage FP43, active against pathogenic and non-pathogenic strains of Escherichia coli , and its ability to inhibit and remove a mixed biofilm of enterohemorrhagic E. coli O157:H7 and O91:H-. Phage FP43 is a member of the Myoviridae family, having a dsDNA genome consisting of 169,248 bp with good stability. It has a short latent period and large burst size. Phage FP43 decreased the formation of biofilm, comprising E. coli O157:H7 and O91:H-, by 82.4% at 30 °C. After incubation for 6 h with FP43, viable counts of E. coli O157:H7 and total cells in the biofilm were reduced by 2.76 and 2.85 log, respectively. In planktonic cells, viable E. coli O157:H7 and total counts decreased by 3.44 and 3.62 log, respectively, after incubation with the phage for 4 h. Moreover, more than 60% of an established, mixed biofilm was removed after a 6 h exposure to the phage, in which E. coli O157:H7 and total viable counts decreased by 2.07 and 1.93 log, respectively. These results suggest that bacteriophage FP43 has broad host range against both pathogenic and non-pathogenic E. coli , and potential to reduce viable counts of both enterohemorrhagic E. coli O157:H7 and E. coli O91 in mixed-strain biofilm. • Lytic phage FP43 against both pathogenic and non-pathogenic E. coli was isolated and characterized. • Phage FP43 inhibited mixed-biofilm formation of enterohemorrhagic E. coli O157:H7 and O91:H- strains. • Phage FP43 can reduce risk of enterohemorrhagic E. coli infection through biofilm in food. [ABSTRACT FROM AUTHOR]