Bacterial behavior in human blood reveals complement evaders with some persister-like features.

Bacterial bloodstream infections (BSI) are a major health concern and can cause up to 40% mortality. Pseudomonas aeruginosa BSI is often of nosocomial origin and is associated with a particularly poor prognosis. The mechanism of bacterial persistence in blood is still largely unknown. Here, we analyzed the behavior of a cohort of clinical and laboratory Pseudomonas aeruginosa strains in human blood. In this specific environment, complement was the main defensive mechanism, acting either by direct bacterial lysis or by opsonophagocytosis, which required recognition by immune cells. We found highly variable survival rates for different strains in blood, whatever their origin, serotype, or the nature of their secreted toxins (ExoS, ExoU or ExlA) and despite their detection by immune cells. We identified and characterized a complement-tolerant subpopulation of bacterial cells that we named "evaders". Evaders shared some features with bacterial persisters, which tolerate antibiotic treatment. Notably, in bi-phasic killing curves, the evaders represented 0.1-0.001% of the initial bacterial load and displayed transient tolerance. However, the evaders are not dormant and require active metabolism to persist in blood. We detected the evaders for five other major human pathogens: Acinetobacter baumannii, Burkholderia multivorans, enteroaggregative Escherichia coli, Klebsiella pneumoniae, and Yersinia enterocolitica. Thus, the evaders could allow the pathogen to persist within the bloodstream, and may be the cause of fatal bacteremia or dissemination, in particular in the absence of effective antibiotic treatments.

Evaluation of the Membrane Damage Mechanism of Chlorogenic Acid against Yersinia enterocolitica and Enterobacter sakazakii and Its Application in the Preservation of Raw Pork and Skim Milk.

Plant-derived antimicrobial agents have adequate antimicrobial effects on food-borne pathogens, which can be used as food preservatives. The purpose of this study was to evaluate the antibacterial mechanism of chlorogenic acid (CA) against Yersinia enterocolitica and Enterobacter sakazakii. The minimum inhibitory concentration (MIC) of CA was determined by employing the broth microdilution method. Then, the cell function and morphological changes of Y. enterocolitica and E. sakazakii treated with CA were characterized. Finally, the growth inhibition models of Y. enterocolitica in raw pork and E. sakazakii in skim milk were constructed through the response surface methodology. The results demonstrated that CA has a satisfactory inhibitory effect against Y. enterocolitica and E. sakazakii with a MIC of 2.5 mg/mL. In addition, CA inhibited the growth of Y. enterocolitica and E. sakazakii via cell membrane damage, such as depolarization of the cell membrane, reduction in intracellular adenosine triphosphate (ATP) and pH levels, and destruction of cell morphology. Moreover, CA reduced two log cycles of Y. enterocolitica in raw pork and E. sakazakii in skim milk at a certain temperature. According to the corresponding findings, CA has the potential to be developed as an effective preservative to control Y. enterocolitica and E. sakazakii-associated foodborne diseases.

Bread Feeding Is a Robust and More Physiological Enteropathogen Administration Method Compared to Oral Gavage.

Oral administration is a preferred model for studying infection by bacterial enteropathogens such as Yersinia spp. In the mouse model, the most frequent method for oral infection consists of oral gavage with a feeding needle directly introduced in the animal stomach via the esophagus. In this study, we compared needle gavage to bread feeding as an alternative mode of bacterial administration. Using bioluminescence-expressing strains of Yersinia pseudotuberculosis and Yersinia enterocolitica, we detected very early upon needle gavage a bioluminescent signal in the neck area together with a signal in the abdominal region, highlighting the presence of two independent sites of bacterial colonization and multiplication. Bacteria were often detected in the esophagus and trachea, as well as in the lymph nodes draining the salivary glands, suggesting that lesions made during needle introduction into the animal oral cavity lead to rapid bacterial draining to proximal lymph nodes. We then tested an alternative mode of bacterial administration using pieces of bread containing bacteria. Upon bread feeding infection, mice exhibited a stronger bioluminescent signal in the abdominal region than with needle gavage, and no signal was detected in the neck area. Moreover, Y. pseudotuberculosis incorporated in the bread is less susceptible to the acidic environment of the stomach and is therefore more efficient in causing intestinal infections. Based on our observations, bread feeding constitutes a natural and more efficient administration method which does not require specialized skills, is less traumatic for the animal, and results in diseases that more closely mimic foodborne intestinal infection.

Antibiofilm activity in the culture supernatant of a marine Pseudomonas sp. bacterium.

In the marine environment, most solid surfaces are covered by microbial biofilms, mainly composed of bacteria and diatoms. The negative effects of biofilms on materials and equipment are numerous and pose a major problem for industry and human activities. Since marine micro-organisms are an important source of bioactive metabolites, it is possible that they synthesize natural ecofriendly molecules that inhibit the adhesion of organisms. In this work, the antibiofilm potential of marine bacteria was investigated using Flavobacterium sp. II2003 as a target. This strain is potentially a pioneer strain of bacteria that was previously selected from marine biofilms for its strong biofilm-forming ability. The culture supernatants of 86 marine heterotrophic bacteria were tested for their ability to inhibit Flavobacterium sp. II2003 biofilm formation and the Pseudomonas sp. IV2006 strain was identified as producing a strong antibiofilm activity. The Pseudomonas sp. IV2006 culture supernatant (SNIV2006) inhibited Flavobacterium sp. II2003 adhesion without killing the bacteria or inhibiting its growth. Moreover, SNIV2006 had no effect on the Flavobacterium sp. II2003 cell surface hydrophilic/hydrophobic and general Lewis acid-base characteristics, but modified the surface properties of glass, making it on the whole more hydrophilic and more alkaline and significantly reducing bacterial cell adhesion. The glass-coating molecules produced by Pseudomonas sp. IV2006 were found to probably be polysaccharides, whereas the antibiofilm molecules contained in SNIV2006 and acting during the 2 h adhesion step on glass and polystyrene surfaces would be proteinaceous. Finally, SNIV2006 exhibited a broad spectrum of antibiofilm activity on other marine bacteria such as Flavobacterium species that are pathogenic for fish, and human pathogens in both the medical environment, such as Staphylococcus aureus and Pseudomonas aeruginosa, and in the food industry, such as Yersinia enterocolitica. Thus, a wide range of applications could be envisaged for the SNIV2006 compounds, both in aquaculture and human health.

Activation of the macroautophagy pathway by Yersinia enterocolitica promotes intracellular multiplication and egress of yersiniae from epithelial cells.

The virulence strategy of pathogenic Yersinia spp. involves cell-invasive as well as phagocytosis-preventing tactics to enable efficient colonisation of the host organism. Enteropathogenic yersiniae display an invasive phenotype in early infection stages, which facilitates penetration of the intestinal mucosa. Here we show that invasion of epithelial cells by Yersinia enterocolitica is followed by intracellular survival and multiplication of a subset of ingested bacteria. The replicating bacteria were enclosed in vacuoles with autophagy-related characteristics, showing phagophore formation, xenophagy, and recruitment of cytoplasmic autophagosomes to the bacteria-containing compartments. The subsequent fusion of these vacuoles with lysosomes and concomitant vesicle acidification were actively blocked by Yersinia. This resulted in increased intracellular proliferation and detectable egress of yersiniae from infected cells. Notably, deficiency of the core autophagy machinery component FIP200 impaired the development of autophagic features at Yersinia-containing vacuoles as well as intracellular replication and release of bacteria to the extracellular environment. These results suggest that Y. enterocolitica may take advantage of the macroautophagy pathway in epithelial cells to create an autophagosomal niche that supports intracellular bacterial survival, replication, and, eventually, spread of the bacteria from infected cells.

Endogenous hepcidin and its agonist mediate resistance to selected infections by clearing non-transferrin-bound iron.

The iron-regulatory hormone hepcidin is induced early in infection, causing iron sequestration in macrophages and decreased plasma iron; this is proposed to limit the replication of extracellular microbes, but could also promote infection with macrophage-tropic pathogens. The mechanisms by which hepcidin and hypoferremia modulate host defense, and the spectrum of microbes affected, are poorly understood. Using mouse models, we show that hepcidin was selectively protective against siderophilic extracellular pathogens (Yersinia enterocolitica O9) by controlling non-transferrin-bound iron (NTBI) rather than iron-transferrin concentration. NTBI promoted the rapid growth of siderophilic but not nonsiderophilic bacteria in mice with either genetic or iatrogenic iron overload and in human plasma. Hepcidin or iron loading did not affect other key components of innate immunity, did not indiscriminately promote intracellular infections (Mycobacterium tuberculosis), and had no effect on extracellular nonsiderophilic Y enterocolitica O8 or Staphylococcus aureus Hepcidin analogs may be useful for treatment of siderophilic infections.

Aporphinoid alkaloids as antimicrobial agents against Yersinia enterocolitica.

Foodborne diseases have become a health issue worldwide, mainly due to the consumption of contaminated foods that are either raw, improperly heat treated or cross-contaminated after adequate heat treatment foods. A group of alkaloids extracted from plants were tested to evaluate their antimicrobial effect against different strains of Yersinia enterocolitica and other foodborne bacteria. The results obtained reveal that oliveridine and pachypodanthine inhibited Y. enterocolitica growth, with MIC values of 25 µmol l-1 and 100 µmol l-1 respectively. The results indicated that both alkaloids are good growth inhibitors, but oliveridine showed greater inhibitory effect with lower MIC values. Inhibitory alkaloids can be developed as potential antimicrobials in food system to prevent or treat foodborne diseases, thus contributing to solve the global issue of contaminated food consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: Alkaloids are abundant secondary metabolites in plants and represent one of the most widespread class of compounds endowed with multiple and varied pharmacological properties. In this work, we propose two aporphinoid alkaloids extracted from plants as new antimicrobial agents. Oliveridine and pachypodanthine inhibited Yersinia enterocolitica growth for up to 96 h of culture. This is the first reported study of the activity of these alkaloids as antimicrobial compounds.

KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate.

Uronates are charged sugars that form the basis of two abundant sources of biomass-pectin and alginate-found in the cell walls of terrestrial plants and marine algae, respectively. These polysaccharides represent an important source of carbon to those organisms with the machinery to degrade them. The microbial pathways of pectin and alginate metabolism are well studied and essentially parallel; in both cases, unsaturated monouronates are produced and processed into the key metabolite 2-keto-3-deoxygluconate (KDG). The enzymes required to catalyze each step have been identified within pectinolytic and alginolytic microbes; yet the function of a small ORF, kdgF, which cooccurs with the genes for these enzymes, is unknown. Here we show that KdgF catalyzes the conversion of pectin- and alginate-derived 4,5-unsaturated monouronates to linear ketonized forms, a step in uronate metabolism that was previously thought to occur spontaneously. Using enzyme assays, NMR, mutagenesis, and deletion of kdgF, we show that KdgF proteins from both pectinolytic and alginolytic bacteria catalyze the ketonization of unsaturated monouronates and contribute to efficient production of KDG. We also report the X-ray crystal structures of two KdgF proteins and propose a mechanism for catalysis. The discovery of the function of KdgF fills a 50-y-old gap in the knowledge of uronate metabolism. Our findings have implications not only for the understanding of an important metabolic pathway, but also the role of pectinolysis in plant-pathogen virulence and the growing interest in the use of pectin and alginate as feedstocks for biofuel production.

Acetoacetate and ethyl acetoacetate as novel inhibitors of bacterial biofilm.

Acetoacetate (AAA) was identified as a biofilm inhibitor in a previous study, where the effect of 190 carbon and nitrogen sources on biofilm amounts by Escherichia coli O157:H7 was determined. With this study, we tested the effect of AAA on growth and biofilm amounts of Cronobacter sakazakii, Serratia marcescens and Yersinia enterocolitica. AAA reduced growth and biofilm amounts of the three pathogens, albeit at rather high concentrations of 10 to 35 mg ml-1 . Acetoacetate at a concentration of 5 mg ml-1 reduced Y. enterocolitica mRNA transcripts of the flagellar master regulator operon flhD, the invasion gene inv, and the adhesion gene yadA. Transcription of the regulator of plasmid-encoded virulence genes virF, the plasmid-encoded virulence gene yopQ, and ymoA were largely unaffected by AAA. Importantly, AAA did not cause an increase in transcription of any of the tested virulence genes. As a more cost efficient homologue of AAA, the effect of ethyl acetoacetate (EAA) was tested. EAA reduced growth, biofilm amounts and live bacterial cell counts up to 3 logs. IC50 values ranged from 0·31 mg ml-1 to 5·6 mg ml-1 . In summary, both AAA and EAA inhibit biofilm, but EAA appears to be more effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial biofilms are communities of bacteria that form on surfaces and are extremely difficult to remove by conventional physical or chemical techniques, antibiotics or the human immune system. Despite advanced technologies, biofilm still contributes to 60 to 80% of human bacterial infections (NIH and CDC) and cause problems in many natural, environmental, bioindustrial or food processing settings. The discovery of novel substances that inhibit biofilm without increasing the virulence of the bacteria opens doors for countless applications where a reduction of biofilm is desired.

Development of a rapid method for the detection of Yersinia enterocolitica serotype O:8 from food.

In this study, a new and alternative method based on monoclonal antibodies (MAbs) for the rapid detection of Yersinia enterocolitica O:8 was developed. This microorganism is an emerging foodborne pathogen causing gastrointestinal disease in humans. The transmission can occur through contaminated food such as raw or undercooked meat, milk and dairy products, water and fresh vegetables. Nine MAbs (46F7, 54B11, 54C11, 62D10, 64C7, 64C10, 72E8, 72E10, 72G6) were characterized and selected versus Y. enterocolitica O:8, and only 2 of them showed also a weak cross-reaction with Campylobacter jejuni. The MAb 54B11 was used for the development of Y. enterocolitica capture-ELISA in food matrices, i.e. meat and dairy products (n = 132). The method was validated by ISO 16140:2003 and compared with the official method for the detection of presumptive pathogenic Y. enterocolitica (ISO 10273:2003). Relative accuracy, sensitivity and specificity corresponded to 100%. The selectivity was evaluated on other food samples (n = 126) showing a lower confidence limit of 90.3% and an upper confidence limit of 100%. The results from this study demonstrated that the developed method was rapid and cheap, specific and sensitive for the screening of the pathogen in food.

Evaluation of the antimicrobial efficacy of neutral electrolyzed water on pork products and the formation of viable but nonculturable (VBNC) pathogens.

The goals of this study were to evaluate the antimicrobial efficacy of Neutral electrolyzed oxidizing (NEO) water on E. coli O157:H7, Salmonella Enteritidis and Yersinia enterocolitica in both pure culture and on inoculated pork chops and skin samples, and to investigate the formation of viable but nonculturable (VBNC) pathogens after treatments. Both the plate count method and flow cytometry were used to evaluate antimicrobial efficacy on pure cultures. Different concentrations of NEO water were prepared by diluting the original NEO water (100%) with sterilized deionized water. The antimicrobial efficacy increased as the concentrations of NEO water increased. The flow cytometry results showed that treating with diluted NEO water led to the formation of VBNC cells. No VBNC cells formed when treating pure cultures with 50% or 100% NEO water. Yersinia cultures were found to be more resistant to NEO treatments than Salmonella and E. coli O157:H7 cultures, with Yersinia cultures showing lower reductions and higher levels of VBNC cells after treatments. The antimicrobial efficacy of NEO water was significantly better on skin samples than on pork chops. The differences in protein content and structure between pork chops and skin samples serve as major factors impacting the NEO water's efficacy.

Control of human pathogenic Yersinia enterocolitica in minced meat: Comparative analysis of different interventions using a risk assessment approach.

This study aimed to evaluate the effect of different processing scenarios along the farm-to-fork chain on the contamination of minced pork with human pathogenic Y. enterocolitica. A modular process risk model (MPRM) was used to perform the assessment of the concentrations of pathogenic Y. enterocolitica in minced meat produced in industrial meat processing plants. The model described the production of minced pork starting from the contamination of pig carcasses with pathogenic Y. enterocolitica just before chilling. The endpoints of the assessment were (i) the proportion of 0.5 kg minced meat packages that contained pathogenic Y. enterocolitica and (ii) the proportion of 0.5 kg minced meat packages that contained more than 10³ pathogenic Y. enterocolitica at the end of storage, just before consumption of raw pork or preparation. Comparing alternative scenarios to the baseline model showed that the initial contamination and different decontamination procedures of carcasses have an important effect on the proportion of highly contaminated minced meat packages at the end of storage. The addition of pork cheeks and minimal quantities of tonsillar tissue into minced meat also had a large effect on the endpoint estimate. Finally, storage time and temperature at consumer level strongly influenced the number of highly contaminated packages.

N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils.

Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution.

Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica.

Many bacterial pathogens subvert mammalian type IA phosphoinositide 3-kinase (PI3K) in order to induce their internalization into host cells. How PI3K promotes internalization is not well understood. Also unclear is whether type IA PI3K affects different pathogens through similar or distinct mechanisms. Here, we performed an RNA interference (RNAi)-based screen to identify components of the type IA PI3K pathway involved in invasin-mediated entry of Yersinia enterocolitica, an enteropathogen that causes enteritis and lymphadenitis. The 69 genes targeted encode known upstream regulators or downstream effectors of PI3K. A similar RNAi screen was previously performed with the food-borne bacterium Listeria monocytogenes The results of the screen with Y. enterocolitica indicate that at least nine members of the PI3K pathway are needed for invasin-mediated entry. Several of these proteins, including centaurin-α1, Dock180, focal adhesion kinase (FAK), Grp1, LL5α, LL5ß, and PLD2 (phospholipase D2), were recruited to sites of entry. In addition, centaurin-α1, FAK, PLD2, and mTOR were required for remodeling of the actin cytoskeleton during entry. Six of the human proteins affecting invasin-dependent internalization also promote InlB-mediated entry of L. monocytogenes Our results identify several host proteins that mediate invasin-induced effects on the actin cytoskeleton and indicate that a subset of PI3K pathway components promote internalization of both Y. enterocolitica and L. monocytogenes.

Antagonistic activities of some Bifidobacterium sp. strains isolated from resident infant gastrointestinal microbiota on Gram-negative enteric pathogens.

The gastrointestinal microbiota contributes to the consolidation of the anti-infectious barrier against enteric pathogens. The purpose of this study was to investigate the influence of Bifidobacterium sp. strains, recently isolated from infant gastrointestinal microbiota on the in vitro growth and virulence features expression of enteropathogenic bacterial strains. The antibacterial activity of twelve Bifidobacterium sp. strains isolated from human feces was examined in vitro against a wide range of Gram negative pathogenic strains isolated from 30 infant patients (3 days to 5 years old) with diarrhea. Both potential probiotic strains (Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium catenulatum, Bifidobacterium breve, Bifidobacterium ruminantium) and enteropathogenic strains (EPEC, EIEC, Klebsiella pneumoniae, Salmonella sp., Yersinia enterocolitica, Pseudomonas aeruginosa) were identified by MALDI-TOF and confirmed serologically when needed. The bactericidal activity, growth curve, adherence to the cellular HEp-2 substratum and production of soluble virulence factors have been assessed in the presence of different Bifidobacterium sp. cultures and fractions (whole culture and free-cell supernatants). Among the twelve Bifidobacterium sp. strains, the largest spectrum of antimicrobial activity against 9 of the 18 enteropathogenic strains was revealed for a B. breve strain recently isolated from infant intestinal feces. The whole culture and free-cell supernatant of B. breve culture decreased the multiplication rate, shortened the log phase and the total duration of the growth curve, with an earlier entrance in the decline phase and inhibited the adherence capacity to a cellular substratum and the swimming/swarming motility too. These results indicate the significant probiotic potential of the B. breve strain.

Transcriptomic Analysis of Yersinia enterocolitica Biovar 1B Infecting Murine Macrophages Reveals New Mechanisms of Extracellular and Intracellular Survival.

Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocolitica biovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26 °C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37 °C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

Absence of YbeY RNase compromises the growth and enhances the virulence plasmid gene expression of Yersinia enterocolitica O:3.

YbeY was recently recognized as an endoribonuclease playing a role in ribosome biosynthesis. In Escherichia coli it functions as a single-strand-specific RNase that processes the 3' end of the 16S rRNA and is crucial for the late-stage 70S ribosome quality control system. Here we report that YbeY is not essential in Yersinia enterocolitica serotype O:3, yet its absence strongly compromised the bacterium. The lack of YbeY resulted in misprocessing of 16S rRNA and a severe decrease of growth rate with complete growth arrest observed at elevated temperatures. Moreover, a ybeY mutation severely disturbed regulation of the Yersinia virulence plasmid (pYV) genes and affected the expression of regulatory small RNA species. Transcription of the pYV genes was upregulated in the ybeY mutant at 22 °C; the same genes were repressed in the wild-type bacterium. Furthermore, ybeY inactivation impaired many virulence-related features, such as resistance to elevated temperature and acid, and hindered utilization of different carbohydrates. In addition, the ybeY mutant strain showed decreased infectivity in a tissue culture infection model, especially at the stage of cell adhesion. Taken together, this study demonstrates the crucial role of YbeY in Y. enterocolitica O:3 physiology and pathogenicity.

Interaction of human mannose-binding lectin (MBL) with Yersinia enterocolitica lipopolysaccharide.

The lipopolysaccharide (LPS) is involved in the interaction between Gram-negative pathogenic bacteria and host. Mannose-binding lectin (MBL), complement-activating soluble pattern-recognition receptor targets microbial glycoconjugates, including LPS. We studied its interactions with a set of Yersinia enterocolitica O:3 LPS mutants. The wild-type strain LPS consists of lipid A (LA) substituted with an inner core oligosaccharide (IC) which in turn is substituted either with the O-specific polysaccharide (OPS) or the outer core hexasaccharide (OC), and sometimes also with the enterobacterial common antigen (ECA). The LPS mutants produced truncated LPS, missing OPS, OC or both, or, in addition, different IC constituents or ECA. MBL bound to LA-IC, LA-IC-OPS and LA-IC-ECA but not LA-IC-OC structures. Moreover, LA-IC substitution with both OPS and ECA prevented the lectin binding. Sequential truncation of the IC heptoses demonstrated that the MBL targets the IC heptose region. Furthermore, microbial growth temperature influenced MBL binding; binding was stronger to bacteria grown at room temperature (22°C) than to bacteria grown at 37°C. In conclusion, our results demonstrate that MBL can interact with Y. enterocolitica LPS, however, the in vivo significance of that interaction remains to be elucidated.

Yersinia adhesin A (YadA)--beauty & beast.

The trimeric autotransporter adhesin Yersinia adhesin A is the prototype of the type Vc secretion systems. It is expressed by enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains, but not by Yersinia pestis. A characteristic trait of YadA is its modular composition and trimeric nature. YadA consists of an N-terminal passenger domain which is exposed on the bacterial cell surface. The translocation of this passenger onto the surface is facilitated by a C-terminal ß-barrel domain which concomitantly anchors YadA into the outer membrane with three YadA monomers contributing to the formation of a single ß-barrel. In Y. enterocolitica, but not Y. pseudotuberculosis, YadA is a decisive virulence factor and its deletion renders the bacteria virtually avirulent in mouse models of infection. This striking importance of YadA in infection may derive from its manifold functions in host cell interaction. Presumably the most important function of YadA is that it mediates adhesion to extracellular matrix components of eukaryotic host cells. Only tight adhesion allows for the injection of "anti-host" effector proteins via a type III secretion system into the host cell cytosol. These effector proteins enable Yersinia to subvert the host immune system in order to replicate and establish infection. YadA is also essential for the survival of Y. enterocolitica upon contact with serum, an important immune-evasion mechanism called serum resistance. To this end, YadA interacts with several components of the host complement system, the first line of immune defense. This review will summarize recent findings about the structure and biogenesis of YadA and its interactions with the host complement system.

Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria.

Lactobacilli are normal microflora of the gastrointestinal (GI) tract and are a heterogeneous group of lactic acid bacteria (LAB). Lactobacillus strains with Probiotic activity may have health Benefits for human. This study investigates the probiotic potential of Lactobacillus strains obtained from the feces of healthy infants and also explores antibacterial activity of Lactobacillus strains with probiotic potential against enteropathogenic bacteria. Fecal samples were collected from 95 healthy infants younger than 18 months. Two hundred and ninety Lactobacillus strains were isolated and assessed for probiotic potential properties including ability to survive in gastrointestinal conditions (pH 2.0, 0.3% oxgall), adherence to HT-29 cells and antibiotic resistance. Six strains including Lactobacillus fermentum (4 strains), Lactobacillus paracasei and Lactobacillus plantarum showed good probiotic potential and inhibited the growth of enteropathogenic bacteria including ETEC H10407, Shigella flexneri ATCC 12022, Shigella sonnei ATCC 9290, Salmonella enteritidis H7 and Yersinia enterocolitica ATCC 23715. These Lactobacillus strains with probiotic potential may be useful for prevention or treatment of diarrhea, but further in vitro and in vivo studies on these strains are still required.