Salmonella-induced changes of the rat intestinal microbiota

The gut microbiome profoundly affects the body functioning: it participates in host protection against pathogenic microorganisms, metabolic events, inhibition of inflammatory responses, formation of innate and adaptive immune response in the intestinal mucosa. One of the causes altering microbiota community is due to antibiotics. Therefore, the processes of antibiotics interaction together with Salmonella enteritidis and Salmonella typhimurium with representatives of normal intestinal microflora are of particular interest. Materials and methods. The quantitative and qualitative analysis of the wall microbiota composition in rats was evaluated by bacteriological method, the statistical data analysis was performed using the software StatSoft Statistica v.12. Results and discussion. Inoculation of vancomycin and S. enteritidis, S. typhimurium in groups II, III, IV resulted in quantitatively decreased E. coli level by 10-, 7- and 110-fold, respectively (p ≤ 0.05). The count of P. aeruginosa decreased markedly only in the group III (p ≤ 0.05). The count of Bacteroides spp. members was profoundly decreased by several thousand times (group II) as well as 70- and 87-fold (groups III and IV), respectively (p ≤ 0.05). The count of E. faecalis and E. faecium decreased by 861-, 6- and several thousand times (groups II, III, IV), respectively (p ≤ 0.05). The count of Proteus spp. markedly decreased in group II by 27-fold and rapidly increased in group IV (p ≤ 0.05). Group III revealed a sharp decline in level of Enterobacter spp. and Klebsiella spp. by 847- and 150-fold, whereas in group II they were increased by 7- and 46-fold, respectively (p ≤ 0.05). The count of Staphylococcus spp. decreased by 10-fold only in group II. The level of Clostridium spp. decreased by several thousand times (group II) and by 5,500 times (group IV) (p ≤ 0.05). The count of Lactobacillus spp. decreased by several thousand times (group II). The count of Bifidobacterium spp. members significantly decreased by 10.9-fold and by several thousand times (groups III, IV). The level of Peptostreptococcus anaerobius profoundly decreased in all three study groups (p ≤ 0.05). The level of Salmonella spp. increased in group II by 49 times, but markedly increased in groups III and IV (p ≤ 0.05). Inoculation of Salmonella after vancomycin pretreatment caused dramatic change in the microbiota composition in groups V and VI, namely: increased count of E. coli by 65- and 105-fold, markedly increased level of P. aeruginosa in group V and VI — by 3-fold. In addition, these groups also showed decreased level of Bacteroides spp. by 9- and 10-fold (p ≤ 0.05). The count of E. faecalis and E. faecium decreased dramatically only in group V (p ≤ 0.05). The count of Proteus spp. decreased by 17 times in group V as well as in group VI (p ≤ 0.05). A sharp increase in level of Enterobacter spp. and Klebsiella spp. members was observed in groups V and VI (p ≤ 0.05). However, representatives of Peptostreptococcus anaerobius in groups V and VI decreased by 20 and 9 times, respectively (p ≤ 0.05). The count of Salmonella spp. decreased only in group V by 7 times (p ≤ 0,05). Inoculating experimental animals with B. fragilis conditioned with S. enteritidis, S. typhimurium and pretreated with vancomycin resulted in markedly decreased level of E. coli in group VII and VIII by 538 times (p ≤ 0.05). The count of P. aeruginosa in groups VII and VIII decreased profoundly, whereas level of Bacteroides spp. members was reciprocally increased (p ≤ 0.05). The level of Lactobacillus spp. decreased by 10.3 times only in group VI. The count of E. faecalis and E. faecium increased by 10 and 19 times in groups VII and VIII, respectively, whereas level of Proteus spp. decreased only in group VII by 322 times (p ≤ 0.05). In addition, a sharp decrease in level of Enterobacter spp. and Klebsiella spp. members (p ≤ 0.05) was found in groups VII and VIII. The count of Peptostreptococcus anaerobius and Lactobacillus spp. members was markedly increased by 7-, 12-, several thousand-fold and 40 times (groups VII and VIII, respectively) (p ≤ 0.05). The count of S. enteritidis and S. typhimurium in groups VII and VIII decreased rapidly (p ≤ 0.05). Conclusion. Inoculation of B. fragilis can be used in treatment of inflammatory bowel diseases or disorders with impaired gut barrier function

Младенческая форма болезни Помпе: клиника, диагностика и лечение

Pompe disease is a rare inherited disease that belongs to lysosomal accumulation diseases and can be considered as cardiac glycogenosistype II, as well as a severe neuromuscular disease or metabolic myopathy. Physicians of different specialties very rarely identify this pathology, which is due to both its rarity and clinical and genetic polymorphism. Infantile Pompe disease is the severest form. It is characterized by a progressive pattern and a fatal outcome during the first year of life. The possibility of performing enzyme replacement therapy for this disease, which can improve the prognosis and quality of life of patients, makes the early diagnosis of Pompe disease urgent. The paper describes the clinical presentation of infantile Pompe disease and current methods for its diagnosis and treatment. The authors give their experience in diagnosing and treating infantile Pompe disease, by demonstrating 3 cases of the disease. The characteristics of each infant, which confirm the clinical and genetic variety of this pathology, are discussed.Болезнь Помпе является редким (орфанным) наследственным заболеванием, которое относится к лизосомным болезням накопления и может рассматриваться как сердечный гликогеноз II типа, а также как тяжелое нервно-мышечное заболевание или метаболическая миопатия. Выявляемость данной патологии среди врачей различного профиля крайне низкая, что обусловлено как редкостью патологии, так и клинико-генетическим полиморфизмом заболевания. Наиболее тяжелая форма болезни Помпе – младенческая (инфантильная). Она характеризуется прогредиентностью течения и летальным исходом в течение первого года жизни. Возможность проведения фермент-заместительной терапии при данном заболевании, позволяющая улучшить прогноз и качество жизни пациентов, определяет актуальность ранней диагностики болезни Помпе. В статье описываются клиника, современные методы диагностики и лечения инфантильной формы болезни Помпе. Представлен собственный опыт диагностики и лечения младенческой формы болезни Помпе на основании демонстрации 3 клинических случаев заболевания. Обсуждаются особенности каждого ребенка, подтверждающие клинико-генетическое разнообразие данной патологии

Salmonella-induced changes in the gut microbiota and immune response genes transcriptome during administration of vancomycin and Bacteroides fragilis

The aim. To study Salmonella-induced changes in the intestinal wall microbiota, the expression of Salmonella effector proteins SipA, SopB, SopE2 and transcriptional activity of genes FFAR2, Foxp3, RORγt in rat GALT during administration of vancomycin and B.fragilis. Methods. Investigations of qualitative and quantitative composition of the microbiota of the wall of the small intestine were carried out, and the expression level of rat genes Foxp3, Rorc (Royt), FFAR2 and Salmonella effector proteins SipA, SopB and SopE2 were determined by RT-PCR, the relationship between groups of microorganisms was established. Results. Administration of B.fragilis against the background pre-treatment with vancomycin and Salmonella infection alters the quantitative composition of the microbiota in the wall of the small intestine contents: a decrease in Salmonella spp., E.coli, P.aeruginosa, Proteus spp., Enterobacter spp., Klebsiella spp. and Shigella spp., as well as increasing Bacteroides spp., E.faecalis, E.faecium and Peptostreptococcus anaerobius. The level of expression of Salmonella effector proteins in animals with the combined administration of vancomycin and S.enteritidis (I group), S.typhimurium (II group) increased: SopB – 101 and 20 times; SopE2 - 80 and 2 times; SipA - 613 times (II group), and also 5-fold decrease was noted in the I group. Relative normalized number of mRNA of genes FFAR2, Foxp3, RORγt in GALT of rats in groups III and IV increased: FFAR2 - 2.7 and 5.4 times; Foxp3 - 2.5 and 85 times, RORγt level decreased by 70% and only in IV group. Conclusions. Using B.fragilis creates conditions for the correction of Salmonella-induced changes of the intestinal microbiome. Pretreatment of animals with vancomycin causes increased transcriptional activity of genes SipA, SopB and SopE2, except SipA after administration of S.enteritidis. Administration of B.fragilis increases the level of mRNA of genes FFAR2 and Foxp3 in GALT and reduces RORγt after infection with S. typhimurium

The definition of neutrophil extracellular traps and the concentration of short-chain fatty acids in salmonella-induced inflammation of the intestine against the background of vancomycin and Bacteroides fragilis

Abstract The aim is to study the features of the formation of neutrophil extracellular traps in the blood and gut-associated lymphoid tissue with salmonella-induced inflammation against the background of the administration of vancomycin and B. fragilis, and to determine the concentration of short-chain fatty acids in the luminal microflora of rats by means of chromatography-mass spectrometry. Methods. Studies were carried out on quantitative counting of Sytox+-neutrophiles and NETs in scrapings of the mucous membrane of the ileum of the intestine and in blood by the method of immunofluorescence microscopy, and also by determination of the concentration of SCFA in the luminal microflora of rats by chromatography-mass spectrometry. Results. The introduction of vancomycin contributed to an increase in the number of Sytox+-cells in scrapings of the intestinal mucosa and in the blood by 55 % and 2.5 times (group II). With the combined administration of vancomycin and S.enteritidis (III group), S. typhimurium (IV group), the mean Sytox+-cell counts in mucosal scrapings increased by 30 % and 2.4 times, and in the blood by 30 % (group IV ), there was also a decrease in the number of NETs by 40 % (group IV). The introduction of B. fragilis against the background of pretreatment with vancomycin and infection with salmonella showed a decrease of Sytox+-cells in the scrapings of the intestinal mucosa by 43 % and 53 %, and in the blood by 46 % and 58 % (V and VI groups), and the number of NETs in scrapings from the intestinal mucosa and in the blood significantly increased by 43 % and 40 % (V group), and by 2.3 and 2.0 times (group VI). When infecting the rats with S. typhimurium against the background of pretreatment with vancomycin and the introduction of B. fragilis, the concentration of acetate in the samples increased by 2 times; propionate – 6 times and butyrate – 3 times. Conclusions. The introduction of B. fragilis in the infection of S. enteritidis and S. typhimurium against the background of vancomycin pretreatment leads to decrease in the number of Sytox+-cells in scrapings from the intestinal mucosa and in the blood, but induces the generation of NETs, and also causes an increase in the concentration of SCFA in the luminal microflora of rats, which helps to reduce salmonella-induced inflammation and restore the integrity of the intestinal epithelium

THE PROCALCITONIN TEST AS A NEARLY CRITERION TO DIAGNOSE SEVERE FORMS OF INTRAUTERINE INFECTIONS AND TO MONITOR ANTIBACTERIAL TREATMENT IN EARLY NEONATAL PERIOD

Background: Research in the field of reliable and available tests to diagnose infectious and inflammatory disorders in newborns in their first two days of life, as well as for determination of indications to antibacterial treatment and its monitoring in the early neonatal period are of utmost importance. Aim: To improve quality of diagnostics of intrauterine infections and to optimize management strategies for newborns with a high risk of infections by means of the procalcitonin test in the early neonatal period.Materials and methods: We assessed 40 normal (on-term) and 10 pre-term newborns born to mothers with infectious and inflammatory urogenital disease. Group 1 (n = 21) included patients with intrauterine pneumonia, group 2 (n = 6), those with intrauterine infection without a clearly defined primary locus, group 3 (n = 13), those with non-infectious disorders and group 4 (n = 10) comprised clinically normal (healthy) newborns. All infants underwent standard clinical and laboratory assessments, including an assessment of procalcitonin level by means of a semi-quantitative procalcitonin express-test (BRAHMS) at days 1, 2 and 3 of life. Results: At day 1, during primary assessment of newborns from group 1, procalcitonin values above 2 ng/mL were measured in 67% (10 of 15) cases; at days 2 and 3 also in 67% (4 of 6). Two patients with low procalcitonin values (below 0.5 ng/mL) had a disease of viral etiology (in 1, enteroviral and in 1, cytomegaloviral). In group 2, procalcitonin values exceeded 2 ng/mL in 3 of 5 newborns. In none of the infants from groups 3 and 4 procalcitonin values exceeded 2 ng/mL during their first 3 days of life. For assessment of efficacy of antibacterial treatment based on procalcitonin levels, all newborns with intrauterine infections were divided into group А (n = 11), where an antibacterial regimen was changed, and group B (n = 16), with no change in antibacterial treatment. During the treatment, 5 newborns from group A (45.5%) had their procalcitonin levels unchanged, whereas in 6 (54.5%) patients it decreased. In group B, 12 (75%) of newborns had their procalcitonin levels unchanged, in 2 (12.5%) it went down and in 2 (12.5%) of patients it went up. Conclusion: A semi-quantitative procalcitonin expresstest is characterized by its high informativity and availability when used in newborns of various gestation ages in the early neonatal period. An increase of procalcitonin level above 2 ng/mL, starting from the first day of life, can be used as an early diagnostic criterion of severe forms of intrauterine infections associated with systemic inflammatory response and symptoms of organ insufficiency. The results of procalcitonin test can be used for monitoring of antibacterial treatment in newborns

Glycogen storage disease type II (Pompe disease) in children

The paper gives the data available in the literature, which reflect the manifestations, diagnosis, and current treatments of the rare (orphan) inherited disease glycogen storage disease type II or Pomp disease in children, as well as its classification. The infant form is shown to be most severe, resulting in death from cardiovascular or pulmonary failure generally within the first year of a child’s life. Emphasis is laid on major difficulties in the differential and true diagnosis of this severe disease. Much attention is given to the new pathogenetic treatment — genetically engineered enzyme replacement drug Myozyme®. The authors describe their clinical case of a child with the juvenile form of glycogen storage disease type II (late-onset Pompe disease). Particular emphasis is laid on the clinical symptoms of the disease and its diagnostic methods, among which the morphological analysis of a muscle biopsy specimen by light and electron microscopies, and enzyme and DNA diagnoses are of most importance. The proband was found to have significant lysosomal glycogen accumulation in the muscle biopsy specimen, reduced lymphocyte acid α-1,4-glucosidase activity to 4,2 nM/mg/h (normal value, 13,0—53,6 nM/mg/h), described in the HGMD missense mutation database from 1000 G>A p.Gly334er of the GAA in homozygous state, which verified the diagnosis of Pompe disease

Serum Cytokine Profile, Beta-Hexosaminidase A Enzymatic Activity and GM2 Ganglioside Levels in the Plasma of a Tay-Sachs Disease Patient after Cord Blood Cell Transplantation and Curcumin Administration: A Case Report

Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder that occurs due to a deficiency of a β hexosaminidase A (HexA) enzyme, resulting in the accumulation of GM2 gangliosides. In this work, we analyzed the effect of umbilical cord blood cell transplantation (UCBCT) and curcumin administration on the course of the disease in a patient with adult TSD. The patient’s serum cytokine profile was determined using multiplex analysis. The level of GM2 gangliosides in plasma was determined using mass spectrometry. The enzymatic activity of HexA in the plasma of the patient was assessed using a fluorescent substrate assay. The HexA α-subunit (HexA) concentration was determined using ELISA. It was shown that both UCBCT and curcumin administration led to a change in the patient’s cytokine profile. The UCBCT resulted in an increase in the concentration of HexA in the patient’s serum and in an improvement in the patient’s neurological status. However, neither UCBCT nor curcumin were able to alter HexA activity and the level of GM2 in patient’s plasma. The data obtained indicate that UCBCT and curcumin administration can alter the immunity of a patient with TSD, reduce the level of inflammatory cytokines and thereby improve the patient’s condition