Protein composition of the phase I Coxiella burnetii soluble antigen prepared by extraction with trichloroacetic acid.

Q fever is a highly infectious, widespread airborne zoonosis caused by Coxiella burnetii bacterium. Humans usually acquire the disease by inhalation of contaminated aerosol produced by infected livestock. Vaccination is the most practical way for prevention and control of the disease in the exposed population. In this work, we reviewed the most important Q-fever outbreaks in Slovakia as well as the progress in vaccine development. One of them represents a soluble antigen complex produced by extraction with trichloroacetic acid from a highly purified C. burnetii phase I strain Nine Mile. It was developed at the Institute of Virology in Bratislava. The protein content of this vaccine was separated by gel electrophoresis and analyzed by mass spectrometry. The study has resulted in the identification of 39 bacterial proteins from which 12 were recognized as immunoreactive. Most of the proteins were involved in bacterium pathogenicity (41.6%) and cell wall maintenance (25%). Four of the immunoreactive proteins may possess the moonlighting activity. Definition of the vaccine components represents a prerequisite for vaccine standardization and approval by governmental authorities.

[Cytolethal distending toxins]. / CDT toxíny.

Cytolethal distending toxins (CDT) are intracellularly acting proteins which interfere with the eukaryotic cell cycle. They are produced by Gram-negative bacteria with affinity to mucocutaneous surfaces and could play a role in the pathogenesis of various mammalian diseases. The functional toxin is composed of three proteins: CdtB entering the nucleus and by its nuclease activity inducing nuclear fragmentation and chromatin disintegration, CdtA, and CdtC, the two latter being responsible for toxin attachment to the surface of the target cell. Cytotoxic effect of CDT leads to the cell cycle arrest before the cell enters mitosis and to further changes (cell distension and death, apoptosis) depending on the cell type. Thus, CDT may function as a virulence factor in pathogenic bacteria that produce it and thus may contribute to the initiation of certain diseases. Most important are inflammatory bowel diseases caused by intestinal bacteria, periodontitis with Aggregatibacter actinomycetemcomitans as the aetiologic agent and ulcus molle where Haemophilus ducreyi is the causative agent.

Genes of intestinal Escherichia coli and their relation to the inflammatory activity in patients with ulcerative colitis and Crohn's disease.

Escherichia coli gene fimA was the most frequent gene that occurred in the intestine of all investigated groups. All subjects with fimA gene had significantly higher values of tumor necrosis factor alpha (TNF-α) and CRP than those with other E. coli genes. There was also a tendency to increased serum interleukin (IL)-6 levels in patients carrying the fimA gene; however, no relation was observed to serum IL-8 and IL-10. Patients with Crohn's disease had significantly higher IL-6 than those with ulcerative colitis (UC) and controls. The highest levels of TNF-α were detected in the UC group. There were no significant differences in serum IL-8 and IL-10 between all three groups. The presence of E. coli gene fimA in the large bowel of patients with IBD is related to the immunological activity of the disease which may be important from the aspect of therapeutical strategy.

High level of quinolone resistance in Escherichia coli from healthy chicken broilers.

The development of resistance to quinolones (nalidixic acid, ciprofloxacin and enrofloxacin) in 2006-2008 was evaluated in 317 strains of Escherichia coli isolated from healthy chicken broilers from various farms. The isolates (2006/2007/2008) showed a high resistance to nalidixic acid (87/85/67 %), ciprofloxacin (CIP) (49/54/29 %) and enrofloxacin (ENR) (52/42/22 %). Nalidixic acid-resistant isolates with low level of MIC for CIP and ENR represented a single mutation; intermediary MIC for CIP and ENR were related to two mutations and high level resistance MIC for CIP (> or =4 mg/L) and ENR (> or =16 mg/L) represented three mutations (two in gyrA and one in parC). There was a correlation between the phenotype reading of high-level resistance and mutations in gyrA (Ser83Leu, Asp87Tyr or Asp87Asn) and parC (Ser80Ile) gene. Plasmid-mediated quinolone-resistance qnrS gene was detected in one Escherichia coli strain with a high level of ciprofloxacin resistance. Our results demonstrate the increase in occurrence of multiresistant E. coli strains with a high level of chromosomal and plasmid resistance to fluoroquinolones.

Enterovirulent E. coli in inflammatory and noninflammatory bowel diseases.

We determined the incidence of enterovirulent E. coli (EVEC; which can to cause gastrointestinal infections) in strains isolated from patients with both of the major inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC) and from patients with noninflammatory bowel diseases (nonIBD). Cell detachment E. coli (CDEC) were detected in 14 % of all strains. A significant difference in the presence of CDEC was found between the groups of strains isolated from UC (24.1 %), nonIBD (11.9 %) and CD (4.7 %). Enteroaggregative E. coli (EAggEC) were detected in 2.5 %, typical enteropathogenic strains (EPEC) in 1.3 % and enterotoxigenic ones (ETEC) in 1.5 %. Enteroinvasive (EIEC) and shigatoxin producing E. coli (STEC) were not detected. Some strains showed a high invasion level in gentamicin-protection assay. These strains could therefore belong to adherent-invasive E. coli (AIEC) because they are free of genes encoding invasins (ipaH, ial) and are equipped with fimA gene. However, complete characterization of these strains and their classification as AIEC will require further tests.

Hemolytic uremic syndrome caused by verotoxin-producing Escherichia coli O26. Case report.

Verotoxin-producing Escherichia coli (VTEC) are nowadays among the most important emerging group of food-borne pathogens (VTEC strains cause gastroenteritis that can be complicated by the hemorrhagic colitis or hemolytic uremic syndrome, HUS). Escherichia coli 026 producing verotoxin 2 was isolated and its identity confirmed by examination of phenotype and genotype; the strain was first described in Slovakia in association with the development of HUS in a 4-year-old girl.

Detection of Shiga toxins, intimin and enterohemolysin in Escherichia coli strains isolated from children in eastern Slovakia.

Fifty Escherichia coli strains isolated from stool samples of 51 healthy children, 143 strains isolated from stool samples of 327 children with diarrhea and 24 strains isolated from stool samples of 21 children with suspected hemolytic uremic syndrome were examined for the presence of Shiga toxin-producing E. coli virulence factors (shiga toxin 1 and 2, intimin and enterohemolysin) and their genes. Vero-cell assay and latex agglutination were used for detection of Shiga toxin 1 and 2, TSB agar with washed erythrocytes was used for detection of enterohemolysin; genes encoding shiga toxin 1 and 2, intimin and enterohemolysin were detected using multiplex PCR. The presence of E. coli strains harboring genes encoding shiga toxin 1 and 2 (12 strains), intimin (34 strains) and enterohemolysin (12 strains) was demonstrated.

Occurrence and genetic association of selected virulence factors in clinical Escherichia coli isolates.

Occurrence of cnf1+ E. coli pathogenic strains among extraintestinal E. coli isolates was evaluated to explain an impact of cytotoxic necrotizing factor type 1 (CNF1) in human infections. A total of 120 E. coli isolates were characterized for presence of virulence factors cnf1- and pap--specific sequences by PCR, and the production of alpha-hemolysin using blood agar-plate test. Different association patterns among the detected virulence factors were obtained by comparison of various groups of clinical E. coli isolates. These differences probably reflect a potential impact of CNF1 in the colonization of vaginal environment.

Detection of pap-, sfa- and afa-specific DNA sequences in Escherichia coli strains isolated from extraintestinal material.

P-fimbriae, S-fimbriae and AFA-adhesins are virulence factors responsible for adherence of Escherichia coli strains to extraintestinal host-cell surface. Detection of pap-, sfa- and afa-specific sequences performed by PCR revealed 74% pap+, 65% sfa+, and 8.3% afa+ strains in a group of 84 extraintestinal E. coli isolates. Detection in a group of fecal strains showed 29% pap+, 21% sfa+ and 4% afa+ strains. pap together with sfa were found as the most frequent combination (56%) among extraintestinal isolates probably due to localization of pap- and sfa-operons on a common pathogenicity island. The occurrence of afa-specific sequence among 56 urine strains was 11%, although no afa+ strain was detected among 28 gynecological isolates. No strains with detected adhesin operons were found among twenty (24%) extraintestinal E. coli strains.

Rare non-albicans Candida species detected in different clinical diagnoses.

In working on the incidence of yeasts we excluded from the tested set C. albicans, C. krusei, C. parapsilosis and C. tropicalis on the basis of morphological and biochemical properties. We found 16 rare species of yeasts: C. claussenii (16), C. guilliermondii (10), C. robusta (9), C. pulcherrima (8), C. zeylanoides (4), C. glabrata (3), C. lusitaniae (3), C. catenulata (2), C. mesenterica (2), C. utilis (2), C. freyschussii, C. intermedia, C. kefyr, C. lipolytica, C. mogii, C. pseudotropicalis (1 for each). These yeasts were detected mainly in cases of premature babies (22) from the nasopharynx (13), from the rectum (4), from the skin (23), from wound drains and from blood (1 for each), with gynaecological diagnoses (15) and rarely other diagnoses, such as malignancy (5), hypertension and respiratory infections (4 for each), kidney transplantation (3), dialysis, haemolytic-uremic syndrome (2 for each), perforation of gastric ulcus, otitis, prostatitis, ulcus cruris, coma, spina bifida, peritonitis and hepatic failure from different clinical material.

Prevalence of Cryptococcus neoformans in clinical specimens.

Cryptococcus neoformans isolated from various clinical materials in 14 cases, was identified by (1) cultivation on Sabouraud glucose agar and CHROMagar Candida, (2) microscopic examination of Indian-ink-stained preparations and (3) determination of biochemical properties (assimilation and fermentation of saccharides, assimilation of KNO3, production of urease and phenol monooxygenase). C. neoformans was determined in five specimens from paediatric patients in the intensive care unit and in nine specimens from adult patients, most frequently from liquor at meningitis (n = 3).

Serum response of Escherichia coli strains causing dyspepsia and urinary tract infection: relation to alpha-hemolysin production and O type.

A total of 109 alpha-hemolytic and 104 nonhemolytic Escherichia coli isolates from children with dyspepsia and urinary tract infections were investigated for resistance to the bactericidal activity of human serum. A significantly higher proportion of serum resistance was found in alpha-hemolytic E. coli isolates than in nonhemolytic isolates (P < 0.01). An association between the titer of alpha-hemolysin produced and serum resistance was found.

Fimbriation, surface hydrophobicity and serum resistance in uropathogenic strains of Escherichia coli.

A total of 80 Escherichia coli strains were examined for expression of P-fimbriae, mannose-sensitive haemagglutination (MSHA) and mannose-resistant haemagglutination (MRHA) of human group A erythrocytes and guinea pig erythrocytes, cell surface hydrophobicity and resistance to serum bactericidal activity. Isolates were obtained from urine of children and adults, either with acute pyelonephritis (n = 15 and n 12) or lower urinary tract infection (UTI) (n = 30 and n = 23, respectively). Results obtained showed that, in E. coli strains isolated both from children and adults with lower UTI, significant differences were not found concerning the incidence of P-fimbriae, cell surface hydrophobicity and serum resistance. In pyelonephritogenic E. coli isolated from children and adults, the incidence of P-fimbriae and cell surface hydrophobicity was associated more frequently with the former (87% vs. 42% and 100% vs. 67%, P < 0.05), while serum resistance was associated with the latter (47% vs. 67%, P < 0.05).

Virulence-associated factors in Escherichia coli strains isolated from children with urinary tract infections.

One hundred and sixty-eight strains of Escherichia coli were isolated from cases of pyelonephritis (24) and lower urinary tract infections (UTI) (144) from hospitalised and outpatient children up to 2 years old. These strains were investigated for the expression of P fimbriae (PF), mannose-resistant and mannose-sensitive haemagglutination, cell-surface hydrophobicity, serum resistance and the production of alpha-haemolysin (AH), colicins and aerobactin. PF, AH, aerobactin production and serum resistance were significantly more frequent amongst strains expressing mannose-resistant haemagglutination. PF and AH production was significantly more frequent in pyelonephritogenic strains than in lower UTI strains. Serotypes O6 and O112 were isolated most frequently and plasmids were found in the majority of strains tested.

Characteristics of Pseudomonas aeruginosa strains isolated from urinary tract infections.

Thirty-three uropathogenic strains of Pseudomonas aeruginosa were investigated for hemolytic activity in both bacterial broth culture filtrates and isolate lyzates, resistance to bactericidal activity of fresh human serum, resistance to six antibiotics and plasmid DNA profile. Twenty-four of the 33 (73%) bacterial filtrates showed lysis of rabbit erythrocytes, as did the three after guinea-pig erythrocyte treatment. Twelve of 33 isolate lysates showed in parallel lysis of both types of erythrocytes used. Serum resistance was found in 17 (52%) isolates, intermediate resistance in 15 (45 %) isolates and only one isolate showed serum sensitivity. Resistance to antibiotics was detected as follows (in %): tetracycline 94, kanamycin 79, chloramphenicol 76, septrin 73, ampicillin 64, streptomycin 45, gentamicin 18. None of the isolates investigated showed resistance to colistine. With the exception of one isolate, plasmid DNA was detected in all P. aeruginosa strains.

[Verotoxin as an important factor in the virulence of E. coli strains]. / Verotoxín ako významný faktor virulencie kmenov E. coli.

Verotoxin as a factor of virulence of E. coli strains is involved in the pathogenesis of hemorrhagic colitis, hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. The most frequent producers of verocytotoxin are E. coli O157:H7 and O157:H-, yet further 57 E. coli serotypes producing verotoxin have been described. The occurrence rate of verocytotoxigenic strains of E. coli is increasing worldwide and should be a matter of concern also in our conditions. (Ref. 56.)

Enterohaemolysin production in Escherichia coli strains.

Enterohaemolysin production was found in 11 (20.3%) out of 54 Escherichia coli strains isolated from stools of infants with dyspepsia and in 3 (2.3%) out of 130 E. coli strains isolated from urinary tract infections. Enterohaemolysin producing E. coli strains isolated from stools belonged to O groups 25 and 111 and the strains from urine to O groups 1 and 15. None of the enterohaemolysin-producing strains isolated from dyspepsia was shown to cause any damage on Vero cells.

[Virulence factors in strains of Escherichia coli isolated in urinary tract infections]. / Faktory virulencie u kmenov Escherichia coli izolovaných pri mocových infekciách.

In 21 strains of E. coli isolated from patients with cystitis and asymptomatic bacteriuria the authors assessed the group, mannososensitive and mannoresistant agglutination of human, bovine, chick, guinea pig, sheep and pig erythrocytes, the production of haemolysis, colicins, aerobactin, the capacity of strains to induce oedema on mouse paws, the lethal effect in mice, the resistance to tetracycline, streptomycin, chloramphenicol, kanamycin, ampicillin and sulfamethoxidine, the transmission of resistance and sensitivity of strains to the action of fresh human serum. In ten strains the authors recorded also the production of haemolysin and the lethality for mice, in four strains the production of colicin V. production of aerobactin and serum resistance. In three strains the aerobactin production was recorded concurrently with the haemolysin production. In none of the samples P. fimbriae were found.

Killing of alpha-haemolytic and non-haemolytic Escherichia coli strains in human serum and polymorphonuclear leucocytes.

The role of alpha-haemolysin (AH) in the resistance of Escherichia coli strains, isolated from patients with extra-intestinal diseases or diarrhoea, to the bactericidal activity of human serum and intracellular killing in polymorphonuclear leucocytes (PMNL) was investigated in vitro. Sets of alpha-haemolytic and non-haemolytic E. coli strains and sets of isogenic E. coli strains, which included wild-type alpha-haemolytic strains and derived strains with a reduced production of AH, were used. Compared with non-haemolytic strains, alpha-haemolytic strains were significantly more resistant to the bactericidal activity of 10% and 100% human serum and to intracellular killing in PMNL. Higher resistance to serum killing and to intracellular killing in PMNL was also found in wild-type alpha-haemolytic E. coli than in isogenic bacteria with reduced production of AH. These results provide evidence that production of AH in E. coli strains counteracts both the bactericidal activity of serum and intracellular killing in PMNL.