Nanostructural and functional properties of Ag-TiO2 coatings prepared by co-sputtering deposition technique.

Ag-TiO2 nanocomposite coatings with varying Ag content were prepared by co-sputtering from two separate sputter sources. This technique allows to prepare coatings not only with a large variation of Ag content and different gradient but also allows much better control of nanocomposite thickness and nanostructure compared with mostly used techniques based on wet chemical approaches. Various thicknesses of nanocomposite layers with different deposition parameters were studied to obtain a better understanding on the growth of Ag nanostructures in the TiO2 films. The metal-volume-fraction was varied between 15% and 47%. Structural and microstructural investigations of the nanocomposite films were carried out by transmission electron microscopy. Special attention was paid to surface segregation of Ag and its suppression. The observed segregation on TiO2 contrasts sharply with the well known embedding tendency of Ag clusters on polymers. Functionality of the Ag-TiO2 nanocomposites was demonstrated via UV-Vis spectroscopy and antibacterial tests. It was shown that a thin layer of TiO2 can be used as an effective barrier to tailor the release behaviour of Ag ions.

Extended-spectrum beta-lactamase production is associated with an increase in cell invasion and expression of fimbrial adhesins in Klebsiella pneumoniae.

Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae strains are suggested to possess higher pathogenic potential than non-ESBL producers. Microbial adherence to and invasion of host cells are critical steps in the infection process, so we examined the expression of type 1 and 3 fimbrial adhesins by 58 ESBL-producing and 152 nonproducing isolates of K. pneumoniae and their abilities to invade ileocecal and bladder epithelial cells. Mannose-sensitive hemagglutination of guinea pig erythrocytes and mannose-resistant hemagglutination of ox erythrocytes were evaluated to determine the strains' abilities to express type 1 and type 3 fimbriae, respectively. Bacterial adhesion to and invasion of epithelial cells were tested by enzyme-linked immunosorbent assay and imipenem killing assay, respectively. The adherence of ESBL- and non-ESBL-producing strains to epithelial cells did not differ significantly (P > 0.05). In contrast, the proportion of strains capable of invading (>5% relative invasion) ileocecal and bladder epithelial cells was significantly higher among ESBL producers (81%, n = 47/58, and 27.6%, n = 16/58, respectively) than among non-ESBL producers (61%, n = 93/152, and 10%, n = 15/152, respectively) (P = 0.0084, odds ratio [OR] = 2.711, 95% confidence interval [CI] = 1.302 to 5.643 and P = 0.0021, OR = 4.79, 95% CI = 1.587 to 7.627). The mean invasion by ESBL producers (5.5% +/- 2.8% and 3.3% +/- 2.7%, respectively) was significantly higher than that by non-ESBL producers (2.9% +/- 2.6% and 1.8% +/- 2%, respectively) (P < 0.0001). Likewise, the proportion of ESBL producers coexpressing both fimbrial adhesins was significantly higher (79.3%; n = 46/58) than that of non-ESBL producers (61.8%; n = 94/152) (P = 0.0214; OR = 2,365; 95% CI = 1.157 to 4.834). Upon acquisition of SHV-12-encoding plasmids, two transconjugants switched on to produce type 3 fimbriae while expression of type 1 fimbriae was not affected. The acquisition of an ESBL plasmid appeared to upregulate the phenotypic expression of one or more genes, resulting in greater invasion ability.

A monoclonal antibody with specificity for the genus Klebsiella binds to a common epitope located in the core region of Klebsiella lipopolysaccharide.

A mouse monoclonal antibody (mAb) which has been obtained after immunization of mice with heat-killed Klebsiella pneumoniae strain R20/O1(-) followed by standard plasmacytoma cell fusion protocols was investigated for its ability to identify various species of the genus Klebsiella. Based on the published observation that the antibody binds to an epitope located in the core region of lipopolysaccharide (LPS) of strain R20/O1(-), we tested whether this epitope is shared and exposed by other species of the genus Klebsiella. The antibody was able to bind to LPS of clinical isolates of K. pneumoniae (n = 77), K. oxytoca (n = 50), K. terrigena (n = 49) and K. planticola (n = 50) in 93%, 98%, 96% and 100%, respectively, but did not bind to LPS of other Gram-negative genera (n = 159) as tested by Western blots and dot blots using proteinase K-digested whole cell lysates as antigens. Western blot analyses indicated that the antibody bound only to those LPS molecules which did not carry an O-antigen and that the antibody is thus different from those already published.

Humoral immune response to Klebsiella capsular polysaccharides in HLA-B27-positive patients with acute anterior uveitis and ankylosing spondylitis.

Klebsiella is suggested to trigger ankylosing spondylitis (AS) and acute anterior uveitis (AAU) in HLA-B27-positive individuals. Previous investigations showed an increased antibody response to the Klebsiella capsular types K26, K36, and K50 in sera from HLA-B27-positive AS patients. In the present study the prevalence and titers of antibodies against Klebsiella capsular antigens were measured by means of an ELISA in 32 sera from HLA-B27-positive AAU patients either with (n = 10) or without AS (n = 22) and compared with sera from HLA-B27-negative AS-patients (n = 13). Sera from either HLA-B27-positive (n = 45) or negative (n = 40) healthy individuals served as control. Sera from HLA-B27-positive AAU with or without AS showed significantly higher antibody prevalence and IgG-titers against capsular antigens of the Klebsiella serotypes K26, K36, and K50 when compared with sera from HLA-B27-negative AS patients or with healthy controls. These results might be taken to indicate the predominance of these serotypes in the HLA-B27-associated AS and AAU.

Inability of encapsulated Klebsiella pneumoniae to assemble functional type 1 fimbriae on their surface.

We screened phase variants of Klebsiella pneumoniae isolates for the expression of capsule and type 1 fimbriae and found that all of the 22 blood isolates were encapsulated and did not express type 1 fimbriae while 10 of 11 urinary tract isolates expressed type 1 fimbriae but were unencapsulated. Phase variants from selected isolates were found to be either unencapsulated and fimbriated or lacked both structures. Variants expressing both structures were not detected. Fimbrial subunits FimH and FimA were localized in the periplasmic space of the parent strain and on the surface of the unencapsulated variants. The results suggest that capsule formation impedes assembly of pre-formed fimbrial subunits on the bacterial surface.

Klebsiella capsular type K7 in relation to toxicity, susceptibility to phagocytosis and resistance to serum.

Klebsiella strains possessing capsule type K7 are found predominantly in respiratory secretions. To investigate the importance of this K antigen in virulence, 13 K7 strains were compared with K2 capsulate isolates which are generally regarded as highly virulent. The toxicity of the strains was determined in a mouse peritonitis model. Generally, K7 isolates were significantly less toxic for mice than K2 strains. In the absence of serum, neither capsule type showed much stimulation of leucocytes, measured as the chemiluminescence (CL) response of human polymorphonuclear leucocytes (PMNL). However, in the presence of normal human serum, CL values with K7 strains increased considerably, whereas the CL response to K2 isolates was unaffected. Correspondingly, intracellular killing by PMNL was observed with K7 strains only, whereas K2 isolates proved to be relatively resistant to phagocytic destruction. No correlation was found between capsule type K7 and serum resistance. These data suggest that, in contrast to K2, capsule type K7 may not be a critical factor in the virulence of K7-capsulate Klebsiella strains nor does it seem to act as an antiphagocytic barrier.

Expression of putative virulence factors by clinical isolates of Klebsiella planticola.

A total of 92 clinical isolates of Klebsiella planticola from man was examined with respect to the production of haemagglutinins and siderophores, serum resistance and distribution of capsular types. For comparison, a group of 207 clinical isolates of K. pneumoniae was also studied. The percentages of K. planticola strains able to express mannose-sensitive haemagglutination, indicating type 1 fimbriae (83%) and mannose-resistant and Klebsiella-like agglutination, indicating type 3 fimbriae (69%), as well as to produce the siderophores enterobactin (100%) and aerobactin (2.2%) were almost identical to those of the K. pneumoniae strains. Similarly, the proportion of serum-resistant strains (30%) was comparable to that of K. pneumoniae (25%). The capsule types most often detected in K. planticola were K14 (13%), K2 (9%) and K70 (9%). The incidence of K2, which is the predominant capsular type in K. pneumoniae, was similar in both species. These findings show that K. planticola, which is being detected with increasing frequency in clinical specimens from man, has the ability to express similar putative virulence factors to K. pneumoniae, suggesting that they may have similar pathogenicity.

Characterisation of Hafnia alvei isolates from human clinical extra-intestinal specimens: haemagglutinins, serum resistance and siderophore synthesis.

Extra-intestinal Hafnia alvei isolates are rarely considered to be pathogenic. To investigate whether such strains are able to produce virulence factors, a total of 70 clinical H. alvei isolates was compared with clinical extra-intestinal isolates of other members of the enterobacterial tribe Klebsiellae (Kiebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens). Whereas mannose-sensitive haemagglutination (MSHA) was less common in H. alvei (59%) than in K. pneumoniae (86%) and E. cloacae (89%) isolates, the incidences of mannose-resistant haemagglutination indicative of type 3 pili (MR/K-HA) and of serum resistance properties were not lower. All H. alvei strains secreted siderophores but, unlike the other enterobacterial species examined, the siderophore type was neither enterobactin nor aerobactin. Although the low pathogenicity of H. alvei isolates could not be attributed to any of the factors investigated, the mean number of factors expressed by each H. alvei isolate was significantly lower than that expressed by K. pneumoniae and E. cloacae isolates but did not differ significantly from that of S. marcescens. Based on these findings, the low pathogenicity of H. alvei appears to be due to its low frequency of expression of virulence factors as compared with clinically significant species such as K. pneumoniae and E. cloacae.

Evaluation of a competitive ELISA method for the determination of Klebsiella O antigens.

Strains of Klebsiella spp. are often inagglutinable by O-specific antisera because of the copious capsule produced by most isolates. A competitive ELISA method based on the observation that bacterial supernates containing homologous O antigen specifically inhibited the reaction of type-specific antisera with purified LPS coated on ELISA plates was used to examine the O antigen of 82 isolates of different Klebsiella species and subspecies. The O antigens O1/2ab (19 isolates), O2ab (13 isolates), O2ac (11 isolates) and O3 (16 isolates) were found to account for > 70% of the O antigenic types. Overall, 65 (79%) of the strains could be assigned to a specific O serogroup. The method is suitable for examining the role of individual O antigens in systemic klebsiella infections such as nosocomial septicaemia and pneumonia.

Molecular signatures of a disturbed nasal barrier function in the primary tissue of Wegener's granulomatosis.

Wegener's granulomatosis (WG) is a complex autoimmune disease of unknown etiology, frequently involving localized inflammation of the nasal mucosa as an early manifestation. The current hypothesis suggests that the disease is triggered by a disturbed interaction between genetic and environmental effects, such as an altered microflora at mucosal layers. In this study, a systematic assessment of 49 transcripts with potential pathophysiological relevance was performed using quantitative real-time PCR in nasal mucosa samples of more than 80 individuals, including normal control (NC) individuals and disease controls. In addition, colonization with Staphylococcus aureus was quantified in the same individuals to assess its impact on transcriptomic signatures. Transcription profiles show an increased heterogeneity in diseased individuals. In all, 10 transcripts were identified to be differentially expressed (P≤0.05, false discovery rate ≤0.05) between patients with WG and NC individuals. These transcripts include antimicrobial peptides (human ß-defensin (DEFB)1: fold-change WG vs. controls: +4.45, lysozyme: -3.4, DEFB4 and S100A7 (S100 calcium-binding protein A7): both "switched on" in WG), innate immune receptors (Toll-like receptor 4: -2.1, NOD-like receptor C3: -2.1, scavenger receptor CD36: +2.9), and cytokines (interferon-γ: -14, transforming growth factor-ß 1: -1.4, interleukin-17D: -2.7). These transcriptional profiles are independent of S. aureus colonization. This study for the first time describes that, on the basis of data obtained from the primary nasal tissue, WG exhibits molecular features that allow its differentiation from other inflammatory disorders with involvement of the nasal mucosa. Further studies based on these findings may enable the identification of subphenotypes, which are currently discussed as an important target for a personalized medicine approach, aiming to reduce side effects and the number of therapy non-responders.

Antimicrobial properties of the equine alpha-defensin DEFA1 against bacterial horse pathogens.

Defensins are small effector molecules of the innate immune system, synthesised by various organisms including plants and animals. The peptides act as endogenous antibiotics with an antimicrobial activity against a broad spectrum of microbes including bacteria, fungi and viruses. alpha-Defensins are a subgroup of the defensin family, their synthesis is limited to some tissues and furthermore to some mammalian species including the horse. Equine DEFA1 is an enteric alpha-defensin exclusively produced in Paneth cells. The peptide showed an activity against a broad spectrum of microbes, but typical pathogens of the horse were not included in the previous antimicrobial studies. Here, we report the antibacterial properties of DEFA1 against clinical isolates of typical horse pathogens including Rhodococcus equi, various streptococci strains, Salmonella choleraesuis, and Pasteurella multocida. The recombinantly expressed DEFA1 peptide exerted potent activity against these pathogenic bacteria. The highest susceptibility showed R. equi. Three genetically different strains of R. equi were killed at low micromolar concentrations, comparable with conventionally used antibiotics.

[Diagnosis, therapy and current research aspects of selected chronic inflammatory diseases with head and neck involvement]. / Diagnostik, Therapie und aktuelle Forschungsaspekte ausgewählter chronisch-entzündlicher Erkrankungen mit Beteiligung im Kopf-Hals-Bereich.

Wegener's granulomatosis, Churg-Strauss syndrome and analgesics intolerance syndrome with polyps demonstrate non-specific manifestations in the head and neck region. These symptoms can often lead to early diagnosis and initiation of the correct therapy. However, symptoms are often ambiguous and many rare differential diagnoses must be borne in mind. This clinical picture presents a challenge for the otorhinolaryngologist, who is commonly the first contacted physician. Diagnostics and therapy have to be carried out in an interdisciplinary approach between rheumatologist, pulmonologist, pathologist, radiologist, ophthalmologist, infection specialist and nephrologist. Despite significant scientific and therapeutic advances, these diseases remain incurable. In recent decades they have lost their life-threatening character (Wegener's granulomatosis) and are now chronically relapsing diseases. Their aetiology, however, is still unclear and treatment leads to a wide spectrum of undesirable effects. Research work is needed to advance diagnostics and therapy in this field. Recent research aspects are presented in this article.

Isolation of Klebsiella terrigena from human feces: biochemical reactions, capsule types, and antibiotic sensitivity.

Colonization of the human intestinal tract by a newly proposed species, K. terrigena, was investigated. 5377 different stool specimens from healthy persons (food handlers) yielded 50 isolates (0.9%). Biochemically, low frequencies in the degradation of urea, dulcitol, and utilization of citrate at 37 degrees C were found when compared to K. pneumoniae. At 30 degrees C, urea hydrolysis was observed twice as often as at 37 degrees C. Apart from ampicillin, K. terrigena was susceptible to 12 other antimicrobial drugs tested. Multiple drug resistance was rare, few isolates being resistant against 2-4 antibiotic agents. Capsule typing revealed 30 different serotypes, K 70 and K 14 were the most frequent. Six strains expressed capsule types K 2 and K 5, which have been reported to be associated with virulence in K. pneumoniae. A possible pathogenic role of K. terrigena is discussed.

Phenotypic properties of Klebsiella pneumoniae and K. oxytoca isolated from different sources.

474 Klebsiella pneumoniae and K. oxytoca strains isolated from different sources (human clinical material, feces of healthy subjects, sewage) were investigated for phenotypic properties. Characteristics analyzed were cultural activities, antimicrobial susceptibilities and capsule types. Comparison of both species revealed differences in adonitol fermentation and resistance to tetracycline, nalidixic and pipemidic acid. Capsule types 2, 7 and 33 were frequently found in K. pneumoniae, but not in K. oxytoca. On the other hand, K 66 was common in K. oxytoca, but not in K. pneumoniae. With regard to the source of isolation, clinical strains of both species proved to be more resistant to mezlocillin, azlocillin and cephalothin than fecal and sewage strains. Similarly, resistances of K. pneumoniae to cotrimoxazole, nalidixic and pipemidic acid were most frequent in clinical strains. Multiple drug resistances were found most often in clinical isolates. Biochemically, different frequencies of positive reactions for urease, lysine decarboxylase activity and acetoin production were found between the groups. Capsule typing demonstrated K2 and K7 in K. pneumoniae and K55 and K66 in K. oxytoca to be more common in clinical and fecal isolates than in sewage strains. While cultural characteristics did not allow discrimination of strains from different sources, capsule typing indicated clinical isolates to be more phenotypically related to strains from feces than to sewage isolates.

Bacteriocin typing of environmental Klebsiella isolates.

A total of 88 environmental Klebsiella pneumoniae and K. oxytoca isolates were typed according to their bacteriocin susceptibility patterns. Bacteriocin typing was performed by a modification of the scrape-and-point method, using a set of 8 producer strains. This method proved to be very applicable to environmental strains of both species; all but one strain could be typed. Twenty-one different patterns were observed. Type 1-3456-8 was most common in both species. In contrast to K. pneumoniae, more than half of the K. oxytoca strains fell into only two bacteriocin susceptibility patterns. Bacteriocin types 1 and 3 showed a very broad spectrum, more than 95% of the isolates being sensitive to one of these bacteriocins.

Hemagglutinins of Klebsiella pneumoniae and K. oxytoca isolated from different sources.

482 Klebsiella pneumoniae and K. oxytoca strains isolated from different sources (human clinical material, feces of healthy subjects, sewage) were investigated for expression of MS-(mannose-sensitive) and MR/K-(mannose-resistant, Klebsiella-like) hemagglutinins. Regardless of the source of isolation, the majority of K. pneumoniae strains induced mannose-sensitive hemagglutination, but very few K. oxytoca isolates produced this type of hemagglutinin. In both species, most strains induced MR/K-hemagglutination. In general, environmental isolates showed a lesser incidence in production of each hemagglutinin and a higher percentage of non-hemagglutinating strains than fecal and clinical strains. These results suggest clinical isolates to be phenotypically more related to fecal than to environmental strains.

Isolation of Klebsiella terrigena from clinical specimens.

In a three-year survey conducted from 1988 to 1990 Klebsiella isolates from human clinical specimens were subjected to additional tests to identify any Klebsiella terrigena strains. Ten strains of Klebsiella terrigena (0.4%) were found among 2355 indole-negative Klebsiella isolates. Most of the isolates were recovered from the respiratory tract. In the API20EC system almost exclusively biotypes no. 1777771 and 1777671 were observed. Serotyping revealed capsule types K2, K5 and K18 in two strains each. In antibiotic susceptibility tests the strains were shown to be comparable in sensitivity to Klebsiella pneumoniae.

Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors.

Bacteria belonging to the genus Klebsiella frequently cause human nosocomial infections. In particular, the medically most important Klebsiella species, Klebsiella pneumoniae, accounts for a significant proportion of hospital-acquired urinary tract infections, pneumonia, septicemias, and soft tissue infections. The principal pathogenic reservoirs for transmission of Klebsiella are the gastrointestinal tract and the hands of hospital personnel. Because of their ability to spread rapidly in the hospital environment, these bacteria tend to cause nosocomial outbreaks. Hospital outbreaks of multidrug-resistant Klebsiella spp., especially those in neonatal wards, are often caused by new types of strains, the so-called extended-spectrum-beta-lactamase (ESBL) producers. The incidence of ESBL-producing strains among clinical Klebsiella isolates has been steadily increasing over the past years. The resulting limitations on the therapeutic options demand new measures for the management of Klebsiella hospital infections. While the different typing methods are useful epidemiological tools for infection control, recent findings about Klebsiella virulence factors have provided new insights into the pathogenic strategies of these bacteria. Klebsiella pathogenicity factors such as capsules or lipopolysaccharides are presently considered to be promising candidates for vaccination efforts that may serve as immunological infection control measures.

Bacteriocin typing of Klebsiella spp. isolated from different sources.

To evaluate whether clinical Klebsiella isolates differ from nonclinical strains with respect to bacteriocin susceptibility patterns, a total of 452 Klebsiella pneumoniae and K. oxytoca strains isolated from different sources were examined. Bacteriocin typing was done by a modification of the scrape-and-point method, using a set of eight producer strains. 96% of the strains were typable. Forty-one different bacteriocin susceptibility patterns were observed. While two thirds of the K. oxytoca isolates belonged to only three different bacteriocin types, the K. pneumoniae strains showed a more heterogeneous distribution of patterns. No differences in pattern distribution were observed between isolates from clinical, fecal, or environmental sources. Certain bacteriocins showed a very broad spectrum of activity; e.g. 93% of all isolates were susceptible to bacteriocin type 3. The results suggest that nonclinical Klebsiella strains do not show other bacteriocin susceptibility types than clinical isolates do.