RESUMEN
Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonas spp. In this study, 12 Pseudomonas phages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30 °C and 4 °C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonas spp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescens and P. putida at 30 °C, 8 °C, and 4 °C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.
Asunto(s)
Pollos , Microbiología de Alimentos , Especificidad del Huésped , Animales , Microbiología del Suelo , Fagos Pseudomonas/fisiología , Fagos Pseudomonas/genética , Pseudomonas/virología , Genoma Viral , Podoviridae/fisiología , Podoviridae/genética , Podoviridae/aislamiento & purificación , Podoviridae/clasificación , Agentes de Control Biológico , ADN Viral/genética , Bacteriófagos/fisiología , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/clasificaciónRESUMEN
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.
Asunto(s)
Bacteriófagos , Escherichia coli O157 , Proteínas de Escherichia coli , Bacteriófagos/genética , Proteína Estafilocócica A , ADN Helicasas , Proteínas de Escherichia coli/genéticaRESUMEN
This study investigated the prevalence, serotype, antimicrobial resistance (AMR), virulence potential, and biofilm formation of Listeria monocytogenes isolated in 2022 in Japan and compared their profiles with those of isolates in 2012 and 2017. A total of 85 chicken samples were randomly collected from different supermarkets in Fukuoka in 2022. L. monocytogenes were isolated by conventional method and characterized by MALDI-TOF MS. Among 85 samples tested in 2022, 9 (10.6%) were positive for L. monocytogenes and 17 strains were isolated from the positive samples. The isolates were serotyped as 1/2b (41.2%), 3a (29.4%), 3b (23.5%) and 1/2a (5.9%). Antimicrobial susceptibility tests of the 2022 isolates showed susceptibility to majority of the antibiotics, except cefoxitin, oxacillin, and fosfomycin. Compared to the previous surveillance results, the prevalence of L. monocytogenes in 2022 (10.6%) was significantly lower (p < 0.05) than those of the isolates in 2017 (24%) and 2012 (52.9%). The distribution of serotypes 1/2a and 1/2b decreased over time, and serotype 4b was not detected in the 2022 isolates. The proportion of multidrug resistant strains in 2022 (16.7%) was significantly lower than those in 2012 (46.7%) and 2017 (82.6%). Moreover, a total of 36 isolates (12 isolates/ year) were used to detect the virulence genes (hlyA, plcA, clpC, and inlA) and biofilm-forming capacity. Most of the isolates from different years harboured four virulence genes. The biofilm formation of the 2022 isolates was significantly weaker (p < 0.05) than those of the 2012 and 2017 isolates. Thus, despite the low rates of contamination in chicken meat and AMR of the isolates, virulent L. monocytogenes contamination in food should still be acknowledged.
Asunto(s)
Antibacterianos , Listeria monocytogenes , Japón/epidemiología , Prevalencia , Virulencia/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Listeria monocytogenes/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
AIMS: Isolation and characterization of Enterococcus phages and application of phage cocktail to control E. faecalis in milk. METHODS AND RESULTS: For phage isolations, double layer agar method was used. Host range of the phages were determined by the spot test. Twelve phages with varying host ranges were isolated. Phages PEF1, PEF7b, and PEF9 with different host ranges and lytic activities were selected for phage cocktails. Compared to two-phages cocktails tested, the cocktail containing all the three phages displayed stronger antibacterial and biofilm removal activities. The cocktail treatment reduced viable E. faecalis in biofilm by 6 log within 6 h at both 30°C and 4°C. In milk, the cocktail gradually reduced the viable count of E. faecalis and the count reached below the lower limit of detection at 48 h at 4°C. CONCLUSION: The strong bactericidal and biofilm removal activities of the phage cocktail suggest the potential of this cocktail as a natural biocontrol agent for combating E. faecalis in milk.
Asunto(s)
Bacteriófagos , Animales , Enterococcus , Leche/microbiología , Especificidad del Huésped , Antibacterianos , Enterococcus faecalisRESUMEN
AIMS: To investigate the antibacterial effects of tea theaflavins and catechins against Bacillus coagulans and the underlying mechanism of antibacterial action. METHODS AND RESULTS: Bactericidal activities of theaflavin and its analogues were evaluated and compared with that of epigallocatechin gallate. Theaflavin derivatives exhibited high bactericidal activity at 50 µmol L-1 , whereas epigallocatechin gallate did not, even at 500 µmol L-1 . Furthermore, we investigated the adsorption of theaflavins to model phospholipid membranes and corresponding effects on membrane fluidity to reveal their effects on the B. coagulans cell surface. Cell membrane fluidity was decreased after treatment with theaflavin derivatives with one or more galloyl moieties. Quartz-crystal microbalance analysis showed a strong affinity of the membrane phosphatidyl glycerol (PG) bilayers for theaflavin derivatives, correlating their bactericidal activity. CONCLUSION: These findings suggest that theaflavins could effectively inhibit B. coagulans by decreasing cell membrane fluidity. SIGNIFICANCE AND IMPACT: Bacillus coagulans is a spore-forming heat-resistant bacterium responsible for spoilage in low-acidic beverages. Natural antimicrobial components in tea-based beverages are central to reducing microbial contamination and product quality deterioration, although mechanisms underlying their antimicrobial action remain obscure. This study highlights the inhibitory action of theaflavins on B. coagulans and their potential application in food and beverage industries.
Asunto(s)
Bacillus coagulans , Biflavonoides , Catequina , Adsorción , Antibacterianos/farmacología , Antioxidantes/farmacología , Bacillus coagulans/metabolismo , Biflavonoides/metabolismo , Biflavonoides/farmacología , Fosfolípidos/farmacología , Té/químicaRESUMEN
Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persist in leafy green vegetables and subsequently produce biofilms. Biofilm formation is an ongoing concern in the food industry as biofilms are relatively resistant to a variety of antimicrobial treatments. In the present study, we evaluated the combined effects of phage FP43 and mild-heated slightly acidic hypochlorous water (SAHW) in reducing established biofilms on lettuce. Prior to the sequential treatments involving phage-SAHW and SAHW-phage for long-term storage, equal inoculum densities of E. coli O157:H7 and E. coli O91:H- were added on iceberg lettuce surfaces and the lettuce samples were stored at 10 °C for 48 h to allow biofilm formation. The sequential treatment with phage FP43 and SAHW significantly decreased the number of adhered cells, especially the combination of phage FP43 at 25 °C for 2 h and mild-heated SAHW, which considerably eliminated E. coli viable biofilm cells to undetectable levels (>3 log CFU/piece). However, the biofilms were not completely removed, as evidenced via SEM observation. Additionally, sequential treatment with SAHW and phage caused continuous reductions in viable counts, decreasing the viability of E. coli O157:H7 and total E. coli to the lower limit of detection after incubation for 5 d. Meanwhile, bacterial regrowth was observed after treatment with SAHW alone. These results indicated that the combination of phage and SAHW could be considered as a promising strategy to control the formation of E. coli O157:H7 biofilms on lettuce.
Asunto(s)
Bacteriófagos , Escherichia coli O157 , Biopelículas , Recuento de Colonia Microbiana , Microbiología de Alimentos , Calor , Lactuca/microbiología , Agua/farmacologíaRESUMEN
The gene encoding LysSTG2, an endolysin from Salmonella-lytic bacteriophage STG2, was cloned, overexpressed, and characterized. LysSTG2 consists of a single domain belonging to the Peptidase_M15 superfamily. LysSTG2 showed strong lytic activity against chloroform-treated S. Typhimurium cells after incubation at 4-50 °C for 30 min, at pH ranging from 7.0 to 11.0, and in the presence of NaCl from 0 to 300 mmol/L. It also showed lytic activity against all the 14 tested Gram-negative strains treated with chloroform, including Salmonella, E. coli, and Pseudomonas aeruginosa, but not against the Gram-positive bacteria tested. In addition, LysSTG2 (100 µg/mL) reduced the viability of S. Typhimurium NBRC 12529 planktonic cells by 1.2 log and that of the biofilm cells after 1-h treatment. Sequential treatment of slightly acidic hypochlorous water (SAHW) containing 40 mg/L available chlorine and LysSTG2 (100 µg/mL) was effective on S. Typhimurium NBRC 12529 biofilm cells, removing more than 99% of biofilm cells. These results demonstrate that LysSTG2 alone can effectively kill S. Typhimurium cells after permeabilization treatment and successfully control S. Typhimurium in biofilms in combination with SAHW, suggesting that the combined use of LysSTG2 and SAHW might be a novel and promising method for combating S. Typhimurium in food industries.
Asunto(s)
Bacteriófagos/enzimología , Biopelículas , Cloro/farmacología , Endopeptidasas/metabolismo , Ácido Hipocloroso/farmacología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/virología , Proteínas Virales/metabolismo , Bacteriófagos/genética , Biopelículas/efectos de los fármacos , Endopeptidasas/genética , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Proteínas Virales/genética , Agua/químicaRESUMEN
The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.
Asunto(s)
Bacteriófagos/fisiología , Campylobacter coli/crecimiento & desarrollo , Campylobacter jejuni/crecimiento & desarrollo , Ácido Edético/farmacología , Salmonella enteritidis/crecimiento & desarrollo , Salmonella typhimurium/crecimiento & desarrollo , Campylobacter coli/efectos de los fármacos , Campylobacter coli/virología , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/virología , Viabilidad Microbiana , Salmonella enteritidis/efectos de los fármacos , Salmonella enteritidis/virología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/virologíaRESUMEN
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 (ΔyafQ), dinJ (ΔdinJ), and tnaA (ΔtnaA) 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.
Asunto(s)
Toxinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/fisiología , Indoles/metabolismo , Termotolerancia/genética , Toxinas Bacterianas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
The pathogenicity of Salmonella Typhimurium, a foodborne pathogen, is mainly attributed to its ability to form biofilm on food contact surfaces. ε-polylysine, a polymer of positively charged lysine, is reported to inhibit biofilm formation of both gram-positive and gram-negative bacteria. To elucidate the mechanism underlying ε-polylysine-mediated inhibition of biofilm formation, the transcriptional profiles of ε-polylysine-treated and untreated Salmonella Typhimurium cells were comparatively analysed. The genome-wide DNA microarray analysis was performed using Salmonella Typhimurium incubated with 0.001% ε-polylysine in 0.1% Bacto Soytone at 30 °C for 2 h. The expression levels of genes involved in curli amyloid fibres and cellulose production, quorum sensing, and flagellar motility were downregulated, whereas those of genes associated with colanic acid synthesis were upregulated after treatment with ε-polylysine. The microarray results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, treatment with ε-polylysine decreased the production of colanic acid in Salmonella Typhimurium. The findings of this study improved our understanding of the mechanisms underlying ε-polylysine-mediated biofilm inhibition and may contribute to the development of new disinfectants to control biofilm during food manufacturing and storage.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Polilisina/farmacología , Salmonella typhimurium/efectos de los fármacos , Adhesión Bacteriana/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Plásticos , Salmonella typhimurium/fisiologíaRESUMEN
Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 °C.Key points⢠A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products. ⢠Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing. ⢠SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus. ⢠Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.
Asunto(s)
Microbiología de Alimentos/métodos , Podoviridae/metabolismo , Fagos de Staphylococcus/aislamiento & purificación , Fagos de Staphylococcus/metabolismo , Staphylococcus aureus/efectos de los fármacos , Animales , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Pollos/virología , Peces/virología , Genoma Viral , Especificidad del Huésped , Nisina/farmacología , Podoviridae/genética , Podoviridae/aislamiento & purificación , Carne de Cerdo/virología , Fagos de Staphylococcus/genética , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/virología , Virulencia/efectos de los fármacosRESUMEN
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.
Asunto(s)
Bacteriófagos/fisiología , Agentes de Control Biológico , ADN Viral/aislamiento & purificación , Escherichia coli Shiga-Toxigénica/crecimiento & desarrollo , Escherichia coli Shiga-Toxigénica/virología , beta-Lactamasas/metabolismo , Animales , Bovinos , Recuento de Colonia Microbiana , ADN Viral/genética , Contaminación de Alimentos/prevención & control , Microbiología de Alimentos , Intestinos/microbiología , Intestinos/virología , Myoviridae/fisiología , Carne Roja/microbiología , Análisis de Secuencia de ADN , Escherichia coli Shiga-Toxigénica/enzimologíaRESUMEN
Shiga toxin producing Escherichia coli (STEC) are dangerous foodborne pathogens. Foods are considered as important sources for STEC infection in human. In this study, STEC contamination of raw meats was investigated and the virulence factors of 120 clinical STEC strains characterized. STEC was detected in 4.4% of tested samples. Among 25 STEC strains from meats, five strains (20%) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80%) were eae-negative. However, 28% of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75% of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32% of these strains. The stx2a gene, a subtype of Shiga toxin gene (stx), was the most prevalent subtype among the identified meat STEC bacteria. None of the meat STEC was O157:H7 serotype. Nevertheless, 92% of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30% and 70% strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8%) and ehxA (96.7%) genes and 92.5% of them showed Stx productivity. Our study shows that some raw meat samples contain non-O157 STEC bacteria and some strains have virulence factors similar to those of clinical strains.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Carne/microbiología , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Factores de Virulencia/genética , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Técnicas de Tipificación Bacteriana , Bovinos , Pollos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Contaminación de Alimentos/análisis , Humanos , Toxina Shiga/metabolismo , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/metabolismo , Subtilisinas/genética , Subtilisinas/metabolismo , Porcinos , Factores de Virulencia/metabolismoRESUMEN
Catechins are a class of polyphenols and have high anti-bacterial activity against various microorganisms. Here, we report the mechanism for antibacterial activity of epigallocatechin gallate (EGCg) against Gram-positive bacteria Bacillus subtilis, which is highly sensitive to EGCg. Transmission electron microscope analysis revealed that deposits containing EGCg were found throughout the cell envelope from the outermost surface to the outer surface of cytoplasmic membrane. Aggregating forms of proteins and EGCg were identified as spots that disappeared or showed markedly decreased intensity after the treatment with EGCg compared to the control by two-dimensional electrophoresis. Among the identified proteins included 4 cell surface proteins, such as oligopeptide ABC transporter binding lipoprotein, glucose phosphotransferase system transporter protein, phosphate ABC transporter substrate-binding protein, and penicillin-binding protein 5. Observations of glucose uptake of cells and cell shape B. subtilis after the treatment with EGCg suggested that EGCg inhibits the major functions of these proteins, leading to growth inhibition of B. subtilis.
Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Catequina/análogos & derivados , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Catequina/química , Catequina/metabolismo , Catequina/farmacología , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Glucosa/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Oxígeno/metabolismo , Esporas Bacterianas/efectos de los fármacosRESUMEN
Subtilase cytotoxin (SubAB) is an important virulence factor of eae-negative Shiga toxin-producing Escherichia coli (STEC). Three variants of SubAB-encoding genes have been reported in the literature; however, the newly described subAB variant (subAB2-2) was found only in STEC strains from deer meat, sheep, and some wild animals. In this study, subAB variants were detected by PCR and DNA sequencing in 5 out of 12 (41.6%) eae-negative STEC strains isolated from patients. Most subAB-positive STEC strains (80%) harbored the subAB1 gene. The subAB2-2 gene was detected for the first time in the clinical STEC O128:H2 strain. Other virulence genes including stx1a, stx1c, stx2b, ehxA, and tia were also detected in this strain. The DNA sequence analyses of the subAB1 and subAB2-2 genes of the clinical STEC strains showed 99% and 100% identity to those of the reference strains 98NK2 and LM27558stx2, respectively. This is the first report on the detection of the subAB2-2 gene in a clinical STEC isolate.
Asunto(s)
Citotoxinas/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/genética , Carne/microbiología , Escherichia coli Shiga-Toxigénica/enzimología , Subtilisinas/genética , Animales , Citotoxinas/metabolismo , Ciervos , Proteínas de Escherichia coli/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , Ovinos , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Subtilisinas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The continuous detection of multi-drug-resistant enterococci in food source environments has aroused widespread concern. In this study, 198 samples from chicken products, animal feces, raw milk, and vegetables were collected in Japan and Egypt to investigate the prevalence of enterococci and virulence characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed for species identification and taxonomic analysis of the isolates. The results showed that the rates of most virulence genes (efaA, gelE, asa1, ace, and hyl) in the Japanese isolates were slightly higher than those in the Egyptian isolates. The rate of efaA was the highest (94.9 %) among seven virulence genes detected, but the cylA gene was not detected in all isolates, which was in accordance with γ-type hemolysis phenotype. In Enterococcus faecalis, the rate of kanamycin-resistant strains was the highest (84.75 %) among the antibiotics tested. Moreover, 78 % of E. faecalis strains exhibited multi-drug resistance. Four moderately vancomycin-resistant strains were found in Egyptian isolates, but none were found in Japanese isolates. MALDI-TOF MS analysis correctly identified 98.5 % (68/69) of the Enterococcus isolates. In the principal component analysis dendrogram, strains isolated from the same region with the same virulence characteristics and similar biofilm-forming abilities were characterized by clustered distribution in different clusters. This finding highlights the potential of MALDI-TOF MS for classifying E. faecalis strains from food sources.
Asunto(s)
Antibacterianos , Biopelículas , Enterococcus , Microbiología de Alimentos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Virulencia , Biopelículas/crecimiento & desarrollo , Enterococcus/genética , Enterococcus/patogenicidad , Enterococcus/efectos de los fármacos , Enterococcus/aislamiento & purificación , Factores de Virulencia/genética , Animales , Egipto , Antibacterianos/farmacología , Verduras/microbiología , Japón , Pollos , Leche/microbiología , Heces/microbiología , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana , Farmacorresistencia Bacteriana Múltiple , Contaminación de Alimentos/análisisRESUMEN
This study focused on the isolation and characterization of bacteriophages with specific activity against toxin-producing and multidrug-resistant strains of Bacillus cereus sensu stricto (B. cereus s. s.). Ten different samples yielded six bacteriophages by utilizing the double-layer agar technique. The most promising phage, vB_BceS-M2, was selected based on its broad host range and robust lytic activity against various B. cereus s. s. strains. The phage vB_BceS-M2 had a circular double-stranded DNA genome of 56,482 bp. This phage exhibited stability over a wide range of temperatures and pH values, which is crucial for its potential application in food matrices. The combined effect of phage vB_BceS-M2 and nisin, a widely used antimicrobial peptide, was investigated to enhance antimicrobial efficacy against B. cereus in food. The results suggested that nisin showed synergy and combined effect with the phage, potentially overcoming the growth of phage-resistant bacteria in the broth. Furthermore, practical applications were conducted in various liquid and solid food matrices, including whole and skimmed milk, boiled rice, cheese, and frozen meatballs, both at 4 and 25 °C. Phage vB_BceS-M2, either alone or in combination with nisin, reduced the growth rate of B. cereus in foods other than whole milk. The combination of bacteriophage and nisin showed promise for the development of effective antimicrobial interventions to counteract toxigenic and antibiotic-resistant B. cereus in food.
Asunto(s)
Antibacterianos , Bacillus cereus , Farmacorresistencia Bacteriana Múltiple , Microbiología de Alimentos , Nisina , Antibacterianos/farmacología , Bacillus cereus/virología , Bacillus cereus/efectos de los fármacos , Fagos de Bacillus/genética , Bacteriófagos , Queso/microbiología , Concentración de Iones de Hidrógeno , Leche/microbiología , Nisina/farmacología , Oryza/microbiología , TemperaturaRESUMEN
Nowadays, the discovery of alternative natural antimicrobial substances such as bacteriophages, essential oils, and other physical and chemical agents is developing in the food industry. In this study, nine bacteriophages were isolated from various parts of raw chickens and exhibited lytic activities against L. monocytogenes and various Listeria spp. The characterization of phage vB_LmoS-PLM9 was stable at 4 to 50 °C and pH range from 4 to 10. Phage vB_LmoS-PLM9 had a circular, double-stranded genomic DNA with 38,345 bp having endolysin but no antibiotic resistance or virulence genes. Among the eight essential oils tested at 10 %, cinnamon bark, and cassia oils showed the strongest antilisterial activities. The combined use of phage vB_LmoS-PLM9 and cinnamon oils indicated higher efficiency than single treatments. The combination of phage (MOI of 10) and both cinnamon oils (0.03 %) reduced the viable counts of L. monocytogenes and inhibited the regrowth of resistant cell populations in broth at 30 °C. Furthermore, treatment with the combination of phage (MOI of 100) and cinnamon oil (0.125 %) was effective in milk, especially at 4 °C by reducing the viable count to less than lower limit of detection. These results suggest combining phage and cinnamon oil is a potential approach for controlling L. monocytogenes in milk.
Asunto(s)
Bacteriófagos , Cinnamomum zeylanicum , Listeria monocytogenes , Leche , Aceites Volátiles , Salmón , Animales , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/virología , Leche/microbiología , Cinnamomum zeylanicum/química , Aceites Volátiles/farmacología , Salmón/microbiología , Microbiología de Alimentos , Aceites de Plantas/farmacología , Conservación de Alimentos/métodos , Pollos , Antibacterianos/farmacologíaRESUMEN
Biofilm formation of Listeria monocytogenes in food processing environments cause potential source of cross-contamination to foodstuffs; hence, the control of biofilm is currently addressed to find effective solutions for preventing biofilm formation or eliminating the established one. Forty-five strains of Listeria monocytogenes isolated from Pangasius fish-processing plants were studied for their capability to form a biofilm on 96-well microtiter plate by using the conventional crystal violet staining. Additionally, the inhibitory effect of biofilm formation by food additives including monascus pigment and ε-polylysine was examined. The average OD value showing biofilm mass of all 45 strains L. monocytogenes increased with an increasing temperature and time (p < 0.05). Monascus pigment and ε-polylysine significantly decreased biofilm formation by 80 ± 5.5% and 20 ± 5.9%, respectively, at the tested concentration (p < 0.05) Further, the effects of lysozyme (0.1 mg/mL) alone or in combination with slightly acidic hypochlorous water (SAHW) with 40 mg/L available chlorine or sodium hypochlorite (NaOCl) with 100 mg/L available chlorine against 7-d established biofilm of L. monocytogenes were investigated. The results indicated that slightly acidic hypochlorous water alone exhibited significant antibacterial activity (p < 0.05), decreasing the viable count by 5.2 ± 0.5 log CFU/mL. It seems that sequential treatment of lysozyme and SAHW showed an additional efficacy against biofilm of L. monocytogenes on polystyrene plate surface, reducing 70% of biomass of biofilm and 7.6 ± 0.3 log of biofilm viable cells (p < 0.05). Additionally, SAHW exhibited greater bactericidal activity against viable biofilm cells than NaOCl did. This result reveals that SAHW is a promising disinfectant agent against L. monocytogenes and the potential alternative to NaOCl in practice.
Asunto(s)
Bagres , Listeria monocytogenes , Animales , Muramidasa/farmacología , Cloro/farmacología , Polilisina/farmacología , Acero Inoxidable , Biopelículas , Agua/farmacología , Recuento de Colonia MicrobianaRESUMEN
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.