Experimental administration of the probiotic Escherichia coli strain Nissle 1917 results in decreased diversity of E. coli strains in pigs.

The strain Escherichia coli Nissle 1917 (EcN) is widely used as an efficient probiotic in therapy and prevention of human infectious diseases, especially of the intestinal system. Concurrently, small adult pigs are being used as experimental omnivore models to study human gastrointestinal functions. EcN bacteria were applied to 6 adult healthy female pigs in a 2-week trial. 6 Control animals remained untreated. Altogether, 164 and 149 bacterial strains were isolated from smear samples taken from gastrointestinal mucosa in the experimental and control group, respectively. Each individual E. coli strain was then tested for the presence of 29 bacteriocin-encoding determinants as well as for DNA markers of A, B1, B2 and D phylogenetic groups. A profound reduction of E. coli genetic variance (from 32 variants to 13 ones, P = 0.0006) was found in the experimental group, accompanied by a lower incidence of bacteriocin producers in the experimental group when compared to control (21.3 and 34.9%, respectively; P = 0.007) and by changes in the incidence of individual bacteriocin types. The experimental administration of EcN strain was not sufficient for stable colonization of porcine gut, but induced significant changes in the enterobacterial microbiota.

Bacteriocinogeny in experimental pigs treated with indomethacin and Escherichia coli Nissle.

AIM: To evaluate bacteriocinogeny in short-term high-dose indomethacin administration with or without probiotic Escherichia coli Nissle 1917 (EcN) in experimental pigs. METHODS: Twenty-four pigs entered the study: Group A (controls), Group B (probiotics alone), Group C (indomethacin alone) and Group D (probiotics and indomethacin). EcN (3.5×10(10) bacteria/d for 14 d) and/or indomethacin (15 mg/kg per day for 10 d) were administrated orally. Anal smears before and smears from the small and large intestine were taken from all animals. Bacteriocin production was determined with 6 different indicator strains; all strains were polymerase chain reaction tested for the presence of 29 individual bacteriocin-encoding determinants. RESULTS: The general microbiota profile was rather uniform in all animals but there was a broad diversity in coliform bacteria (parallel genotypes A, B1, B2 and D found). In total, 637 bacterial strains were tested, mostly Escherichia coli (E. coli). There was a higher incidence of non-E. coli strains among samples taken from the jejunum and ileum compared to that of the colon and rectum indicating predominance of E. coli strains in the large intestine. Bacteriocinogeny was found in 24/77 (31%) before and in 155/560 (28%) isolated bacteria at the end of the study. Altogether, 13 individual bacteriocin types (out of 29 tested) were identified among investigated strains. Incidence of four E. coli genotypes was equally distributed in all groups of E. coli strains, with majority of genotype A (ranging from 81% to 88%). The following types of bacteriocins were most commonly revealed: colicins Ia/Ib (44%), microcin V (18%), colicin E1 (16%) and microcin H47 (6%). There was a difference in bacteriocinogeny between control group A (52/149, 35%) and groups with treatment at the end of the study: B: 31/122 (25%, P=0.120); C: 43/155 (28%, P=0.222); D: 29/134 (22%, P=0.020). There was a significantly lower prevalence of colicin Ib, microcins H47 and V (probiotics group, P<0.001), colicin E1 and microcin H47 (indomethacin group, P<0.001) and microcins H47 and V (probiotics and indomethacin group, P=0.025) compared to controls. Escherichia fergusonii (E. fergusonii) was identified in 6 animals (6/11 isolates from the rectum). One strain was non-colicinogenic, while all other strains of E. fergusonii solely produced colicin E1. All animals started and remained methanogenic despite the fact that EcN is a substantial hydrogen producer. There was an increase in breath methane (after the treatment) in 5/6 pigs from the indomethacin group (C). CONCLUSION: EcN did not exert long-term liveability in the porcine intestine. All experimental pigs remained methanogenic. Indomethacin and EcN administered together might produce the worst impact on bacteriocinogeny.

Small intestinal bacterial overgrowth syndrome.

Human intestinal microbiota create a complex polymicrobial ecology. This is characterised by its high population density, wide diversity and complexity of interaction. Any dysbalance of this complex intestinal microbiome, both qualitative and quantitative, might have serious health consequence for a macro-organism, including small intestinal bacterial overgrowth syndrome (SIBO). SIBO is defined as an increase in the number and/or alteration in the type of bacteria in the upper gastrointestinal tract. There are several endogenous defence mechanisms for preventing bacterial overgrowth: gastric acid secretion, intestinal motility, intact ileo-caecal valve, immunoglobulins within intestinal secretion and bacteriostatic properties of pancreatic and biliary secretion. Aetiology of SIBO is usually complex, associated with disorders of protective antibacterial mechanisms (e.g. achlorhydria, pancreatic exocrine insufficiency, immunodeficiency syndromes), anatomical abnormalities (e.g. small intestinal obstruction, diverticula, fistulae, surgical blind loop, previous ileo-caecal resections) and/or motility disorders (e.g. scleroderma, autonomic neuropathy in diabetes mellitus, post-radiation enteropathy, small intestinal pseudo-obstruction). In some patients more than one factor may be involved. Symptoms related to SIBO are bloating, diarrhoea, malabsorption, weight loss and malnutrition. The gold standard for diagnosing SIBO is still microbial investigation of jejunal aspirates. Non-invasive hydrogen and methane breath tests are most commonly used for the diagnosis of SIBO using glucose or lactulose. Therapy for SIBO must be complex, addressing all causes, symptoms and complications, and fully individualised. It should include treatment of the underlying disease, nutritional support and cyclical gastro-intestinal selective antibiotics. Prognosis is usually serious, determined mostly by the underlying disease that led to SIBO.

Value of amniotic fluid interleukin-8 for the prediction of histological chorioamnionitis in preterm premature rupture of membranes.

OBJECTIVE: To determine whether amniotic fluid levels of interleukin-8 (IL-8) are of value in the antenatal diagnosis of acute histological chorioamnionitis (HCA) in preterm premature rupture of membranes (PPROM). SETTING: Department of Obstetrics and Gynaecology, Charles University, Medical School and University Hradec Kralove, Czech Republic. METHODS: We compared amniotic fluid IL-8 levels in twenty-nine pregnant women with preterm premature rupture of membranes between 24th and 36th gestational weeks with presence and absence acute histological chorioamnionitis or/and microbial invasion in the amniotic cavity using nonparametric tests (Mann-Whitney test), given the non-normal distribution of analyte. Comparisons of proportions were performed with Shapiro-Wilk normality test. RESULTS: Patients with HCA had a significantly higher median amniotic fluid IL-8 concentration than patients without the histological signs of chorioamnionitis (1867 pg/mL, 826-5577 versus 1045 pg/mL, 60-4133, p=0.013). Patients with MIAC had a significantly higher median amniotic fluid level than patients without invasion (1888 pg/mL, 519-5577 versus 1225 pg/mL, 60-2766, p= 0.017). Women with HCA and MIAC had a significantly higher median amniotic fluid IL-8 level than women without histological signs of chorioamnionitis and microbial invasion (3117 pg/mL, 826-5577 versus 1468 pg/mL, 394-2766, p=0.034). CONCLUSIONS: HCA or/and MIAC are associated with a significant increase of amniotic fluid interleukin-8 levels. Amniotic fluid IL-8 seems to be a marker of intraamniotic inflammation.

[Infections of the oral mucosa caused by herpes simplex virus]. / Infekce ústní sliznice vyvolané herpes simplex virem.

OBJECTIVES: Recapitulation of our knowledge dealing with diagnosis and therapy of oral mucosal diseases caused by HSV. MATERIAL AND METHODS: Analysis of a sample of 135 patients coming from Department of Dentistry, Faculty of Medicine in Hradec Králové within 11 years. Statistical evaluation of the data and related associations (clinical diagnosis, age, gender, general health status, treatment mode) were realized by ANOVA analysis, t-test, and Fisher's exact test. RESULTS: A total sample of 135 individuals (81 women and 54 men) consisted of 49 individuals with primary herpetic gingivostomatitis (37 %) and recurrences with clinical signs of herpetic stomatitis in 68 patients (50 %) and labial herpes simplex in 18 patients (13 %) aged from 1 to 90 years, mean age 40.4 year, with maximal occurrence in the 3rd and 7th decades. The mean age of individuals with herpetic gingivostomatitis was significantly lower (p = 0.0001). The occurrence of herpetic stomatitis was associated with severe concomitant diseases (p = 0,0001). Topical treatment was applied in 73 individuals (54 %), combined mode in 58 individuals (43 %), only systemic therapy in 4 individuals (3 %). In most of individuals with herpetic gingivostomatitis only topical treatment was used (80 %, in most of herpetic recurrences combined therapy was applied (62 %). CONCLUSIONS: The age of individuals with primary infection and herpetic recurrences was significantly different. Herpetic stomatitis occurred often as an opportunistic viral infection in seriously ill and older patients. Antiseptics with antiviral properties and their combinations with systemic administration of antiviral drugs proved successfully in the treatment.

[Considering the role of the PCR method in routine diagnosis of toxoplasmosis in peripheral blood tests in immunocompetent patients]. / Zamyslemí nad ,,postavenim" metody PCR v rutinní diagnostice toxoplazmózy pri vysetrení periferní krve u imunokompetentnich nemocných.

Infections caused by pathogenic protozoan Toxoplasma gondii in our geographic area is the most frequent parasitic infection; Czech Republic declares seroprevalence approx. 30 %. Diagnosis of toxoplasmosis is mostly based on serological methods are used (EIA IgM, IgA, IgE, IgG and avidity in IgG, Western Blot, complement fixation). According to positive results of these tests diagnosis of acute toxoplasmosis is established. In our retrospective study we tried to evaluate results of T. gondii DNA positivity from blood samples by PCR compared with positive markers of acute infection in patients before specific therapy was initiated. In accordance with literature we concluded, that in routine examination of immunocompetent outpatients of Clinic of Infectious Diseases from the moment of 4 weeks after lymphatic nodes swelling protozoan DNA detection in blood sample is not possible.

Clinical aspects and luteal phase assessment in patients with recurrent vulvovaginal candidiasis.

OBJECTIVE: This study was undertaken to characterize the patients with recurrent vulvovaginal candidiasis. STUDY DESIGN: Basic data of personal history and history of recurrent vulvovaginal candidiasis, lower genital tract symptoms and signs in 50 patients were analyzed in this longitudinal follow-up study including the determination of midluteal serum progesterone and urinary pregnanediol levels during the luteal phase in 84 cycles (recurrent vulvovaginal candidiasis) and 60 cycles (healthy controls). RESULTS: All patients suffered primary idiopathic form of recurrent vulvovaginal candidiasis. Frequently, there was a striking discrepancy between severe symptoms and clinical finding, which was often negligible or normal. There was no redness and no or minimum discharge in 52% of culture documented attacks. In contrast to the healthy controls, the patients had significantly lower levels of progesterone (p<0.01) as well as those of urinary pregnanediol (p<0.05). CONCLUSION: Culture positive attacks in patients with recurrent vulvovaginal candidiasis represented rather a form of vulvovaginal discomfort than attacks of vulvovaginal candidiasis with typical inflammatory changes. Significantly lower progesterone levels in the RVVC patients as compared to the healthy controls suggest a link between an altered hormonal status and one of possible causes of RVVC in these women.

[Malaria-an unusual cause of fever in the infant.].

Malaria is the fourth most frequent cause of death in African children. Connected with perinatal diseases as well as gastrointestinal and respiratory infections malaria has been still a serious health problem of that region. Occurrence of tropical malaria in infants reported in European countries is relatively rare. Not only from that reason, the assesment of diagnosis in children under one year of age seems to be obviously more difficult. The authors report the malaria in five-month-old infant from Cameroon who became ill during his stay in the Czech Republic. Non-specific symptoms, high level of parasitemia and impairment of blood coagulation were the main features of the emergent infection. On conclusion, the lack of suitable forms of childrens antimalarial drugs both for profylaxis and treatment is mentioned.

The serum levels of calcium, magnesium, iron and zinc in patients with recurrent vulvovaginal candidosis during attack, remission and in healthy controls.

The real cause of recurrent vulvovaginal candidosis (RVVC) is concealed and the etiopathogenesis of this disease remains to be determined. In a cohort study, concentrations of metals in 44 patients with RVVC and 30 healthy age-matched women were measured and compared. The concentrations of serum calcium (Ca), magnesium (Mg) and iron (Fe) were measured photometrically, the zinc (Zn) levels were determined using flame atomic absorption spectrometry. For statistical analysis were used the Student's t-tests (paired analysis for attack vs. remission; non-paired analysis for patient vs. control). Although all measured metals were within normal ranges the patients with RVVC had in contrast to the healthy controls significantly lower levels of serum Ca, Mg and Zn and insignificantly higher levels of Fe. These relative changes may contribute to the development of attacks in patients with RVVC.

[Experience with the PCR method in chlamydia detection.].

PURPOSE OF THE STUDY: The study was intended to summarize and evaluate the results in patients with a suspected infection by the genus Chlamydia, investigated with an in-house method of nested PCR (polymerase chain reaction). The study worked with data from patients living in eastern Bohemia, who were examined in the years 2001-2003 at the Dept. of Molecular Biology, a research laboratory shared by the Institute of Clinical Microbiology and the Institute of Clinical Biochemistry and Diagnostics. MATERIAL AND METHODS: 291 explorations were done in 2001, in 2002 already 562 and in 2003 their figure reached 760. The total number of samples received for investigation during that period was 1 613. 1 587 were actually investigated, 26 were unsuitable and could not be used. More than 70 % of all investigations were done with three types of material: urine (41.8 % of all the investigated samples), BAL (15.3 % of all the investigated samples) and whole blood (14.9 % of all the investigated samples). The investigations were carried out with the in-house nested PCR method, which uses primers from the MOMP(ompA) area of the genus Chlamydia spp. RESULTS: Total positivity was 5.67 %, in 1.26 % of the samples the resulted was considered uncertain and 93.07 % of the investigated samples were negative. In men PCR positivity was 6.11 %, in women 5.35 %. The major proportion of positive samples was from the age groups 70-79 years (11.67 %), 10-19 years (6.51 %) and 40-49 years (6.45 %). Overall positivity in smears from the urogenital system was 6.48 %, from urine 3.92 %, from BAL 10.70 % and from whole blood 5.51 %. KEYWORDS: nested PCR, Chlamydia spp., detection.

[Comparison of the detection of anaerobic bacteria in haemoculture systems BACTEC Lytic/10 Anaerobic/F, BacT/Alert FAN Anaerobic and BacT/Alert FN Anaerobic]. / Srovnání detekce anaerobních bakterií v hemokultivacních systémech BACTEC Lytic/10 Anaerobic/F, BacT/Alert FAN Anaerobic a BacT/Alert FN Anaerobic.

The purpose of this study is to deal with the problem of anaerobic cultivation of clinical specimens and consider the possibility of using semi-automated blood culture instruments. The Bactec Lytic, BacT/Alert FAN Anaerobic and BacT/Alert FN Anaerobic bottles were inoculated with Bacteriodes fragilis, Clostridium perfringens, Peptostreptococcus anaerobius and Feingoldia magna strains. The times to detection (TTD) for positive bottles were evaluated with reference to the number of inoculated bacteria. Inoculation with the same suspension of equal bacterial strain resulted in shorter TTD values for all the Bactec Lytic bottles as compared with the BacT/Alert FAN Anaerobic or BacT/Alert FN Anaerobic bottles, respectively. Statistically significant differences were recorded (p=0,05) by Bacteroides fragilis and Clostridium perfringens species. Feingoldia magna was submitted to culture in the Bactec Lytic alone. The anaerobic blood culture bottles are deemed acceptable for application in the culture of anaerobc isolates drawn from other samples except for blood (namely when prompt delivery of the sample to the laboratory is impossible and the sample can suffer damage), however, only a limited range of microorganisms can be taken into account for the detection purposes. The Bactec Lytic bottles are more appropriately designed for the detection of anaerobic bacterial species compared to the BacT/Alert FAN Anaerobic and BacT/Alert FN Anaerobic bottles. They achieve faster growth parametres and provide more successful readings of anaerobic bacteria culture and detection.

[Diagnostics of larval toxocariasis]. / Diagnostika larvální toxokarózy.

PURPOSE OF THE STUDY: The paper, conceived as a retrospective study, resumes the results of our own investigations of the presence of serum IgG antibodies against Toxocara canis (as a crossover also against T. cati) in the sera of patients suspected of presenting larval toxocariasis in the region of Eastern Bohemia of the Czech Republic in the years 1997-2002. Our aim was to determine the immunity rate of IgG, its relationship to age and sex, further the whole range of diagnoses, the frequency of demands for such an examination by physicians and the evaluation of our experience with the detection of low avidity IgG. MATERIAL AND METHODS: With a commercial diagnostic kit the ELISA method was used to detect the presence of IgG antibodies against T. canis in 2 994 sera. IgG avidity was determined in 92 positive samples with an avidity solution, which is included in the diagnostic kit and has been used in our laboratory since 2001. RESULTS: The immunity rate of larval toxocariasis in our set was 20.4 % (612 samples). With age this rate increases (in individual decades of life - 11.9; 8.3; 11.4; 17.9; 26.1; 29.8 and 34.2%). Up to adolescence positive findings predominate among boys (males 0-10 years 84.2 %; 11-20 years 76 %). In the third decade we see a dramatic change in favour of women (women 21-30 years - 74.6 %). In absolute figures the number of positive findings stagnates in men after the age of 30 years, while among women positive findings continue to grow - yet expressed in percentages there is signifinct change in the male-female ratio (beginning with the 4th decade we find among the women 61.2; 69.7; 72.6 and 64.8 %). Physicians' interest in the diagnosis of toxocariasis in the years 1997-2002 is confirmed by the significantly higher number of investigated samples (135; 446; 617; 422; 558; 816). This, however, does not go hand in hand with a higher proportion of IgG positivity. IgG avidity was investigated in 92 positive samples - low avidity was confirmed in six of them (6.5 %); the appropriate case histories are not a part of the study. CONCLUSIONS: Our results tally with the data published by other departments in the Czech Republic. They confirm that the methods used have, as before, their place in the diagnostic range of microbiological laboratories.

[Comparison of the results of in vitro antifungal susceptibility testing using the Fungitest kit and the disc diffusion test]. / Srovnání výsledku testování citlivosti hub k antifungálním látkám in vitro pomocí soupravy Fungitest a diskového difúzního testu.

AIM OF STUDY: Substantial progress has been achieved in the field of in vitro antifungal susceptibility testing, including the interpretation of the test results towards the clinical practice. Reference methods for antifungal testing of yeasts (M27-A) and filamentous fungi (M38-P) are available now. There are several alternatives to these standard protocols as the potential candidates for a routine laboratory testing of antifungal drugs. The aim of the study is to compare the results of antifungal susceptibility testing obtained by Fungitest and disk diffusion method. MATERIALS AND METHODS: From 1997 to 2000, 134 strains of 16 yeast species from the patients with suspected or proven mycosis were evaluated in the mycology laboratory of the Department of Clinical Microbiology, Teaching Hospital in Hradec Krlové. All these strains were tested by both disc test on Sabouraud agar (pH = 5.6) and Fungitest. The agreement of the results of the both methods was determined for all antimycotics and fungal species tested in terms of the categories susceptible, intermediate and resistant strain. RESULTS: Overall, there was only 60.3 % agreement between these methods for all antifungal drugs with the best results for flucytosine (71.4 % agreement), further for ketoconazole (66.2 %), amphotericin B (60.2 %), fluconazole (57.9 %), itraconazole (52.6 %), and miconazole (50.5 %). The best overall agreement between the results for individual species was found with C. lusitaniae (73.9 %), C. pelliculosa (73.5 %), C. parapsilosis (71.7 %), and partly with C. albicans (67.5 %). The poorest results were associated with fluconazole-resistant yeasts Saccharomyces cerevisiae (30.0 %), C. krusei (32.5 %) and C. glabrata (47.7 %). Regressive analysis revealed a relative close relationship between the results of these methods for miconazole (R = 0.49, P < 0.001), fluconazole (R = 0.45, P < 0.001), itraconazole (R = 0.42, P < 0.001) and flucytosine (R= 0.37, P < 0.001), but not for amphotericin B (R = 0.10) and ketoconazole (R = 0.16). CONCLUSION: The comparison of the results of the disk test and Fungitest showed some discrepancies which were dependent on an antifungal drug, a yeast species and the test conditions. Sabouraud agar and the criteria for interpretation of the results of the disk test seemed to be main sources of the disagreement. A modified Mueller-Hinton agar (2 % glucose, methylene blue) as a recommended test medium in a proposed agar diffusion method M44-P is the way to the improvement of standardization and reproducibility of the test.