Enterobacterial Infection Research Articles

A two-step activation mechanism enables mast cells to differentiate their response between extracellular and invasive enterobacterial infection

Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human mast cells triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

Implementing Alfred60AST in a clinical lab: Clinical impact on the management of septic patients and financial analysis

Sepsis is an important cause of morbidity and mortality. An accelerated microbiology diagnosis is crucial in order to reduce the time to initiate targeted antibiotic therapy. The Alfred60AST system is able to provide phenotypic Antimicrobial Susceptibility Testing (AST) results within hours. This study has two objectives: assess the clinical impact of this technology and determine its cost-effectiveness. During a ten-week period, all new enterobacterial or enterococcal bloodstream infection was analyzed with the Alfred60AST system, in parallel with routine methods. Its impact on the clinician's therapeutic strategy was studied. In order to assess the financial and practical aspects of the method, an analysis of the extracosts and a survey of the technical staff were conducted. Fifty-three cases of bacteriemia were included. For the Enterobacteriaceae bacteriemias, a clinical impact was shown in 18.9% of the cases (e.g, treatment modification). The financial analysis highlighted an increase in costs (+38% for Enterobacteriaceae, +50% for Enterococci), compared to the theoretical costs reported by the firm, due to the workflow and the volumes of samples used. Finally, results of the technical staff survey were favorable in terms of ease of use of the system. In addition to its ease of use, the Alfred60AST system is able to provide an AST in a record time. This study shows a real interest of the technique in the therapeutic management of patients with enterobacterial sepsis. However, its routine implementation requires an increase of the analyzed volumes as well as a 24/7 organization of the laboratory in order to be profitable.

Antimicrobial peptide expression in the cockroach gut during enterobacterial infection is specific and influenced by type III secretion.

ABSTRACTOmnivorous synanthropic cockroaches, such as the German cockroach (Blattella germanica), are reservoirs and vectors of enteric bacterial pathogens. A lifestyle conducive to frequent encounters with high loads of diverse bacteria may have led to the evolution of unique innate immune systems in these insects. The innate immune response of insects relies largely on generalized mechanisms to sense and eliminate foreign microbes. However, analyses of the genomes of common synanthropic cockroaches previously revealed a repertoire of pathogen associated molecular pattern (PAMP) receptors and antimicrobial peptides (AMPs) that is significantly expanded relative to most holometabolous insect models and vectors, supporting the intriguing possibility that cockroaches may encode enhanced recognition within their immune system and may possess an enhanced capacity to fine tune innate immune responses. Investigating how cockroaches respond to infection with enterobacteria provides the opportunity to expand our fundamental knowledge of the regulation of insect innate immunity in a context that is biologically and medically relevant. German cockroaches can harbor both Salmonella enterica serovar Typhimurium and Escherichia coli in their gut without experiencing pathogenesis. The former colonizes the gut and replicates while the latter persists only transiently. We hypothesized that differences in the innate immune response may contribute to or result from the difference in infection dynamics between the two enterobacteria. To test this hypothesis, we used qRT-PCR to analyze expression of five genes encoding representative AMPs (Attacins, Blattellicin, Defensins) in the gut of German cockroaches 1 and 24 h after ingestion of live or heat-killed enterobacteria. We found that robust AMP expression was induced in response to ingestion of a live wild-type strain of S. Typhimurium, but not in response to live E. coli, heat-killed S. Typhimurium, or a live mutant strain of S. Typhimurium lacking type III secretion systems. These results indicate that the cockroach immune system does not respond to stimulation with high levels of ingested bacterial PAMPs such as peptidoglycan. Rather, AMP expression in the gut appears to be induced by active bacterial colonization involving type III secretion. We speculate that this form of regulation may have evolved to prevent over activation of the immune system from frequent ingestion of innocuous, non-colonizing, or non-viable bacteria. While additional work is needed to delineate the molecular mechanisms underlying our observations, our findings provide significant novel insight into the immunological adaptation of cockroaches to life in septic environments as well as the factors that regulate bacterial pathogen transmission by these insects.

Pathogen-specific risk of seizure in children with moderate-to-severe diarrhoea: Case control study with follow-up.

To determine the pathogen-specific risk of seizure in under-five children hospitalised with moderate-to-severe diarrhoea (MSD) in rural settings. This was a prospective case-control study with follow-up, conducted in a sentinel facility of Global Enteric Multicenter Study in Mirzapur, a rural community of Bangladesh between 2007 and 2010. Children aged 0-59months who presented with MSD and seizure constituted the cases whereas those who did not have seizure comprised the controls. MSD was defined if the episodes were associated with dehydration or dysentery or required hospitalisation with diarrhoea or dysentery. All enrolled children were followed up at home within 50-90days of enrolment. A total of 64 cases and 128 randomly selected controls formed the analysable dataset. The result of logistic regression analysis after adjusting for potential confounders revealed that shigellosis (Shigella species, OR=5.34, 95% CI=2.37-12.04) particularly S. flexneri (OR=3.34, 95% CI=1.48-7.57), S. flexneri 6 (OR=23.24, 95% CI=2.79-193.85), S. sonnei (OR=6.90, 95% CI=2.34-19.85); norovirus (OR=6.77, 95% CI=1.69-27.11), fever (OR=16.75, 95% CI=1.81-154.70) and loss of consciousness (OR=35.25, 95% CI=1.71-726.20) were the independent risk factors for seizure in MSD children. At enrolment, cases had lower WHZ (P=0.006) compared to their peers, follow-up anthropometrics showed significant improvement in WHZ (P<0.001) and WAZ (P<0.05), whereas deterioration in HAZ (P<0.001) in both cases and controls. Childhood MSD episodes particularly due to Shigella and norovirus are often associated with seizure. Prompt identification and appropriate management of children with shigellosis may reduce occurrence and adverse consequences of seizure linked with MSD.

Two Cases of Meningitis Associated with Gram Negative Bacteremiae and Strongyloidiasis

Background: Strongyloidiasis is a neglected tropical disease and is widely distributed. HTLV-1 coinfection is frequent, and this disease increases the risk of hyper infestation. Disseminated strongyloidiasis often causes severe enterobacteria infection. Case Presentation: We report the cases of two patients presenting with meningial syndrome. The meningitis was associated with a Gram negative bacteremiae. The investigations performed proved an infection or a previous contact with Strongyloides stercoralis. The infectious outcome was favorable after antibiotic treatment and antiparasitic treatment but one of them was also co-infected with HTLV-1 and died after a lymphoma occurred. Discussion: S. stercoralis infects the intestinal mucosa, and chronic infection of this pathogen induces inflammation of the intestinal mucosa. Enteric bacteria can gain systemic access and be responsible of organ involvement, including Central Nervous System. HTLV-1 infection increases the prevalence of strongyloidiasis, the rate of treatment failure, and the risk of hyperinfestation. Conclusions: Strongyloidiasis should not be underdiagnosed, as a specific treatment with antiparasitic drugs is available. We should perform direct microscopy of the stool to detect S. stercoralis in patients who develop severe enterobacterial infection when patients are from an endemic area.

Enterobacterial infection in Saudi Arabia: First record of Klebsiella pneumoniae with triple carbapenemase genes resistance.

Carbapenemase producing Enterobacteriaceae are emerging as important pathogens worldwide with serious effects on patients' outcome. The study aimed to investigate the emergence of carbapenemases associated with enterobacterial infection in Western region of Saudi Arabia. Clinical isolates from suspected patients with enterobacterial infection were investigated over a one-year period from four tertiary care hospitals of Makkah, Saudi Arabia. All isolates were identified using Vitek-2 system and then screened for potential carbapenemase production using disk diffusion test. Suspected isolates with reduced susceptibility to carbapenems were further investigated for blaNDM-1, blaKPC and blaOXA-48 resistant genes. Out of 120 confirmed Enterobacteriaceae isolates, Klebsiella pneumoniae and Escherichia coli comprised the largest proportion (35% and 34.2%, respectively) of encountered infections. Twenty-six (21.7%) isolates showed resistance to carbapenems, the majority of which (21/26) were K. pneumoniae. Remarkably, 17 isolates carried triple resistant genes KPC/NDM-1/OXA-48 while the other 4 carried double resistant genes (KPC/OXA-48) or (NDM-1/OXA-48). The current study revealed that the mentioned triple resistance genes have the higher incidence with significant association risk among males (COR 4.5; CI: 1.9-17.3; P = 0.018), non-Saudi nationalities (COR 4.9; CI: 1.5-19.3; P = 0.003), ICU-obtained specimens (COR 3.6; CI: 1.5-8.4; P = 0.002) and blood specimens (COR 2.8; CI: 1.1-6.9; P = 0.02). Multidrug-resistant Enterobacteriaceae isolates in particular K. pneumoniae co-harboring KPC, NDM-1 and OXA-48 genes are emerging in Western region, Saudi Arabia. This is the first record of triple carbapenemase genes co-producing K. pneumoniae associated with enterobacterial infection.

New pathogen-specific immunoPET/MR tracer for molecular imaging of a systemic bacterial infection

The specific and rapid detection of Enterobacteriaceae, the most frequent cause of gram-negative bacterial infections in humans, remains a major challenge. We developed a non-invasive method to rapidly detect systemic Yersinia enterocolitica infections using immunoPET (antibody-targeted positron emission tomography) with [64Cu]NODAGA-labeled Yersinia-specific polyclonal antibodies targeting the outer membrane protein YadA. In contrast to the tracer [18F]FDG, [64Cu]NODAGA-YadA uptake co-localized in a dose dependent manner with bacterial lesions of Yersinia-infected mice, as detected by magnetic resonance (MR) imaging. This was accompanied by elevated uptake of [64Cu]NODAGA-YadA in infected tissues, in ex vivo biodistribution studies, whereas reduced uptake was observed following blocking with unlabeled anti-YadA antibody. We show, for the first time, a bacteria-specific, antibody-based, in vivo imaging method for the diagnosis of a Gram-negative enterobacterial infection as a proof of concept, which may provide new insights into pathogen-host interactions.

The Antibacterial Activity of Silver Nanoparticles Produced in the Plant Sesamum indicum Seed Extract: A Green Method Against Multi-Drug Resistant Escherichia coli

Background: The nanoparticles synthesis through biological processes is evolving a new era of research interests in nanotechnology. In usual methods silver nanoparticles are synthesized through chemical methods, having extremely toxic and flammable natures. Objectives: The aim of the present study was to synthesize silver nanoparticles, through the green method of utilizing Sesamum indicum (S. indicum) extract and to determine the potential antibacterial effects of the product against multi-drug resistant Escherichia coli (E. coli). Materials and Methods: The formation and characterization of AgNPs (silver nanoparticles) were confirmed by UV-vis spectroscopy, energy-dispersive spectroscopy (EDX), X-ray diffraction (XRD) and transmission electron microscope (TEM). All 30 strains of E. coli were isolated from urine cultures of hospitalized patients (Amir Al-Momenin Hospital, Zabol, South-Eastern Iran) with urinary tract infection, 2011-2012. The minimum inhibitory (MIC) concentrations were investigated by microdilution method. Results: The results showed that isolated E. coli were resistant to four different antimicrobial agents including ceftazidime (26.6%), cefixime (40%), tetracycline (63.3%) and erythromycin (56.6%). The highest MIC value for produced nano silver in S. indicum seed extract, was 200 ppm, against five isolates of E. coli. Conclusions: Considering the sufficient antimicrobial activities of nanoparticles tested in this study, they are suggested for enterobacterial infection treatment, especially in hospital environment.

Stability of orally administered immunoglobulin in the gastrointestinal tract

Oral administration of immunoglobulin in the colostrum or egg yolk has been considered an effective tool for preventing enterobacterial infection via passive immunization. During this process, the transmission and residence of the active immunoglobulin are the most important conditions for successful protection. We investigated the stability of encapsulated colostrum and egg yolk immunoglobulin for the effective transmission of immunoglobulin in the gastrointestinal (GI) tract. First, we measured GI transit time. Contrast media passed through and reached the stomach within 10min, the small intestine within 3.5h, and the cecum within 5h. Both the encapsulated colostrum containing anti-hepatitis A virus (HAV) antibody (IgG) and egg yolk with anti-rotavirus antibody (IgY) showed lower antibody activity than the non-encapsulated colostrum did in the stomach after administration; however, significantly higher antibody activities were observed in the encapsulated groups than in the non-encapsulated groups in the small intestine 3.5h after the administration. In the large intestine, the antibody activities of the encapsulated groups were maintained or slightly increased in a time-dependent manner; however, the titers of each non-capsulated control were as low as the negative controls. Therefore, this encapsulation is considered a useful tool for the delivery of active antibody through the GI tract.

Regulation of Toll-like Receptor 5 Gene Expression and Function on Mucosal Dendritic Cells

Toll-like receptor (TLR) 5 has been shown to maintain intestinal homeostasis and regulate host defense against enterobacterial infection. However, how TLR5 expression is regulated and its function in the intestine have not been fully elucidated. Here we demonstrate that mucosal dendritic cells (DCs), but not splenic DCs, express high levels of TLR5 protein. Alternatively spliced Tlr5 transcripts were identified but it did not explain the selective expression of TLR5 on mucosal DCs. Treatment with various bacterial ligands downregulated BMDC TLR5 expression, while retinoic acid and host stromal cell-derived signals promoted TLR5 expression in a TGF-β-independent mechanism. Signaling through TLR5 restrained regulatory T (Treg) cell generation, and accordingly, TLR5−/− mice displayed increased frequencies of Foxp3+ Treg cells in the intestinal lamina propria. Our data indicate that bacterial and host factors differentially regulate DC TLR5 expression. TLR5 signaling regulates immune responses towards the microbiota via modulation of the Treg/effector T cell balance.

Infection-Induced Intestinal Oxidative Stress Triggers Organ-to-Organ Immunological Communication in Drosophila

Local infections can trigger immune responses in distant organs, and this interorgan immunological crosstalk helps maintain immune homeostasis. We find that enterobacterial infection or chemically and genetically stimulating reactive oxygen species (ROS)-induced stress responses in the Drosophila gut triggers global antimicrobial peptide (AMP) responses in the fat body, a major immune organ in flies. ROS stress induces nitric oxide (NO) production in the gut, which triggers production of the AMP Diptericin, but not Drosomycin, in the fat body. Hemocytes serve as a signaling relay for communication between intestinal ROS/NO signaling and fat body AMP responses. The induction of AMP responses requires Rel/NF-κB activation within the fat body. Although Rel-mediated Drosomycin induction is repressed by the AP-1 transcription factor, this repressor activity is inhibited by intestinal ROS. Thus, intestinal ROS signaling plays an important role in initiating gut-to-fat body immunological communication in Drosophila.

Analysis of Salmonella enteritidis Outer Membrane Proteins and Lipopolysaccharide Profiles with the Detection of Immune Dominant Proteins

Problem statement: Salmonella infection is a serious medical and veterinary problem world-wide and causes great concern in the human health and food safety. Salmonella enteritidis outer membrane proteins and lipopolysaccharide play important role in the virulence and immunological properties of bacteria. Prophylaxis of enterobacterial infection has proven difficult to achieve. Vaccination is an effective tool for the prevention of Salmonella infection. Approach: Therefore, a study was conducted on S. enteritidis isolates from chicken meat samples for Outer Membrane Proteins (OMPs) and Lipopolysaccharide (LPS) profile were analyzed by SDS-PAGE electrophoresis and immunodominent antigen was detected by western blot analysis. Results: S. enteritidis with different OMPs bands were exhibited with molecular weight ranging from 5-90 kDa. The major outer membrane protein profiles of all S. enteritidis isolates were homogenous with different expression in intensity of protein was observed. LPS of different S. enteritidis isolates exhibited doublet band was observed and identified as R type of strains. The immunoblotting results at 14.4 and 24 KDa proteins were good immunogen. Conclusion: The 14.4 and 24 KDa proteins were immune response protein. This proteins can use for vaccine development.

Immune responses of TLR5+ lamina propria dendritic cells in enterobacterial infection

Toll-like receptors (TLRs) recognize distinct microbial components and induce innate immune responses. TLR5 has been shown to recognize bacterial flagellin. Unlike other TLRs, TLR5 is not expressed on conventional dendritic cells or macrophages. By contrast, TLR5 is mainly expressed on intestinal CD11c(+) lamina propria cells (LPCs), which do not express TLR4. These cells detect pathogenic bacteria and secreted proinflammatory cytokines, mainly in a TLR5-dependent manner. Notably, transport of pathogenic Salmonella typhimurium from the intestinal tract to mesenteric lymph nodes was impaired in Tlr5 (-/-) mice, suggesting that CD11c(+) LPCs expressing TLR5 are used by S. typhimurium for systemic infection. CD11c(+) LPCs consist of four subsets distinguished by differential expression patterns of CD11c and CD11b. CD11c(hi)CD11b(hi) LPDCs have been identified as TLR5-expressing cells. In response to flagellin, these LPDCs induce the differentiation of naive B cells into IgA(+) plasma cells via a mechanism independent of gut-associated lymphoid tissue (GALT), and trigger the differentiation of antigen-specific Th17 and Th1 cells. These LPDCs have unique properties in that they can induce acquired immunity as well as innate immunity via TLR5 in the intestine.

Extended-spectrum β-lactamase (ESBL)-producing enterobacteria: factors associated with infection in the community setting, Auckland, New Zealand

We aimed to document the epidemiology of extended-spectrum beta-lactamase (ESBL)-producing enterobacteria in the Auckland community and identify factors associated with infection using a case-control study design. ESBL-producing enterobacteria were isolated from 107 infected patients, for which demographic and clinical data were available for 98 cases (92%). Escherichia coli was the predominant organism (82%), with urine as the commonest source (97%). Compared with a control group infected with ESBL-negative enterobacteria, factors significantly associated with infection on univariate analysis were: living in a residential care home (RCH); recent admission to hospital 'M'; recent antibiotic use; older age (>75 years); presence of a urinary catheter; and a history of comorbid chronic obstructive pulmonary disease (COPD), cardiovascular disease, neurological disease or recurrent urinary tract infection. On multivariate analysis, residence in RCH and COPD remained significant associations. Pulsed-field gel electrophoresis typing of the ESBL-producing E. coli identified a common strain. We concluded that residence in RCH and a history of COPD are significant associations with ESBL-producing enterobacterial infection in the Auckland community. Several spatial clusters in RCHs and a common strain suggest point-source outbreaks. A substantial number of community cases did not live in an RCH nor had been recently hospitalised, suggesting the independent generation of ESBL-producing enterobacteria in the broader community.

Control of Pseudomonas aeruginosa Skin Infections in Mice Is Mast Cell-Dependent

Mast cells (MCs) have recently been shown to be essential for the elicitation of efficient immune responses in murine sepsis. To explore whether MCs also contribute to the control of bacterial skin infections, we studied skin lesions induced by Pseudomonas aeruginosa (PA) in genetically MC-deficient Kit(W)/Kit(W-v) mice, normal Kit(+/+) mice, and MC-reconstituted Kit(W)/Kit(W-v) mice. PA injections resulted in strikingly (>2-fold) larger skin lesions in Kit(W)/Kit(W-v) mice than in Kit(+/+) mice, which exhibited pronounced MC degranulation at infection sites. In addition, neutrophil recruitment following PA injections and bacterial clearance from sites of infection was significantly impaired in Kit(W)/Kit(W-v) mice compared with Kit(+/+) mice. Notably, the adoptive transfer of MCs to the skin of Kit(W)/Kit(W-v) mice before PA infection resulted in normal neutrophil accumulation as well as skin lesions comparable with those in Kit(+/+) mice in both bacterial burden and size. These findings demonstrate for the first time that activated MCs are crucial for the induction of protective innate immune responses to bacterial skin infections.

HLA-B27-restricted T cells from patients with ankylosing spondylitis recognize peptides from B*2705 that are similar to bacteria-derived peptides.

Ankylosing spondylitis (AS) is an inflammatory systemic disease affecting the spine, sacroiliacal and peripheral joints. Although the aetiology of AS remains unknown, the strong association with the HLA-B27 allele might reflect directly a detrimental effect of the HLA-B27 molecule itself, resulting from its potential capability to present 'arthritogenic' peptides to CD8+ T cells. Because some forms of SpA are triggered by enterobacterial infection, such arthritogenic peptides might originate from autologous and/or bacterial proteins triggering cross-reactive CD8+ T cell clones. Intriguingly, two peptides from the second extracellular domain of HLA-B*2705 share sequence homologies with several enterobacterial antigens, exhibit the HLA-B27-binding-motif, and are presented by HLA-B*2705 itself. The objective of this study was to examine the clonal T cell reactivity against these peptides in patients with AS. To this end, we screened peripheral blood lymphocytes (PBL) of 26 patients with AS and 24 healthy donors for TNF-alpha-producing cells using ELISPOT assays. PBL and synovial fluid-derived lymphocytes (SFL) of peptide-responsive patients were then stimulated and cultured with the relevant peptide and control peptides in vitro. Antigen-specific T cell lines (TCL) were identified by standard chromium release assays. Clonal analysis was performed subsequently applying TCRB-CDR3 spectratyping. Among eight peptides tested, only the HLA-B27 168-176 peptide LRRYLENGK was recognized by PBL from B27+ AS patients but not from B27+ healthy controls (P=0.001). LRRYLENGK-specific T cell clones used preferentially the TCRBV5S1 and the BV14 segment. These results suggest that an HLA-B27-derived peptide with homology to bacterial peptides may play a role in AS.

Mast cell modulation of the innate immune response to enterobacterial infection.

Although mast cells have been associated with many biological responses (e.g. inflammation, tissue repair and angiogenesis) and clinical conditions (e.g. rheumatoid arthritis, interstitial cystitis, scleroderma, psoriasis, neurofibromatosis and Crohn’s disease), they remain one of the least understood cells of the immune system. By virtue of their high affinity IgE/F ceRI-receptors and their intrinsic capacity to release large amounts of inflammatory mediators, mast cells are primary known as major effector cells of allergy or type 1 hypersensitivity reactions (Marone et al 1997). In spite of these deleterious properties, mast cells have been preserved through evolution even among the lowest orders of animals indicating that these cells must be serving a valuable function in the body. A number of factors suggest a role for mast cells in modulating the innate immune response against bacteria. These include the fact that mast cells are present in large numbers under the epithelia of various mucosal surfaces and skin. They also have the intrinsic capacity to mobilize a rapid and vigorous inflammatory response in the host upon activation and mast cells have already been shown to contribute to certain aspects of the IgE-mediated adaptive immune response to parasites (Levy &

Quinolones et fluoroquinolones dans l'univers bactérien

Due to their narrow range of activity which is limited to Gram-negative bacteria and to their specific pharmacokinetic properties which result in high urinary concentrations, the first-generation quinolones, the most important of which is nalidixic acid, have basically remained drugs for the treatment of urinary infections in the adult with enterobacterial infection. During the 1980s the fluoroquinolones were introduced for use as therapeutic drugs, in particular pefloxacin, ofloxacin, and ciprofloxacin, which are active against staphylococci and to a certain extent against Pseudomonas aeruginosa. The 1990s were marked by the appearance of new fluoroquinolones which had developed an activity at low concentrations against pneumococci, including both those that were sensitive and resistant to penicillin G, thereby permitting their range to be extended to the treatment of respiratory infections. In the past 40 years, the quinolone group has increased and gained ground over the bacterial environment. The latter has developed defense mechanisms, i.e., bacterial resistance. The role of the practitioner is to survey the use of these new antimicrobial agents so that their therapeutic effect is maintained or even reinforced. The essential condition for this is respect for good medical practice. As in all things, it is abuse rather than judicious use that creates problems.

Protective roles of mast cells against enterobacterial infection are mediated by Toll-like receptor 4.

Toll-like receptors (TLRs) are mammalian homologues of the Drosophila Toll receptors and are thought to have roles in innate recognition of bacteria. We demonstrated that TLR 2, 4, 6, and 8 but not TLR5 were expressed on mouse bone marrow-derived mast cells (BMMCs). Using BMMCs from the genetically TLR4-mutated strain C3H/HeJ, we demonstrated that functional TLR4 was required for a full responsiveness of BMMCs to produce inflammatory cytokines (IL-1beta, TNF-alpha, IL-6, and IL-13) by LPS stimulation. TLR4-mediated stimulation of mast cells by LPS was followed by activation of NF-kappaB but not by stress-activated protein kinase/c-Jun NH2-terminal kinase signaling. In addition, in the cecal ligation and puncture-induced acute septic peritonitis model, we demonstrated that genetically mast cell-deficient W/W(v) mice that were reconstituted with TLR4-mutated BMMCs had significantly higher mortality than W/W(v) mice reconstituted with TLR4-intact BMMCs. Higher mortality of TLR4-mutated BMMC-reconstituted W/W(v) mice was well correlated with defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Taken together, these observations provide definitive evidence that mast cells play important roles in exerting the innate immunity by releasing inflammatory cytokines and recruitment of neutrophils after recognition of enterobacteria through TLR4 on mast cells.

A Study of the Incidence and Significance of Intramammary Enterobacterial Infections Acquired During the Dry Period

We assessed the incidence of enterobacterial infection of the mammary glands of 629 cows, from six commercial herds in Somerset, during the nonlactating period; samples were collected from all clinical quarters of these cows during the subsequent lactation. A rise in the incidence of intramammary enterobacterial infection was detected between drying off and before calving. Quarters infected with an enterobacterial organism during the dry period were more likely to develop mastitis due to that pathogen than were uninfected quarters. Of all enterobacterial mastitis occurring in the first 100 d of lactation, 52.6% arose in quarters previously infected, during the dry period, with the same strain of bacteria, as identified by DNA fingerprinting using enterobacterial repetitive intergenic consensus primers. When compared with unsampled controls, quarters sampled during the dry period did not show a higher incidence of infection at calving or of subsequent clinical mastitis. These findings suggest that chronic infections are important in the epidemiology of enterobacterial mastitis and that environmental management during the dry period may greatly impact the incidence of enterobacterial mastitis in the subsequent lactation.