RESUMO
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.
Assuntos
Antibacterianos , Bacillus cereus , Farmacorresistência Bacteriana Múltipla , Microbiologia de Alimentos , Nisina , Antibacterianos/farmacologia , Bacillus cereus/virologia , Bacillus cereus/efeitos dos fármacos , Fagos Bacilares/genética , Bacteriófagos , Queijo/microbiologia , Concentração de Íons de Hidrogênio , Leite/microbiologia , Nisina/farmacologia , Oryza/microbiologia , TemperaturaRESUMO
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.
Assuntos
Antibacterianos , Biofilmes , Enterococcus , Microbiologia de Alimentos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Virulência , Biofilmes/crescimento & desenvolvimento , Enterococcus/genética , Enterococcus/patogenicidade , Enterococcus/efeitos dos fármacos , Enterococcus/isolamento & purificação , Fatores de Virulência/genética , Animais , Egito , Antibacterianos/farmacologia , Verduras/microbiologia , Japão , Galinhas , Leite/microbiologia , Fezes/microbiologia , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana , Farmacorresistência Bacteriana Múltipla , Contaminação de Alimentos/análiseRESUMO
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.
Assuntos
Bacteriófagos , Cinnamomum zeylanicum , Listeria monocytogenes , Leite , Óleos Voláteis , Salmão , Animais , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/virologia , Leite/microbiologia , Cinnamomum zeylanicum/química , Óleos Voláteis/farmacologia , Salmão/microbiologia , Microbiologia de Alimentos , Óleos de Plantas/farmacologia , Conservação de Alimentos/métodos , Galinhas , Antibacterianos/farmacologiaRESUMO
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.
Assuntos
Galinhas , Microbiologia de Alimentos , Especificidade de Hospedeiro , Animais , Microbiologia do Solo , Fagos de Pseudomonas/fisiologia , Fagos de Pseudomonas/genética , Pseudomonas/virologia , Genoma Viral , Podoviridae/fisiologia , Podoviridae/genética , Podoviridae/isolamento & purificação , Podoviridae/classificação , Agentes de Controle Biológico , DNA Viral/genética , Bacteriófagos/fisiologia , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bacteriófagos/classificaçãoRESUMO
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.
Assuntos
Bacteriófagos , Animais , Enterococcus , Leite/microbiologia , Especificidade de Hospedeiro , Antibacterianos , Enterococcus faecalisRESUMO
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.