Model systems allowing quantification of sensitivity to disinfectants and comparison of disinfectant susceptibility of persistent and presumed nonpersistent Listeria monocytogenes

Aims:  To determine if Listeria monocytogenes persistent strains differ from presumed nonpersistent strains in disinfection susceptibility and to examine the influence of attachment and NaCl on susceptibility.
Methods and Results:  Two model-systems that allowed quantitative inter-strain comparison of disinfectant sensitivity were developed. Persistent L. monocytogenes were not more tolerant to the disinfectants Incimaxx DES and Triquart SUPER than presumed nonpersistent isolates. When calibrating the systems with respect to presence of biological material and cell density, attached bacteria were as sensitive to disinfectants as were planktonic bacteria. Growth with 5% NaCl increased the tolerance of planktonic cells to Incimaxx DES. All strains of spot inoculated L. monocytogenes survived well 20 h of drying when protected by growth media and 5% NaCl, but were not protected by NaCl against disinfection.
Conclusions:  Persistent strains of L. monocytogenes are as susceptible to disinfectants as are presumed nonpersistent strains and attachment does not render the strains more tolerant to disinfectants. Growth with NaCl affected the susceptibility of the planktonic L. monocytogenes to Incimaxx DES and protected spot inoculated cells during drying.
Significance and Impact of the Study:  Attachment to surfaces does not per se offer protection to L. monocytogenes against disinfectants and disinfection tolerances do not appear to influence the ability of a strain to persist.     

HasF, a TolC-homolog of Serratia marcescens, is involved in energy-dependent efflux

A tolC-like gene (hasF) was identified upon scanning the incomplete database of the S. marcescens genome. This gene was amplified using PCR and cloned in the pUC18 vector to yield pUCHF. Sequencing of the S. marcescens tolC-like hasF gene and subsequent amino acid sequence prediction revealed approximately 80% amino acid homology with the Escherichia coli TolC. A tolC-deficient strain of E. coli (BL923) containing pUCHF/hasF was analyzed for susceptibility to fluoroquinolones (ciprofloxacin, norfloxacin, and ofloxacin), chloramphenicol, sodium dodecyl sulfate (SDS), and ethidium bromide. Antibiotic susceptibility assays of the E. coli tolC-deficient mutant BL923 demonstrated a 64-fold increase in resistance to SDS and ethidium bromide upon introduction of the S. marcescens tolC-like hasF gene. No change was observed for susceptibility to fluoroquinolones and chloramphenicol. Ethidium bromide accumulation assays performed using E. coli BL923:pUCHF established the role of the S. marcescens hasF gene product in proton gradient-dependent efflux.     

Antimicrobial activity of Bacillus subtilis and Bacillus pumilus during the fermentation of African locust bean (Parkia biglobosa) for Soumbala production

Aims:  To examine predominant isolates of Bacillus subtilis and B. pumilus isolated from Soumbala for their antimicrobial activity against indicator microorganisms as Micrococcus luteus , Staphyloccocus aureus , Bacillus cereus , Enterococus facium , Listeria monocytogenes , Escherichia coli , Salmonella typhimurium , Shigella dysenteriae , Yersinia enterocolitica , Aspergillus ochraceus and Penicillium roqueforti .
Methods and Results:  Growth inhibition of indicator microorganisms by cells and supernatants of three B. subtilis and two B. pumilus strains was investigated using agar diffusion tests. Inactivation of indicator microorganisms was investigated in laboratory broth and during the fermentation of African locust bean for Soumbala production. The Bacillus isolates showed variable ability of inhibition and inactivation according to the indicator microorganism. The supernatants of pure cultures of B. subtilis inhibited one strain of B. cereus , one of Staph. aureus and E. coli and caused abnormal germination of Aspergillus ochraceus . The supernatant of mixed cultures of B. subtilis and indicators inhibited all the indicators. A treatment with protease eliminated the inhibitions. Isolates of B. subtilis inactivated all the indicators organisms during the fermentation of African locust bean as well as in laboratory broth with about five to eight decimal reduction.
Conclusion:  Bacillus isolates from Soumbala inhibit and inactivate Gram-positive and Gram-negative bacteria as well as ochratoxin A producing fungi during both laboratory cultivation and natural fermentation.
Significance and Impact of the Study:  Selection of starter cultures of Bacillus spp. for controlled production of Soumbala.     

The antibacterial mechanism of carvacrol and thymol against Escherichia coli

Aims:  To investigate the antibacterial mechanism of carvacrol and thymol against Escherichia coli.
Methods and Results:  The time-kill curve results showed that carvacrol and thymol at 200 mg l⁻¹ could inhibit the growth of E. coli . Flow cytometry and fluorescent dyes were used to explore the effect of two components on membrane permeability and membrane potential. In membrane permeability experiment, the mean fluorescence intensity of cells treated with 200 mg l⁻¹ carvacrol or thymol were lower than nonexposed cells. The ratio of red to green fluorescence intensity of DiOC₂(3) reflected the change of membrane potential. Carvacrol and thymol at 200 mg l⁻¹ caused the ratio of red/green decreasing from 0·42 of control to 0·08 and 0·07, respectively.
Conclusions:  Carvacrol and thymol had desired antimicrobial effect on E. coli . The antibacterial effects were attributed to their ability to permeabilize and depolarize the cytoplasmic membrane.
Significance and Impact of the Study:  This study showed the potential use of flow cytometry as a suitable method to investigate the mode of antibacterial action of essential oil components.     

Effects of concentrated supernatants recovered from Lactobacillus plantarum on Escherichia coli growth and on the viability of a human promyelocytic cell line

Aims:  The ability of concentrated supernatants from Lactobacillus plantarum to produce a disruption of plasma membrane in eukaryotic and prokaryotic cells has been examined.
Methods and Results:  A strain of Lact. plantarum (tolerant to acid and bile salts and resistant to several antibiotics) was used. It inhibited the growth of pathogenic Escherichia coli and L. monocytogenes . Supernatants from Lact. plantarum were concentrated by centrifugation. Either E. coli or HL-60 cells (a human promyelocytic cell line) were treated in the presence of the concentrated supernatants. The effect of concentrated supernatants from Lact. plantarum on E. coli growth demonstrated a bacteriostatic activity and a loss of cell viability measured by sytox green staining. Concentrated supernatants were capable of disturbing plasma membrane in E. coli and of promoting a cytotoxic and lyctic action on HL-60 cells and on human erythrocytes, respectively.
Conclusions:  These results suggest that Lact. plantarum release an effective compound responsible for an important effect in the disruption of E. coli plasma membrane and for a cytototoxic activity on promyelocytic leukaemia cells.
Significance and Impact of the Study:  This is the first in vitro study about the antimicrobial and biological activities of concentrated supernatants from Lact. plantarum .     

Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin

The growing resistance against antibiotics demands the search for alternative treatment strategies. Photodynamic therapy is a promising candidate. The natural intermediate of chlorophyll biosynthesis, protochlorophyllide, was produced, purified and tested as a novel photosensitizer for the inactivation of five model organisms including Staphylococcus aureus, Listeria monocytogenes and Yersinia pseudotuberculosis , all responsible for serious clinical infections. When microorganisms were exposed to white light from a tungsten filament lamp (0.1 mW cm⁻²), Gram-positive S. aureus, L. monocytogenes and Bacillus subtilis were photochemically inactivated at concentrations of 0.5 mg L⁻¹ protochlorophyllide. Transmission electron microscopy revealed a disordered septum formation during cell division and the partial loss of the cytoplasmic cell contents. Gram-negative Y. pseudotuberculosis and Escherichia coli were found to be insensitive to protochlorophyllide treatment due to the permeability barrier of the outer membrane. However, the two bacteria were rendered susceptible to eradication by protochlorophyllide (10 mg L⁻¹) upon addition of polymyxin B nonapeptide at 50 and 20 mg L⁻¹, respectively. The release of DNA and a detrimental rearrangement of the cytoplasm were observed.     

Relationship between the susceptibility of various bacteria to active oxygen species and to intracellular killing by macrophages

The susceptibilities of six micro-organisms to active oxygen species generated in the xanthine oxidase-mediated bactericidal system were as follows: Escherichia coli 81 greater than or equal to Listeria monocytogenes EGD greater than or equal to Salmonella typhimurium HKB-1 greater than or equal to Staphylococcus aureus Smith much greater than Mycobacterium tuberculosis H37Rv approximately equal to Candida albicans NIH A207 (the last two organisms were essentially resistant to this system). The H2O2-Fe-mediated halogenation system exhibited a higher microbicidal activity. When the micro-organisms were compared for their sensitivity to bactericidal activity of resident mouse peritoneal macrophages (M phi s), C. albicans, Staph. aureus and E. coli were killed rapidly, whereas M. tuberculosis, L. monocytogenes and S. typhimurium were more resistant. In tests for the ability to trigger an oxidative burst in mouse peritoneal M phi s (as measured by chemiluminescence), Staph. aureus showed the highest activity followed by the other organisms in the following order: C. albicans greater than E. coli greater than L. monocytogenes congruent to M. tuberculosis. S. typhimurium exhibited no triggering activity. The high susceptibility of Staph. aureus and E. coli to M phi bactericidal activity, and the partial resistance of L. monocytogenes and M. tuberculosis, correlated with their susceptibility to active oxygen and the H2O2-Fe-mediated halogenation reaction.     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

Sensitivity of Escherichia coli cell membranes to various classes of detergents

The mechanisms of interaction between non-ionic or cationic surfactants with Escherichia coli K-12 cell membranes were studied using an approach based on the registration of changes in the membrane permeability to ethidium bromide, a fluorescent dye for nucleic acids. Triton X-100, a non-ionic detergent, was shown to exert no effect on the permeability of intact cell membranes. Triton X-100 interacted with the bacteria only after treatment with EDTA, a complexing agent for bivalent cations. Cetyltrimethyl ammonium bromide increased the permeability to ethidium bromide and the action of this cationic detergent did not require the pretreatment with the complexing agent. SDS, an anionic detergent, damaged E. coli K-12 and this could be registered by the lowering of intensity of light scattering by the bacterial suspension. The surface charge of E. coli K-12 cells was shown to influence the interaction of ionic detergents with bacterial cell membranes. Its variation by changing the pH of the incubation medium did not make E. coli K-12 sensitive to Triton X-100.     

Activation ofStreptomyces viridochromogenes spores by detergents

Constitutively dormant spores ofStreptomyces viridochromogenes germinate rapidly following treatment with 1.0% of the detergents Tween 80, sodium dodecyl sulfate (SDS), or sodium heptadodecyl sulfate. Six other detergents did not activate the spores. Activation by SDS was studied further. The spores were not activated following treatment with 0.09% or less of SDS for 60 min at 37°C. Activation was complete within 1 to 2 min of treatment with 1.0% SDS. the SDS-activated spores became deactivated during incubation in buffer. Deactivation was slow at 4°C and complete after incubation for 12 h at 25°C or 6 h at 37°C. The endogenous respiratory rate of the spores was increased 3-fold by SDS activation.     

In vitro inhibition activity of different bacteriocin-producing Escherichia coli against Salmonella strains isolated from clinical cases

Aims:  To compare in vitro the inhibitory activity of four bacteriocin-producing Escherichia coli to a well-characterized panel of Salmonella strains, recently isolated from clinical cases in Switzerland.
Methods and Results:  A panel of 68 nontyphoidal Salmonella strains was characterized by pulsed-field gel electrophoresis analysis and susceptibility to antibiotics. The majority of tested strains were genetically different, with 40% resistant to at least one antibiotic. E. coli Mcc24 showed highest in vitro activity against Salmonella (100%, microcin 24), followed by E. coli L1000 (94%, microcin B17), E. coli 53 (49%, colicin H) and E. coli 52 (21%, colicin G) as revealed using a cross-streak activity assay.
Conclusions:  Escherichia coli Mcc24, a genetically modified organism producing microcin 24, and E. coli L1000, a natural strain isolated from human faeces carrying the mcb -operon for microcin B17-production, were the most effective strains in inhibiting in vitro both antibiotic resistant and sensitive Salmonella isolates.
Significance and Impact of the Study:  Due to an increasing prevalence of antibiotic resistant Salmonella strains, alternative strategies to fight these foodborne pathogens are needed. E. coli L1000 appears to be a promising candidate in view of developing biotechnological alternatives to antibiotics against Salmonella infections.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.     

Activities of Urease and Nickel Uptake of Helicobacter pylori Proteins are Media- and Host-Dependent

Background:  Nickel-dependent urease activity and nickel supply are essential for successful colonization of Helicobacter pylori in the acidic environment of the human stomach. A comparison of media effects on these two activities have never been carried out. Additionally to H. pylori we cultivated an Escherichia coli strain expressing the urease and the nickel transporter NixA of H. pylori on the same four media and measured in all cases urease and nickel uptake activity.
Aim:  To compare nickel uptake and urease activity on an inter- and intraspecies level.
Results:  In H. pylori nickel uptake (four to 200 times) and urease activities (400 to 30,000 times) were found to be much higher in comparison to the tested E. coli strain after growth on all media. These differences could not be explained by reduced protein amounts in the heterologous host E. coli . On which media the two bacteria extracted most of the nickel were organism-dependent: E. coli on Brucella Broth, H. pylori on Trypticase Soy Broth, and Minimal Media.
Conclusion:  H. pylori took nickel much more efficiently up than E. coli . The observed differences in urease activity are most likely due to additional protein components absent in the recombinant E. coli strain. The observed variety in nickel uptake and urease activities on the different media in the same organism depended on the intrinsic nickel content and chelating capacities of media components. Different culture conditions may lead to varying results; generalizations should be concluded only after excluding their media dependence.     

Interactions between humic matter and bacteria when disinfecting water with UV light

Aims:  To investigate the impact of aquatic humic matter on the inactivation of Escherichia coli and Bacillus subtilis by ultraviolet (UV) light.
Methods and Results:  A bench-scale study investigated the potential for Aldrich^® humic acid (AHA) and Suwannee River natural organic matter (SR-NOM) to coat the surface of E. coli and B. subtilis and offer protection from low-pressure UV light. UV doses of 5 and 14 mJ cm⁻² were applied using a collimated beam at four concentrations of humic matter (0, 10, 50 and 120 mg l⁻¹) in reagent grade water. Both AHA and SR-NOM were found to offer statistically significant protection of both E. coli and B. subtilis at concentrations of 50 and 120 mg l⁻¹ for a UV dose of 14 mJ cm⁻².
Conclusions:  Both E. coli and B. subtilis are susceptible to coating by humic matter which can reduce the sensitivity of the cells to UV light.
Significance and impact of the study:  Micro-organisms in the environment may acquire characteristics through interaction with humic matter that render them more resistant to UV disinfection than would be predicted based on laboratory inactivation studies using clean cells.     

Screening and characterization of monoclonal antibodies to the surface antigens of Listeria monocytogenes serotype 4b

Aims:  This study aims to develop and characterize monoclonal antibodies (Mabs) with high specificity and affinity for surface antigens of an epidemiologically important serotype 4b of Listeria monocytogenes .
Methods and Results:  Hybridoma clones were derived from B lymphocytes of mice immunized with L. monocytogenes serotype 4b and screened against this strain by an enzyme-linked immunosorbent assay. Twenty-nine clones secreting Mabs reactive with formalin-killed bacteria were obtained; 15, 8, 5 and 1 Mabs were immunoglobulin subclasses IgG2a, IgG2b, IgM and IgG1, respectively. Immuofluoresence or immunogold labelling demonstrated all except five IgM and one IgG2a Mabs bound to the surface of a live L. monocytogenes serotype 4b. The majority of the 23 surface-binding Mabs recognized linear epitopes on a 77-kDa protein. These surface-binding Mabs exhibited little or no cross-reactivity with non-4b serotypes (1/2a, 1/2b, 3a, etc.) of L. monocytogenes , five other Listeria species, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.
Conclusions:  The Mabs recognizing a 77-kDa surface protein are novel antibodies with specificity and affinity for L. monocytogenes serotype 4b.
Significance and Impact of the Study:  These anti-77 kDa surface protein Mabs may be explored as reagents for the development of Mabs-based diagnostic immunoassays for L. monocytogenes serotype 4b strains.     

Comparison of types and antimicrobial susceptibility of Staphylococcus from conventional and organic dairies in west-central Minnesota, USA

Aims:  To assess whether conventional and organic dairy management practices are associated with differences in the susceptibility of Staphylococcus to antimicrobial agents.
Methods and Results:  Staphylococcus was isolated from milk samples collected from conventional and organic dairies in west-central Minnesota. Isolates were categorized as (1) coagulase-positive, (2) novobiocin-sensitive coagulase-negative or (3) novobiocin-resistant coagulase-negative. Novobiocin-resistant coagulase-negative Staphylococcus (CNS) was more common on conventional farms and novobiocin-sensitive CNS predominated the isolates from organic farms. Overall, a larger proportion of isolates from organic rather than conventional farms were susceptible to erythromycin, pirlimycin and tetracycline. However, for pirlimycin and tetracycline, different patterns of susceptibility were observed among Staphylococcus categories.
Conclusion:  In this study, organic dairy management was associated with more overall antimicrobial susceptibility among Staphylococcus than was conventional management. However, different patterns of susceptibility among Staphylococcus categories suggest that multiple management practices, including some unrelated to antimicrobial use, may contribute to the observed differences in susceptibility.
Significance and Impact of the Study:  This study adds to our understanding of the implications of dairy management choices.     

Growth and reducing capacity of Listeria monocytogenes under different initial redox potential

Aims:  To determine the reducing capacity of Listeria monocytogenes and to highlight the effect of redox potential on its growth parameters.
Methods and Results:  The reducing capacity of L. monocytogenes was monitored in Brain Heart Infusion Broth media at different initial redox potential (Eh) and pH at 37°C. The effect of Eh obtained by gas flushing (air, N₂ and N₂-H₂) or by adding potassium ferricyanide and dithiotreitol in concentration from 1 to 10 mmol l⁻¹on L. monocytogenes growth parameters at pH 6·0, 7·0 and 8·0 was investigated. A total change of 539 mV (±44 mV) from an initial redox value of +330 ± 8 mV to a more negative potential in redox curves was observed resulting from L. monocytogenes growth at pH 7·0 at 37°C. A significant influence of pH and redox potential on L. monocytogenes lag phase of growth was shown ( P  < 0·05).
Conclusions:  Listeria monocytogenes exhibited longer lag phase in reducing conditions and at pH 6·0. The method used to modify the redox potential was shown to have no effect on growth parameters at pH 7·0.
Significance and Impact of the Study:  The provided information on the extending lag time and the possible delayed growth of this major pathogen in reducing conditions might be useful for its control in foods.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment.

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.     

Identification, cloning and sequencing of Escherichia coli strain χ1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran

Aims:  To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain χ1378 isolated from Iranian poultry and to predict its protein product, Iss.
Methods and Results:  The iss gene from E. coli strain χ1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain χ1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains.
Conclusions:  The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC).
Significance and Impact of the study:  Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.