Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7

AIMS: To quantify the antibacterial properties of five essential oils (EO) on a non-toxigenic strain of Escherichia coli O157:H7 in the presence and absence of a stabilizer and an emulsifier and at three different temperatures. METHODS AND RESULTS: Five EOs known to exhibit antibacterial properties were screened by disc diffusion assay and the most active were selected for further study in microdilution colorimetric assays. Oregano (Origanum vulgare) and thyme (Thymus vulgaris; light and red varieties) EO had the strongest bacteriostatic and bactericidal properties, followed by bay (Pimenta racemosa) and clove bud (Eugenia caryophyllata synonym: Syzygium aromaticum) EO. Oregano oil was colicidal at 625 microl l(-1) at 10, 20 and 37 degrees C. The addition of 0.05% (w/v) agar as stabilizer reinforced the antibacterial properties, particularly at 10 degrees C, whereas 0.25% (w/v) lecithin reduced antibacterial activity. Scanning electron micrographs showed extensive morphological changes to treated cells. CONCLUSIONS: Oregano and thyme EO possess significant in vitro colicidal and colistatic properties, which are exhibited in a broad temperature range and substantially improved by the addition of agar as stabilizer. Bay and clove bud EO are less active. Lecithin diminished antibacterial properties. The bactericidal concentration of oregano EO irreversibly damaged E. coli O157:H7 cells within 1 min. SIGNIFICANCE AND IMPACT OF THE STUDY: Oregano and light thyme EO, particularly when enhanced by agar stabilizer, may be effective in reducing the number or preventing the growth of E. coli O157:H7 in foods.     

Antimicrobial Effects of Mustard Flour and Acetic Acid against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Serovar Typhimurium

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22°C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (10⁶ to 10⁷ CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22°C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5°C, was S. enterica serovar Typhimurium (1 day) < E. coli O157:H7 (3 days) < L. monocytogenes (9 days). The food-borne pathogens tested were reduced much more rapidly at 22°C than at 5°C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of E. coli O157:H7 and L. monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E. coli O157:H7 and L. monocytogenes. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.     

Multiplex PCR and DNA array for the detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. targeting virulence-related genes

A multiplex PCR and DNA array for quick detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. was developed using specific genetic markers derived from virulence-related genes. The genetic markers of cytK, sei, prfA, rfB, and hilA gene specifically amplified DNA fragments of 320 bp, 500 bp, 700 bp, 1.0 kb and 1.2 kb from B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., respectively. These markers are specific for the detection of the corresponding target pathogens. The sensitivity of the genetic markers was down to ～0.5 fg genomic DNA and ～10¹ CFU/ml (one bacterial cell per reaction) of bacterial culture. The combination of mPCR and DNA macroarray hybridization sensitively and specifically detected B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., in complex mixed cultures and food matrices. Thus, this mPCR and macroarray-based approach serves as rapid and reliable diagnostic tool for the detection of these five pathogens.     

Fundamental Characteristics of Deep-UV Light-Emitting Diodes and Their Application To Control Foodborne Pathogens

Low-pressure mercury UV (LP-UV) lamps have long been used for bacterial inactivation, but due to certain disadvantages, such as the possibility of mercury leakage, deep-UV-C light-emitting diodes (DUV-LEDs) for disinfection have recently been of great interest as an alternative. Therefore, in this study, we examined the basic spectral properties of DUV-LEDs and the effects of UV-C irradiation for inactivating foodborne pathogens, including Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes, on solid media, as well as in water. As the temperature increased, DUV-LED light intensity decreased slightly, whereas LP-UV lamps showed increasing intensity until they reached a peak at around 30°C. As the irradiation dosage and temperature increased, E. coli O157:H7 and S. Typhimurium experienced 5- to 6-log-unit reductions. L. monocytogenes was reduced by over 5 log units at a dose of 1.67 mJ/cm². At 90% relative humidity (RH), only E. coli O157:H7 experienced inactivation significantly greater than at 30 and 60% RH. In a water treatment study involving a continuous system, 6.38-, 5.81-, and 3.47-log-unit reductions were achieved in E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, at 0.5 liter per minute (LPM) and 200 mW output power. The results of this study suggest that the use of DUV-LEDs may compensate for the drawbacks of using LP-UV lamps to inactivate foodborne pathogens.     

Assessment of Photodynamic Destruction of Escherichia coli O157:H7 and Listeria monocytogenes by Using ATP Bioluminescence

Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

     

Antibacterial activity of chitosan tripolyphosphate nanoparticles loaded with various metal ions

The aim of this study was to prepare and select chitosan nanoparticles loaded metal ions with high antibacterial activities. Chitosan nanoparticles were prepared based on ionic gelation between chitosan and sodium tripolyphosphate. Then, Ag⁺, Cu²⁺, Zn²⁺, Mn²⁺, or Fe²⁺ was individually loaded onto chitosan nanoparticles. Their particle sizes and zeta potentials were measured. Their antibacterial activities were evaluated by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli 25922, Salmonella choleraesuis ATCC 50020 and Staphylococcus aureus 25923 in vitro. Results showed that antibacterial activity was significantly enhanced by the metal ions loaded, except for Fe²⁺. Especially for chitosan nanoparticles loaded Cu²⁺, the MIC and MBC against E. coli 25922, S.choleraesuis ATCC 50020 and S. aureus 25923 were 21–42 times lower than that of Cu²⁺, respectively. Moreover, it was found that antibacterial activity was directly proportional to zeta potential.     

Antibacterial activity and mechanism of action of ε-poly-l-lysine

ε-Poly-l-lysine (ε-PL)² is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS) ³ levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR) ⁴ indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response) ⁵ regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence.     

The Pepsin Hydrolysate of Bovine Lactoferrin Causes a Collapse of the Membrane Potential in Escherichia coli O157:H7

In the present study, the ability of bovine lactoferrin hydrolysate (LfH) to disrupt the cytoplasmic membrane of Escherichia coli O157:H7 was investigated. Lactoferrin and LfH antimicrobial activities were compared against E. coli O157:H7 and E. coli O157:H7 spheroplasts. The effect of LfH on the cytoplasmic membrane of E. coli O157:H7 cells was determined by evaluating potassium efflux (K⁺), dissipation of ATP and membrane potential (ΔΨ). LfH produced a rapid efflux of potassium ions, a decrease in intracellular levels of ATP coupled with a substantial increase in extracellular ATP levels and a complete dissipation of the ΔΨ. The results suggest that LfH causes a collapse of the membrane integrity by pore formation in the inner membrane, leading to the death of the cell. Moreover, the mechanism of action of LfH on E. coli O157:H7 appears to involve an interference with the inner membrane integrity based on experiments using E. coli O157:H7 spheroplasts.     

Association of Escherichia coli O157:H7 with Houseflies on a Cattle Farm

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 × 10¹ to 1.5 × 10⁵ CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.     

Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O-Antigen Linked with Maltose-Binding Protein Elicits Humoral and Cellular Responses

Glycoconjugate is one of the most efficacious and safest vaccines against bacterial pathogens. Previous studies of glycoconjugates against pathogen E. coli O157:H7 focused more on the humoral responses they elicited. However, little was known about their cellular responses. In this study, we exploited a novel approach based on bacterial protein N-linked glycosylation system to produce glycoconjugate containing Escherichia coli O157:H7 O-antigen linked with maltose-binding protein and examined its humoral and cellular responses in BALB/c mice. The transfer of E. coli O157:H7 O-antigen to MBP was confirmed by western blot and MALDI-TOF MS. Mice injected with glycoconjugate O-Ag-MBP elicited serum bactericidal antibodies including anti-E. coli O157:H7 O-antigen IgG and IgM. Interestingly, O-Ag-MBP also stimulated the secretion of anti-E. coli O157:H7 O-antigen IgA in intestine. In addition, O-Ag-MBP stimulated cellular responses by recruiting Th1-biased CD4⁺ T cells, CD8⁺ T cells. Meanwhile, O-Ag-MBP induced the upregulation of Th1-related IFN-γ and downregulation of Th2-related IL-4, and the upregulation of IFN-γ was stimulated by MBP in a dose-dependent manner. MBP showed TLR4 agonist-like properties to activate Th1 cells as carrier protein of O-Ag-MBP. Thus, glycoconjugate vaccine E. coli O157:H7-specific O-Ag-MBP produced by bacterial protein N-linked glycosylation system was able to elicit both humoral and Th1-biased cellular responses.     

Interaction between the Microbial Community and Invading Escherichia coli O157:H7 in Soils from Vegetable Fields

The survival of Escherichia coli O157:H7 in soils can contaminate vegetables, fruits, drinking water, etc. However, data on the impact of E. coli O157:H7 on soil microbial communities are limited. In this study, we monitored the changes in the indigenous microbial community by using the phospholipid fatty acid (PLFA) method to investigate the interaction of the soil microbial community with E. coli O157:H7 in soils. Simple correlation analysis showed that the survival of E. coli O157:H7 in the test soils was negatively correlated with the ratio of Gram-negative (G⁻) to Gram-positive (G⁺) bacterial PLFAs (G⁻/G⁺ ratio). In particular, levels of 14 PLFAs were negatively correlated with the survival time of E. coli O157:H7. The contents of actinomycetous and fungal PLFAs in the test soils declined significantly (P, <0.05) after 25 days of incubation with E. coli O157:H7. The G⁻/G⁺ ratio declined slightly, while the ratio of bacterial to fungal PLFAs (B/F ratio) and the ratio of normal saturated PLFAs to monounsaturated PLFAs (S/M ratio) increased, after E. coli O157:H7 inoculation. Principal component analysis results further indicated that invasion by E. coli O157:H7 had some effects on the soil microbial community. Our data revealed that the toxicity of E. coli O157:H7 presents not only in its pathogenicity but also in its effect on soil microecology. Hence, close attention should be paid to the survival of E. coli O157:H7 and its potential for contaminating soils.     

Survival of Escherichia coli O157:H7 in Soils from Jiangsu Province,China

Escherichia coli O157:H7 (E. coli O157:H7) is recognized as a hazardous microorganism in the environment and for public health. The E. coli O157:H7 survival dynamics were investigated in 12 representative soils from Jiangsu Province, where the largest E. coli O157:H7 infection in China occurred. It was observed that E. coli O157:H7 declined rapidly in acidic soils (pH, 4.57 – 5.14) but slowly in neutral soils (pH, 6.51 – 7.39). The survival dynamics were well described by the Weibull model, with the calculated t_d value (survival time of the culturable E. coli O157:H7 needed to reach the detection limit of 100 CFU g⁻¹) from 4.57 days in an acidic soil (pH, 4.57) to 34.34 days in a neutral soil (pH, 6.77). Stepwise multiple regression analysis indicated that soil pH and soil organic carbon favored E. coli O157:H7 survival, while a high initial ratio of Gram-negative bacteria phospholipid fatty acids (PLFAs) to Gram-positive bacteria PLFAs, and high content of exchangeable potassium inhibited E. coli O157:H7 survival. Principal component analysis clearly showed that the survival profiles in soils with high pH were different from those with low pH.     

Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7

Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 × 10⁻⁸ ml/min and 1.91 × 10⁻⁸ ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10⁻² CFU/ml for S. Typhimurium and 4.58 × 10⁻⁵ CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

Efficacy of Essential Oils on Fungi Isolated from Archaeological Objects in Saqqara Excavation,Egypt

The archeological objects constitute an important part of the worldwide cultural heritage. The impact of the fungal activity on the deterioration of cultural heritage is a global problem and their preservation over time is a challenging task. Antifungal activities of 12 essential oils (EOs) (black cumin, castor, cinnamon, clove, cumin, garlic, geranium, lavender, lemongrass, menthe, olive, and thyme) were examined against 16 fungal species isolated from three tested archaeological objects (wall painting stone, wooden statue, and pottery coffin) from Saqqara stores in Egypt. Molecular identification was carried out for the highly frequent species (Aspergillus niger, A. flavus and Rhizopus oryzae) in the three tested archaeological samples. Antifungal activity and minimal inhibitory concentration (MIC) of the tested EOs with different concentrations (0.125, 0.25, 0.5, 0.75, 1?μl/ml) were measured.

The most efficient EOs were thyme (MIC ranged from 0.25–0.75μl/ml) followed by clove (MIC ranged from 0.25–1?μl/ml) and geranium, (MIC ranged from 0.5–1?μl/ml). Thymol (37.1%) and p-Cymene (26.32%) were the active constituents of thyme, while Triacetin (69.36%) and eugenol (28.67) were the most efficient components of clove oil followed by geranium active components (à-Citronellol 20.62% and Geraniol 14.43%). Aspergillus niger was the most resistance species, while Fusarium oxysporum and Penicilium citrinium were the most susceptible ones.     

Effect of Dietary Stress on Fecal Shedding of Escherichia coli O157:H7 in Calves

Two groups of calves were subjected to dietary stress by withholding of food beginning 1 or 14 days after inoculation with 10¹⁰ CFU of Escherichia coli O157:H7. Following treatment, neither group had a significant increase in fecal shedding of E. coli O157:H7. A third group of calves had food withheld for 48 h prior to inoculation with 10⁷ CFU of E. coli O157:H7. These calves were more susceptible to infection and shed significantly more E. coli O157:H7 organisms than calves maintained on a normal diet.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.