Structural basis of bacterial defense against g-type lysozyme-based innate immunity.

Gram-negative bacteria can produce specific proteinaceous inhibitors to defend themselves against the lytic action of host lysozymes. So far, four different lysozyme inhibitor families have been identified. Here, we report the crystal structure of the Escherichia coli periplasmic lysozyme inhibitor of g-type lysozyme (PliG-Ec) in complex with Atlantic salmon g-type lysozyme (SalG) at a resolution of 0.95 Å, which is exceptionally high for a complex of two proteins. The structure reveals for the first time the mechanism of g-type lysozyme inhibition by the PliG family. The latter contains two specific conserved regions that are essential for its inhibitory activity. The inhibitory complex formation is based on a double 'key-lock' mechanism. The first key-lock element is formed by the insertion of two conserved PliG regions into the active site of the lysozyme. The second element is defined by a distinct pocket of PliG accommodating a lysozyme loop. Computational analysis indicates that this pocket represents a suitable site for small molecule binding, which opens an avenue for the development of novel antibacterial agents that suppress the inhibitory activity of PliG.

Effect of egg washing on the cuticle quality of brown and white table eggs.

Egg washing is currently not permitted within the European Union, with few exceptions. This is mainly because there are concerns that cuticle damage could occur during or after the washing process, as a result of a suboptimal operation. In this study, the cuticle coverage levels of 400 washed or unwashed eggs, derived from either a brown or a white egg-laying flock at the end of lay, were compared. The eggs from older hens inherently have poorer cuticle coverage and as a result arguably constitute a greater risk to consumer safety if they are then washed. Thus, the effects of the washing procedure used in this study on cuticle quality were tested under the worst-case scenario. A standard Swedish egg washing process was used. The cuticle coverage of the eggs was assessed by a colorimeter by quantifying the color difference before and after staining with Tartrazine and Green S. The cuticle of an additional 30 eggs from each of the four groups was then visually assessed by scanning electron microscopy. The staining characteristics of the cuticle varied greatly within each group of eggs and showed that the washing process did not lead to cuticle damage. Scanning electron microscopy confirmed that there was no irreversible damage to the cuticle of the washed eggs and that it was not possible to correctly assign the treatment (washed or not) based on a visual assessment. In conclusion, no evidence could be found to suggest that the washing procedure used in this investigation irreversibly changed the quality of the cuticle.

Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention.

Vibrio anguillarum, also known as Listonella anguillarum, is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. In both aquaculture and larviculture, this disease is responsible for severe economic losses worldwide. Because of its high morbidity and mortality rates, substantial research has been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. This review summarizes the current state of knowledge pertaining to V. anguillarum, focusing on pathogenesis, known virulence factors, diagnosis, prevention and treatment.

Effects on Salmonella shell contamination and trans-shell penetration of coating hens' eggs with chitosan.

Chitosan is a biopolymer with antimicrobial activity and film-forming properties. In this study, the effects on Salmonella shell contamination and trans-shell penetration of coating hens' eggs with chitosan was evaluated. A chitosan was selected from eight types (four non-commercial and four commercial) based on its antimicrobial activity against Salmonella enterica serovar Enteritidis (S. Enteritidis). For this purpose, a contact plate method was developed and chitosans were applied at a concentration of 0.25% (w/v). A commercial type with a molecular weight of 310-375 kDa and a deacetylation degree of 75% that reduced S. Enteritidis by 0.71 log(10) colony forming units compared to the control (without chitosan) was selected for further studies. The chitosan was shown to have antimicrobial activity against other egg borne bacteria, i.e., Acinetobacter baumannii, Alcaligenes sp., Carnobacterium sp., Pseudomonas sp., Serratia marcescens and Staphylococcus warneri, and against S. enterica serovar Typhimurium, Escherichia coli and Listeria monocytogenes. The effects of various concentrations of the selected chitosan (0.25%, 1% and 2%) on Salmonella shell contamination and trans-shell penetration were assessed using the agar molding technique. Effective reduction of eggshell contamination could not be demonstrated, but trans-shell penetration was significantly reduced in the presence of a 2% chitosan eggshell coating, with only 6.1% of the eggs being penetrated compared to 24.5% of the uncoated eggs. It was concluded that the 2% chitosan coating has the potential to reduce contamination of egg contents resulting from trans-shell penetration by S. Enteritidis.

Molecular basis of bacterial defense against host lysozymes: X-ray structures of periplasmic lysozyme inhibitors PliI and PliC.

Lysozymes play a key role in the innate immune system of vertebrates and invertebrates by hydrolyzing peptidoglycan, a vital component of the bacterial cell wall. Gram-negative bacteria produce various types of lysozyme inhibitors that allow them to survive the bactericidal action of lysozyme when their outer membrane is permeabilized. So far, three lysozyme inhibitor families have been described: the Ivy (inhibitor of vertebrate lysozyme) family, the MliC/PliC (membrane-associated/periplasmic lysozyme inhibitor of C-type lysozyme) family, and the PliI (periplasmic lysozyme inhibitor of I-type lysozyme) family. Here, we report high-resolution crystal structures of Salmonella typhimurium PliC (PliC-St) and Aeromonas hydrophila PliI (PliI-Ah). The structure of PliI-Ah is the first in the recently discovered PliI family of lysozyme inhibitors, while the structure of PliC-St is the first structure of a periplasmic lysozyme inhibitor from the PliC/MliC family. Using small-angle X-ray scattering, we demonstrate that both PliC-St and PliI-Ah form stable dimers in solution. The functional dimer architecture of PliC-St is very different from that of the recently described MliC from Pseudomonas aeruginosa (MliC-Pa), despite the close resemblance of their monomers. Furthermore, PliI-Ah has distinctly different monomer and dimer folds compared to PliC, MliC, and Ivy proteins. Site-directed mutagenesis suggests that the inhibitory action of PliI-Ah proceeds via an insertion of a loop containing the conserved SGxY motif into the active center of I-type lysozymes. This motif is related to the functional SGxxY motif found in the MliC/PliC family.

Identification of a bacterial inhibitor against g-type lysozyme.

Lysozymes are antibacterial effectors of the innate immune system in animals that hydrolyze peptidoglycan. Bacteria have evolved protective mechanisms that contribute to lysozyme tolerance such as the production of lysozyme inhibitors, but only inhibitors of chicken (c-) and invertebrate (i-) type lysozyme have been identified. We here report the discovery of a novel Escherichia coli inhibitor specific for goose (g-) type lysozymes, which we designate PliG (periplasmic lysozyme inhibitor of g-type lysozyme). Although it does not inhibit c- or i-type lysozymes, PliG shares a structural sequence motif with the previously described PliI and MliC/PliC lysozyme inhibitor families, suggesting a common ancestry and mode of action. Deletion of pliG increased the sensitivity of E. coli to g-type lysozyme. The existence of inhibitors against all major types of animal lysozyme and their contribution to lysozyme tolerance suggest that lysozyme inhibitors may play a role in bacterial interactions with animal hosts.

Biofilm formation and the food industry, a focus on the bacterial outer surface.

The ability of many bacteria to adhere to surfaces and to form biofilms has major implications in a variety of industries including the food industry, where biofilms create a persistent source of contamination. The formation of a biofilm is determined not only by the nature of the attachment surface, but also by the characteristics of the bacterial cell and by environmental factors. This review focuses on the features of the bacterial cell surface such as flagella, surface appendages and polysaccharides that play a role in this process, in particular for bacteria linked to food-processing environments. In addition, some aspects of the attachment surface, biofilm control and eradication will be highlighted.

Lysozyme inhibitor conferring bacterial tolerance to invertebrate type lysozyme.

Invertebrate (I-) type lysozymes, like all other known lysozymes, are dedicated to the hydrolysis of peptidoglycan, the major bacterial cell wall polymer, thereby contributing to the innate immune system and/or digestive system of invertebrate organisms. Bacteria on the other hand have developed several protective strategies against lysozymes, including the production of periplasmic and/or membrane-bound lysozyme inhibitors. The latter have until now only been described for chicken (C-) type lysozymes. We here report the discovery, purification, identification and characterization of the first bacterial specific I-type lysozyme inhibitor from Aeromonas hydrophila, which we designate PliI (periplasmic lysozyme inhibitor of the I-type lysozyme). PliI has homologs in several proteobacterial genera and contributes to I-type lysozyme tolerance in A. hydrophila in the presence of an outer membrane permeabilizer. These and previous findings on C-type lysozyme inhibitors suggest that bacterial lysozyme inhibitors may have an important function, for example, in bacteria-host interactions.

Inactivation of Escherichia coli and Shigella in acidic fruit and vegetable juices by peroxidase systems.

AIMS: To study the bactericidal properties of the lactoperoxidase (LPER)-thiocyanate and soybean peroxidase (SBP)-thiocyanate systems at low pH, their efficiency for inactivation of Escherichia coli and Shigella in acidic fruit and vegetable juices, their effect on colour stability of the juices and interaction with ascorbic acid. METHODS AND RESULTS: Three-strain cocktails of E. coli and Shigella spp. in selected juices were supplemented with the LPER or SBP system. Within 24 h at 20 degrees C, the LPER system inactivated both cocktails by > or = 5 log10 units in apple, 2-5 log10 units in orange and < or = 1 log10 unit in tomato juices. In the presence of SBP, browning was significant in apple juice and white grape juice, slight in pink grape juice and absent in orange or tomato juice. Ascorbic acid protected E. coli and Shigella against inactivation by the LPER system, and peroxidase systems significantly reduced the ascorbic acid content of juices. CONCLUSIONS: Our results suggest a different specificity of LPER and SBP for SCN-, phenolic substrates of browning and ascorbic acid in acidic juices. The LPER system appeared a more appropriate candidate than the SBP system for biopreservation of juices. SIGNIFICANCE AND IMPACT OF THE STUDY: This work may open perspectives towards the development of LPER or other peroxidases as biopreservatives in acidic foods.

Validation of predictive growth models describing superatmospheric oxygen effects on Pseudomonas fluorescens and Listeria innocua on fresh-cut lettuce.

Two microbial growth models predicting the growth of Pseudomonas fluorescens and Listeria innocua at superatmospheric oxygen and carbon dioxide concentrations at 7 degrees C were validated on fresh-cut butterhead lettuce. Cut lettuce was inoculated with the same strain of L. innocua as the in vitro experiments. The P. fluorescens strain was tagged with a gene encoding green fluorescent protein (GFP) in order to distinguish the inoculated strain from contaminating Pseudomonaceae. Also growth of aerobic mesophilic and lactic acid bacteria was monitored during the experiments. The suggested P. fluorescens model was appropriate to predict growth on cut lettuce. L. innocua on the other hand, grew considerably slower under in vivo circumstances than predicted. CO(2) had a growth promoting effect on L. innocua growing on cut lettuce, whereas in vitro an inhibiting effect was observed. Validation parameters are calculated and hypotheses to explain the discrepancy between predicted and observed growth of L. innocua are provided.