Critical roles of NLRP3 inflammasome in IL-1ß secretion induced by Corynebacterium pseudotuberculosis in vitro.

Corynebacterium pseudotuberculosis is a prominent human and animal pathogen causing chronic inflammatory diseases. Interleukin-1ß (IL-1ß) is involved in the response to such pathogenic infections. However, the mechanism by which IL-1ß is secreted during C. pseudotuberculosis infection remains unclear. This study aimed to investigate the mechanism underlying IL-1ß secretion by macrophages infected with C. pseudotuberculosis. Herein, we firstly revealed that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 (Casp1) play critical roles in IL-1ß secretion rather than IL-1ß precursor (pro-IL-1ß) expression in C. pseudotuberculosis-infected macrophages. Toll like receptor 4 (TLR4) is partially involved in IL-1ß secretion, while absent in melanoma 2 (AIM2) is not involved in IL-1ß secretion by C. pseudotuberculosis-infected macrophages. In addition, nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinases (p38 MAPK) inhibitors almost attenuated IL-1ß secretion, implying that NF-κB and p38MAPK pathway are involved in IL-1ß secretion in C. pseudotuberculosis-infected macrophages. Furthermore, C. pseudotuberculosis were significantly more numerous in Nlrp3-/-, Asc-/-, and Casp-1-/- macrophages than in WT macrophages at 24 h after infection (P < 0.05), indicating that NLRP3 inflammasome components limit C. pseudotuberculosis replication in macrophages. Together, these data provide novel insights into the mechanisms underlying IL-1ß secretion in C. pseudotuberculosis-infected macrophages and further the current understanding of the host pro-inflammatory immune response against this pathogen.

Oxytetracycline concentrations in interstitial fluid from tissue chambers inoculated with Corynebacterium pseudotuberculosis after intramuscular or intrachamber administration in sheep.

OBJECTIVE: To determine oxytetracycline concentrations in plasma and in fluid from Corynebacterium pseudotuberculosis (CPT)-inoculated tissue chambers (used as experimental abscess models) and uninoculated (control) tissue chambers in sheep after IM or local administration of the drug and to investigate whether CPT growth was reduced or eliminated by these treatments. ANIMALS: 10 clinically normal female sheep. PROCEDURES: Sterile tissue chambers were surgically implanted in both paralumbar fossae of each sheep; ≥ 2 weeks later (day -6), 1 randomly selected chamber was inoculated with CPT, and the opposite chamber was injected with sterile growth medium. Sheep received oxytetracycline IM (n = 5) or by percutaneous injection into CPT-inoculated (4) or uninoculated (1) chambers on day 0. Tissue fluid from each chamber and venous blood samples for plasma collection were obtained at predetermined times over 6 days for bacterial counts (tissue chambers) and analysis of oxytetracycline concentrations (tissue chambers and plasma). Sheep were euthanized on day 6. Regional lymph nodes were collected bilaterally from each sheep for culture. RESULTS: Measurable concentrations of oxytetracycline were present in each chamber throughout the study, regardless of administration route or presence of CPT. No CPT growth was detected after the 48-hour time point in inoculated chambers injected with oxytetracycline; however, CPT was isolated from all inoculated chambers throughout the study after IM drug administration. One regional lymph node (ipsilateral to a CPT-inoculated, oxytetracycline-injected chamber with no CPT growth after 48 hours) was culture positive for CPT. CONCLUSIONS AND CLINICAL RELEVANCE: Intralesional administration of oxytetracycline may eliminate growth of CPT locally, but complete elimination of the organism remains difficult.

Encefalopatía hiperamoniémica secundaria a infección urinaria por germen productor de ureasa: Caso clínico pediátrico / Hyperammonemic encephalopathy due to urinary tract infection by urea splitting bacteria: A pediatric case report

El incremento del amonio en sangre, hiperamoniemia, es pasible de provocar compromiso neurológico al atravesar la barrera hematoencefálica. La causa más frecuente y conocida de hiperamoniemia es la alteración en la función hepática. Sin embargo, se deben considerar otras patologías, de menor frecuencia y poco conocidas. La infección del tracto urinario por gérmenes productores de ureasa debe ser contemplada a pesar de ser infrecuente en pediatría. Se reporta el caso de un niño con encefalopatía aguda grave, con niveles elevados de amonio en sangre, en quien, luego de descartar otros diagnósticos diferenciales, se asumió el cuadro como hiperamoniemia secundaria a infección del tracto urinario por Corynebacterium riegelii, un germen productor de ureasa. Se implementaron medidas generales de tratamiento para la encefalopatía hiperamoniémica y tratamiento antibiótico específico, con buena evolución el paciente.

Elevated level of ammonia in the blood, defined as hyperammonemia, is feasible to cause neurological symptoms when crossing the blood-brain barrier. The most frequent and studied cause of hyperammonemia is liver failure. Nevertheless, other less frequent and known etiologies must be considered. Urinary tract infection caused by urea-splitting bacteria, despite being unusual in pediatric patients, must be taken into account. We report a pediatric patient with severe acute encephalopathy and high levels of ammonia in blood. After ruling out other causes of hyperammonemia, it was assumed secondary to urinary tract infection by Corynebacterium riegelii, a ureasplitting bacteria. General treatment for hyperammonemic encephalopathy was established, as well as specific treatment with antibiotics. The patient evolved favorably.

[Hyperammonemic encephalopathy due to urinary tract infection by urea splitting bacteria. A pediatric case report]. / Encefalopatía hiperamoniémica secundaria a infección urinaria por germen productor de ureasa. Caso clínico pediátrico.

Elevated level of ammonia in the blood, defined as hyperammonemia, is feasible to cause neurological symptoms when crossing the blood-brain barrier. The most frequent and studied cause of hyperammonemia is liver failure. Nevertheless, other less frequent and known etiologies must be considered. Urinary tract infection caused by urea-splitting bacteria, despite being unusual in pediatric patients, must be taken into account. We report a pediatric patient with severe acute encephalopathy and high levels of ammonia in blood. After ruling out other causes of hyperammonemia, it was assumed secondary to urinary tract infection by Corynebacterium riegelii, a ureasplitting bacteria. General treatment for hyperammonemic encephalopathy was established, as well as specific treatment with antibiotics. The patient evolved favorably.

El incremento del amonio en sangre, hiperamoniemia, es pasible de provocar compromiso neurológico al atravesar la barrera hematoencefálica. La causa más frecuente y conocida de hiperamoniemia es la alteración en la función hepática. Sin embargo, se deben considerar otras patologías, de menor frecuencia y poco conocidas. La infección del tracto urinario por gérmenes productores de ureasa debe ser contemplada a pesar de ser infrecuente en pediatría. Se reporta el caso de un niño con encefalopatía aguda grave, con niveles elevados de amonio en sangre, en quien, luego de descartar otros diagnósticos diferenciales, se asumió el cuadro como hiperamoniemia secundaria a infección del tracto urinario por Corynebacterium riegelii, un germen productor de ureasa. Se implementaron medidas generales de tratamiento para la encefalopatía hiperamoniémica y tratamiento antibiótico específico, con buena evolución el paciente.

[PATHOGENICITY FACTORS OF CORYNEBACTERIUM NON DIPHTHERIAE].

Pathogenicity factors of Corynebacterium non diphtheriae - pili, microcapsule, cell wall, pathogenicity enzymes, toxins, that determine the ability of microorganisms to consequentially interact with epithelium of entry gates of the organism, replicate in vivo, overcome cell and hu- moral mechanisms of protection, are examined in the review. Particular attention in the paper is given to species of non-diphtheria corynebacteria, that are pathogenic for human and able to produce toxins - Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. Mechanisms of expression regulation of PLD-exotoxins, its interaction with immune system cells are described.

Proteomic characterization of a natural host-pathogen interaction: repertoire of in vivo expressed bacterial and host surface-associated proteins.

Interactions between a host and a bacterial pathogen are mediated by cross-talk between molecules present on, or secreted by, pathogens and host binding-molecules. Identifying proteins involved at this interface would provide substantial insights into this interaction. Although numerous studies have examined in vitro models of infection at the level of transcriptional change and proteomic profiling, there is virtually no information available on naturally occurring host-pathogen interactions in vivo. We employed membrane shaving to identify peptide fragments cleaved from surface-expressed bacterial proteins and also detected proteins originating from the infected host. We optimized this technique for media-cultured Corynebacterium pseudotuberculosis, a sheep pathogen, revealing a set of 247 surface proteins. We then studied a natural host-pathogen interaction by performing membrane shaving on C. pseudotuberculosis harvested directly from naturally infected sheep lymph nodes. Thirty-one bacterial surface proteins were identified, including 13 not identified in culture media, suggesting that a different surface protein repertoire is expressed in this hostile environment. Forty-nine host proteins were identified, including immune mediators and antimicrobial peptides such as cathelicidin. This novel application of proteolytic shaving has documented sets of host and pathogen proteins present at the bacterial surface in an infection of the native host.

Serological proteome analysis of Corynebacterium pseudotuberculosis isolated from different hosts reveals novel candidates for prophylactics to control caseous lymphadenitis.

Caseous lymphadenitis (CLA) is a highly prevalent disease in goats and sheep worldwide, which is caused by Corynebacterium pseudotuberculosis. Although several prophylactic methods against CLA have been proposed previously, the identification of new C. pseudotuberculosis proteins that are really produced during the infectious process is still needed to improve efficiency and accuracy in vaccines and diagnostics. In this study, we used optimized conditions for serological proteome analysis (SERPA) in order to identify new immune-reactive proteins in C. pseudotuberculosis culture supernatants of two strains, 1002 and C231, isolated from goats and sheep, respectively. Using a sheep and goat serum pool, 13 novel immune-reactive exoproteins common to the two strains were identified. Four of these proteins present known functions and were already described as immune-reactive proteins in other microorganisms, whereas the other nine are of unknown function and show low similarity with proteins from other bacterial species. These data reveal promising targets for immunoprophylactic methods against CLA.

Behavioral and immune responses to infection require Gαq- RhoA signaling in C. elegans.

Following pathogen infection the hosts' nervous and immune systems react with coordinated responses to the danger. A key question is how the neuronal and immune responses to pathogens are coordinated, are there common signaling pathways used by both responses? Using C. elegans we show that infection by pathogenic strains of M. nematophilum, but not exposure to avirulent strains, triggers behavioral and immune responses both of which require a conserved Gαq-RhoGEF Trio-Rho signaling pathway. Upon infection signaling by the Gαq pathway within cholinergic motorneurons is necessary and sufficient to increase release of the neurotransmitter acetylcholine and increase locomotion rates and these behavioral changes result in C. elegans leaving lawns of M. nematophilum. In the immune response to infection signaling by the Gαq pathway within rectal epithelial cells is necessary and sufficient to cause changes in cell morphology resulting in tail swelling that limits the infection. These Gαq mediated behavioral and immune responses to infection are separate, act in a cell autonomous fashion and activation of this pathway in the appropriate cells can trigger these responses in the absence of infection. Within the rectal epithelium the Gαq signaling pathway cooperates with a Ras signaling pathway to activate a Raf-ERK-MAPK pathway to trigger the cell morphology changes, whereas in motorneurons Gαq signaling triggers behavioral responses independent of Ras signaling. Thus, a conserved Gαq pathway cooperates with cell specific factors in the nervous and immune systems to produce appropriate responses to pathogen. Thus, our data suggests that ligands for Gq coupled receptors are likely to be part of the signals generated in response to M. nematophilum infection.

Splenic amyloidosis: a rare cause of spontaneous splenic rupture.

A 62 year-old woman who presented with an atraumatic acute abdomen was discovered to have haemoperitoneum with splenic rupture on urgent computed tomography and was immediately referred for life-saving emergency splenectomy. Histopathological examination revealed secondary splenic amyloidosis. The patient was later found to be suffering from infective endocarditis secondary to her permanent cardiac pacemaker. This report describes a patient who could have suffered from a long-standing infected vegetation on a permanent cardiac pacemaker, which led to splenic amyloidosis and spontaneous splenic rupture.

Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis.

Caseous lymphadenitis (CLA) of sheep, caused by Corynebacterium pseudotuberculosis, has been a significant disease in the majority of sheep-rearing regions for over a century. Because of the chronic and often sub-clinical nature of the infection, it has proved difficult to control and prevalence is high in many parts of the world, which in turn leads to significant economic losses for farmers. This review describes the important characteristics of C. pseudotuberculosis and examines the pathogenesis and epidemiology of the infection in sheep. The review also discusses the immune response to infection and describes the methods that have been developed to control CLA, with particular emphasis on the use of vaccination and serological testing.

Successful treatment of Corynebacterium urealyticum encrusted cystitis: a case report and literature review.

Encrusted cystitis is a very rare chronic inflammatory disease of the bladder characterized by precipitation and incrustation of phosphate and ammonium-magnesium salts on the vescical mucosa, caused by urinary infection due to urolithic microorganisms. Corynebacterium urealyticum or Corynebacterium group D2, a multiple antibiotic-resistant urea-splitting bacterium, is the most frequently incriminated aetiology. We report a case of a 57-year-old man affected by systemic erythematosus lupus with a long history of dysuria and suprapubic pain who underwent percutaneous nephrostomy drainage with urethral stenting for lupoid obstructive uropathy. Before the diagnosis of encrusted cystitis by Corynebacterium urealyticum was established, the patient underwent five cystoscopies to remove the plaques and multiple unsuccessful antibiotic treatment courses. Eventually the infection was definitively cured after a two-week course with intramuscular teicoplanin.

Upregulation of galectin-3 by Corynebacterium kutscheri infection in the rat lung.

Corynebacterium (C) kutscheri and Staphylococcus aureus were isolated from two Sprague-Dawley (SD) rats with a hemisected spinal cord. Grossly, gray-white bulging foci and abscesses were distributed throughout the parenchyma of the lung. Pathologically, severe necrotizing lobar pneumonia with abscesses and fibrinous pleuritis were observed. Immunohistochemical analysis found accumulation of galectin-3 in alveolar macrophages and the alveolar interstitial region. No other viral or bacterial pathogens were detected in these animals. In addition, similar pathogenic changes and accumulation of galectin-3 were observed in the lungs of SD rats experimentally infected with C. kutscheri. Using northern blot analysis, the relative galectin-3 and GAPDH mRNA levels were 4.6 to 9.3 times higher in C. kutscheri-infected lung than in uninfected controls. These results demonstrate that a single C. kutscheri infection can induce the upregulation of galectin-3 in the lung and that this molecule may have an important pathogenic role in C. kutscheri infections in rats.

Bacterial conjunctivitis in Muc1 null mice.

PURPOSE: In contrast to wild-type mice, genetically engineered Mucin1 (Muc1) null animals display a marked propensity for development of blepharitis and conjunctivitis. Molecular approaches confirmed the presence of Muc1 mRNA and protein in the conjunctival tissue of wild-type mice and identified the bacterial species in Muc1 null symptomatic mice. METHODS: Muc1 null animals housed in a conventional facility were examined for visually apparent inflammation of the eye and surrounding tissue. Blood taken from overtly affected animals was assayed for antibodies to common murine viral agents. Swabs of infected eyes and whole eye preparations were used to detect and speciate bacterial pathogens. Frozen sections of whole eye, lid margin, and Harderian gland were immunostained with antibodies to Muc1 and cytokeratin 14, both epithelial cell markers. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were performed on RNA isolated from conjunctiva and Harderian gland of wild-type mice to compare relative levels of transcript. RESULTS: Student's unpaired t-test performed on the eye inflammation frequency of Muc1 null mice confirmed a statistical significance (P < 0.01) when compared to wild-type background animals housed in the same room. Analysis of blood samples from affected Muc1 null animals detected no common murine viral pathogens. Bacterial analysis of conjunctival swabs and whole eye preparations demonstrated the presence of coagulase-negative Staphylococcus, Streptococcus type alpha, and Corynebacterium group G2. Muc1 antibody staining of wild-type sections revealed the presence of Muc1 on conjunctival goblet and non-goblet cells and on the epithelium of the Harderian gland. Serial sections stained with cytokeratin 14 antibody confirmed the epithelial nature of cells expressing the Muc1 protein. RNA from conjunctiva and Harderian gland subjected to RT-PCR and northern blot analysis showed an abundance of Muc1 transcript in these tissues. CONCLUSIONS: Muc1 mRNA and protein are present in murine conjunctival and Harderian gland epithelia. Animals lacking Muc1 mRNA and protein are predisposed to developing eye inflammation when compared to wild-type animals with an intact Muc1 gene. Muc1 appears to play a critical protective role at the ocular surface, presumably by acting as a barrier to infection by certain bacterial strains.

Complement receptor type 3 plays an important role in development of protective immunity to primary and secondary Corynebacterium pseudotuberculosis infection in mice.

The present study was performed to investigate the role of CR3, the type 3 complement receptor, in host defense against primary and secondary Corynebacterium (C.) pseudotuberculosis infection in mice. Treatment of mice with 5C6, an anti-CR3 monoclonal antibody (mAb), resulted in unrestricted multiplication of bacteria in the organs and dramatically increased mortalities of the infected mice. Histological examinations showed the inflammation, degeneration and necrosis of organs and revealed that the infection-enhancing effect of 5C6 mAb was associated with the failure of mice to focus mononuclear phagocytes at sites of bacterial multiplication. These results suggest that CR3 plays an important role in host defense against primary as well as secondary C. pseudotuberculosis infection in mice.

Evaluation of the RapID CB Plus system for identification of Corynebacterium species and other gram-positive rods.

Due to the difficulty of identifying Corynebacterium spp. with standard methods, we compared them with the RapID CB Plus system (Remel, Lenexa, Kans. [formerly Innovative Diagnostic Systems, Norcross, Ga.]), which consists of 4 carbohydrate and 14 preformed enzyme tests, for the identification of 98 clinical isolates of Corynebacterium sp., other coryneforms, Listeria monocytogenes, and 17 ATCC strains. Forty (95%) of 42 strains of Corynebacterium spp. were accurately identified to the species level by the RapID CB Plus system, and two additional strains of C. striatum were identified with one additional conventional test for lipid requirement. Twenty-seven (75%) of the 36 coryneform strains tested were identified correctly to the species level. However, three of four strains of Brevibacterium sp. and all seven of the L. monocytogenes strains were identified to the genus level only. Actinomyces strains had variable results, and the one strain of Arcanobacterium haemolyticum tested was not identified. Overall, the RapID CB Plus system compared favorably with the conventional methods, was easy to inoculate and interpret, and is promising as a new method for identification of gram-positive bacilli.

Acute-phase proteins in perinatal human plasma.

Plasma from eight newborns (4 pre-term and 4 full-term) with early-onset (< 72 h) sepsis and six apparently healthy controls was analyzed. The presence of spots identified as haptoglobin and serum amyloid A protein was the electrophoretic result most consistently associated with disease. Time course monitoring showed rises, peaks and declines of spot intensity as expected for acute-phase proteins induced by transient stimuli. Haptoglobin beta chains appear to be undersialated in pre-term newborns, whereas post-translational modifications of alpha chains and serum amyloid A protein are similar to those observed in adults. The undersialation of beta chain and occurrence of alpha chain phenotypes different from those found in maternal serum indicate that perinatal haptoglobin originates from neonatal synthesis.

Local production of tumor necrosis factor-alpha in corynebacterial pulmonary lesions in sheep.

Corynebacterium pseudotuberculosis infection is a common cause of pyogranulomas in ovine lungs and often occurs as a dual infection with lentiviruses. This coinfection usually leads to the development of chronic pneumonia and cachexia that is similar to the clinical syndrome seen in human beings with AIDS-related pneumonias. Recent in vitro studies indicate that monokines such as tumor necrosis factor-alpha (TNF alpha) are induced by C. pseudotuberculosis, suggesting that TNF alpha is involved in the pathogenesis of corynebacterial lesions in vivo. To substantiate in vitro observations concerning bacterial induction of TNF alpha in ovine pulmonary macrophages, immunohistochemical labeling techniques were used in combination with in situ hybridization to identify TNF-producing cells in corynebacterial lesion sites in vivo. TNF alpha message and translation product were found in macrophages comprising pyogranulomas that were induced by naturally acquired and experimental pulmonary C. pseudotuberculosis infections.