[Show abstract][Hide abstract] ABSTRACT: Dairy products, in particular soft cheeses, pose a major concern to the dairy industry and public health authorities as they are the leading source of listeriosis outbreaks, a severe foodborne infection affecting pregnant women, children, elderly and immunocompromised people, with a high (20–30%) mortality rate. Cheeses offer a suitable environment for the survival and growth of Listeria monocytogenes, allowing this pathogen to display tolerance responses that can favour its presence in cheese and persistence in dairy processing plants. Extensive food safety regulations in the EU towards prevention of contamination of dairy products with L. monocytogenes have been implemented. However due to the specific abilities of this pathogen to overcome the processing hurdles, its control remains a challenge. Compliance with the Good Manufacturing Practices, observation of Hazard Analysis Critical Control Points (HACCP) and the surveillance of the pathogen in the cheese processing environment are crucial to provide consumers with a safe product. This review aims to provide an overview on the current knowledge about the potential for the transmission of L. monocytogenes in cheese and its abilities to overcome the challenging processing conditions and implications for the behaviour of the pathogen in the host.
Food Research International 11/2014; · 3.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gastrointestinal system poses different stresses to the foodborne pathogen, Listeria monocytogenes, including the low pH of the stomach and the presence of bile and the high osmolality of the intestinal fluid. The present study evaluated how previous exposure of three L. monocytogenes dairy isolates (C882 and T8, serovar 4b isolates and A9 serovar 1/2a or 3b isolate) to a cheese-simulated medium (pH5.5 and 3.5% NaCl [w/v], adapted cultures) affected subsequent survival in a simulated gastrointestinal system. Listerial cultures exposed to the cheese-simulated medium at pH7.0, with no added NaCl, were considered non-adapted. To investigate the main events involved in listerial survival during the gastric and intestinal subsequent challenge, a proteomic approach was used. All L. monocytogenes strains were able to survive the deleterious effects of the gastrointestinal fluids and no significant differences were observed between adapted and non-adapted cells. However the L. monocytogenes strains showed a different protein pattern in response to the gastrointestinal stress. Data indicated that synthesis of stress related proteins is more pronounced in non-adapted cells. Although, a significant number of enzymes involved in glycolysis and energy production were also consistently over-produced by the three strains. These findings provided new insights into the means used by L. monocytogenes to overcome the gastrointestinal system and allow the pathogen to move to the next phase of the infectious process.
International journal of food microbiology 03/2013; 163(2-3):51-63. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conditions of electroporation to transform encapsulated strains of Streptococcus pneumoniae with plasmid DNA have been defined. For a heavily encapsulated strain, an electroporation solution of 10–20% (v/v) glycerol and 3.2 kV cm-1 field strength, 1000 Ω resistance and 25 μF capacitance were optimal. For lightly encapsulated or non-encapsulated strains, optimal conditions were a sucrose-based electroporation solution and 12.5 kV cm-1 field strength, 200 Ω resistance and 25 μF capacitance.
[Show abstract][Hide abstract] ABSTRACT: During food processing, and particularly in cheese manufacturing processes, Listeria monocytogenes may be exposed routinely to environments of low pH or high salt concentration. It has been suggested that these environmental conditions may contribute to bacterial adherence to abiotic surfaces and increased resistance to disinfection. In this study strains isolated from the environment of artisanal cheese-making dairies were used to investigate the behaviour of L. monocytogenes in response to acid and salt stress and clear differences between strains was observed. In planktonic culture, strains varied in resistance to low pH or high NaCl concentration and in the occurrence of an adaptive response to moderate acid or NaCl. There was dislocation in responses to salt and acid. Strains resistant, or adaptive, to acid were not resistant or adaptive to NaCl. The reverse also was observed. Exposure to moderate acid did not promote adherence to polystyrene but survival, at low pH or high NaCl concentration, of cells adherent to stainless steel was increased, even for strains that had no adaptive response planktonically, but the detail of these observations varied between strains. In contrast to acid adaptation, with some strains salt adaptation enhanced adherence of L. monocytogenes to polystyrene but this was not true for all strains. For some strains salt- or acid adaptation may enhance the survival of sessile cells exposed to hypochlorite disinfection.
International Journal of Food Microbiology 04/2008; 123(1-2):142-50. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have shown previously that surfactant protein D (SP-D) binds and agglutinates Streptococcus pneumoniae in vitro. In this study, the role of SP-D in innate immunity against S. pneumoniae was investigated in vivo, by comparing the outcome of intranasal infection in surfactant protein D deficient (SP-D-/-) to wildtype mice (SP-D+/+). Deficiency of SP-D was associated with enhanced colonisation and infection of the upper and lower respiratory tract and earlier onset and longer persistence of bacteraemia. Recruitment of neutrophils to inflammatory sites in the lung was similar in both strains mice in the first 24 hrs post-infection, but different by 48 hrs. T cell influx was greatly enhanced in SP-D-/- mice as compared to SP-D+/+ mice. Our data provides evidence that SP-D has a significant role to play in the clearance of pneumococci during the early stages of infection in both pulmonary sites and blood.
Respiratory research 02/2005; 6:126. · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diseases caused by Streptococcus pneumoniae include pneumonia, septicaemia and meningitis. All these are associated with high morbidity and mortality. The pneumococcus can colonize the nasopharynx, and this can be a prelude to bronchopneumonia and invasion of the vasculature space. Proliferation in the blood can result in a breach of the blood-brain barrier and entry into the cerebrospinal fluid (CSF) where the bacteria cause inflammation of the meningeal membranes resulting in meningitis. The infected host may develop septicaemia and/or meningitis secondary to bronchopneumonia. Also septicaemia is a common precursor of meningitis. The mechanisms surrounding the sequence of infection are unknown, but will be dependent on the properties of both the host and bacterium. Treatment of these diseases with antibiotics leads to clearance of the bacteria from the infected tissues, but the bacteriolytic nature of antibiotics leads to an acute release of bacterial toxins and thus after antibiotic therapy the patients can be left with organ-specific deficits. One of the main toxins released from pneumococci is the membrane pore forming toxin pneumolysin. Here we review the extensive studies on the role of pneumolysin in the pathogenesis of pneumococcal diseases.
[Show abstract][Hide abstract] ABSTRACT: The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied. Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2-3.5% (w/v) sodium chloride, also were studied. The effect of temperature on pH and sodium chloride sensitivity was also determined. The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data. Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant. A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride. All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported. Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride. Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5). The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride). In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.
International Journal of Food Microbiology 08/2003; 84(2):207-16. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the adhesion of Listeria monocytogenes 10403S to 17 different, food-use approved materials representing metals, rubbers and polymers.
Adhesion assays were conducted by placing 'coupons' of the materials in planktonic cultures at 30 degrees C, and then immediately withdrawing them ('short contact') or leaving them submerged in the cultures for 2 h. Adherent cells were recovered by sonication. In the short contact experiments, the logarithm of the mean viable counts ranged from 3.67 +/- 0.43 to 4.78 +/- 0.38. After 2 h contact time, the numbers of adherent cells had increased significantly for all materials with the exception of polypropylene. The highest count (6.33 +/- 0.31) recorded was for stainless steel 405.
Adhesion to a wide range of materials was time-dependent and characterized by reversible and irreversible stages.
Adhesion test protocols must account for cell carry-over and cells which are only weakly bound. Material selection may only have a limited role in reducing food contamination by listeria.
Journal of Applied Microbiology 07/2001; 90(6):1000-5. · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pneumococcus is the principle cause of bacterial pneumonia and also a major cause of bacterial meningitis. The mechanisms and sites of pneumococcal adherence and invasion of the respiratory tract in vivo are not clear however. We have made pneumococci expressing green fluorescent protein (GFP) and used it to trace pneumococcal adherence and invasion in vivo. By using GFP pneumococci we have shown bacterial adherence and invasion of broncho-epithelial cells in vivo by 4 h post-infection, with increases in pneumococcal invasiveness by 24 h. Using confocal image analysis we have shown varying levels of pneumococcal penetration and internalisation into host cells, as well as translocation through epithelial layers. To our knowledge this is the first report of pneumococcal invasion and cellular translocation in vivo.
[Show abstract][Hide abstract] ABSTRACT: From a panel of nine inbred mice strains intranasally infected with Streptococcus pneumoniae type 2 strain, BALB/c mice were resistant and CBA/Ca and SJL mice were susceptible to infection. Further investigation revealed that BALB/c mice were able to prevent proliferation of pneumococci in the lungs and blood, whereas CBA/Ca mice showed no bacterial clearance. Rapidly increasing numbers of bacteria in the blood was a feature of CBA/Ca but not BALB/c mice. In the lungs, BALB/c mice recruited significantly more neutrophils than CBA/Ca mice at 12 and 24 h postinfection. Inflammatory lesions in BALB/c mice were visible much earlier than in CBA/Ca mice, and there was a greater cellular infiltration into the lung tissue of BALB/c mice at the earlier time points. Our data suggest that resistance or susceptibility to intranasal pneumococci may have an association with recruitment and/or function of neutrophils.
Infection and Immunity 02/2001; 69(1):426-34. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.
Journal of Biological Chemistry 02/2001; 276(8):5714-9. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this paper we describe reconstructions by electron cryo-microscopy of two oligomeric states of the pore-forming toxin pneumolysin. The results are interpreted by the fitting of atomic models of separated domains to the 3-dimensional electron density maps, revealing two steps in the mechanism of pore formation by the family of cholesterol-binding toxins. We briefly describe the observation of the toxin pore in model membranes and contrast the apparent mechanism of pneumolysin with that of other pore-forming toxins.
International Journal of Medical Microbiology 11/2000; 290(4-5):389-94. · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae was shown to contain two types of superoxide dismutase, MnSOD and FeSOD. Levels of MnSOD increased during growth in an aerobic environment. The sodA gene, encoding MnSOD, of virulent S. pneumoniae type 2 strain D39 was inactivated to give mutant D39HY1. Aerobically, D39HY1 had a lower growth rate than the wild type and exhibited susceptibility to the redox-active compound paraquat, but anaerobic growth of D39HY1 was identical to that of the wild type. Virulence studies showed that the median survival time of mice infected intranasally with D39HY1 was significantly longer than that of mice infected with the wild-type pneumococcus. In contrast to the wild type, D39HY1 did not multiply in lungs during the first 24 h but thereafter grew at the same rate as the wild type. Appearance in the bloodstream was also delayed, but growth in the blood was unimpaired by the sodA mutation. The pattern of inflammation in lungs infected with D39HY1 differed from that in wild-type-infected mice. After infection with D39HY1, neutrophils were densely packed around bronchioles, in contrast to the wild-type infection, where neutrophils were more diffusely localized.
Infection and Immunity 06/2000; 68(5):2819-26. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H(2)O(2)) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 10(8) CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H(2)O(2) at a concentration as low as 100 microM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H(2)O(2) (10 microM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H(2)O(2) to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H(2)O(2), and in the presence of 10(8) CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H(2)O(2) release from the brain slice. Coincubation with catalase converted the H(2)O(2) produced by the pneumococci to H(2)O. Penicillin-induced lysis of bacteria dramatically reduced H(2)O(2) production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H(2)O(2), which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H(2)O(2) released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H(2)O(2)-induced cell toxicity, indicating a role for H(2)O(2) in the response. There is also a slight additive effect of pneumolysin and H(2)O(2) on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.
Infection and Immunity 04/2000; 68(3):1557-62. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although there is substantial evidence that pneumolysin is an important virulence factor in pneumococcal pneumonia, relatively little is known about how it influences cellular infiltration into the lungs. We investigated how the inability of mutant pneumococci to produce pneumolysin altered the pattern of inflammation and cellular infiltration into the lungs. The effect on bacterial growth in the lungs also was assessed. There were three phases of growth of wild-type bacteria in the lungs: a decline followed by a rapid increase and then stasis or decline. The absence of pneumolysin was associated with a more rapid early decline and then a much slower increase in numbers. The pattern of inflammatory-cell accumulation also had distinct stages, and the timing of these stages was influenced by the presence of pneumolysin. Neutrophils began to accumulate about 12 to 16 h after infection with wild-type pneumococci. This accumulation occurred after the early decline in pneumococcal numbers but coincided with the period of rapid growth. Following infection with pneumococci unable to make pneumolysin, neutrophil influx was slower and less intense. Coincident with the third stage of pneumococcal growth was an accumulation of T and B lymphocytes at the sites of inflammation, but the accumulation was not associated with an increase in the total number of lymphocytes in the lungs. Lymphocyte accumulation in the absence of pneumolysin occurred but was delayed.
Infection and Immunity 03/2000; 68(2):492-501. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Densely ciliated ependymal cells cover the ventricular surface of the brain and cerebral aqueducts separating cerebrospinal fluid, which is infected in meningitis, from neuronal tissue. We have established an ex vivo model that allows measurement of ependymal ciliary beat frequency, using high-speed video analysis, during incubation with bacterial toxins. Ciliated ependyma, from Wistar rats, was exposed to the pneumococcal toxin, pneumolysin, and a mutant form with markedly reduced cytotoxic activity (;0.1%). Wild-type pneumolysin (1500 HU/ml and 150 HU/ml: 10 and 1 microg/ml) caused rapid ciliary stasis (30-150 s), sloughing of cilia and cytoplasmic extrusion. Ciliary slowing before stasis was seen at 15 HU/ml (0.1 microg/ml); however, no effect on ciliary beat frequency was seen at lower concentrations (1.5 HU/ml and 0.15 HU/ml: 0.01 and 0.001 microg/ml). Mutant pneumolysin, 99.9% deficient in haemolytic activity, caused rapid ciliary stasis at 10 microg/ml but no effect was seen at lower concentrations (1-0.1 microg/ml). Pneumolysin, at levels which may be produced during severe pneumococcal meningitis, may cause rapid ependymal ciliary stasis.
[Show abstract][Hide abstract] ABSTRACT: Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation. Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.
Journal of Molecular Biology 12/1999; 293(5):1145-60. · 3.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.