Bacteroides fragilis: A whole MALDI-based workflow from identification to confirmation of carbapenemase production for routine laboratories.

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level. The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well. The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections.

Structure and function of proteins of the phosphotransferase system and of 6-phospho-beta-glycosidases in gram-positive bacteria.

New information about the proteins of the phosphotransferase system (PTS) and of phosphoglycosidases of homofermentative lactic acid bacteria and related species is presented. Tertiary structures were elucidated from soluble PTS components. They help to understand regulatory processes and PTS function in lactic acid bacteria. A tertiary structure of a membrane-bound enzyme II is still not available, but expression of Gram-positive genes encoding enzymes II can be achieved in Escherichia coli and enables the development of effective isolation procedures which are necessary for crystallization experiments. Considerable progress was made in analysing the functions of structural genes which are in close vicinity of the genes encoding the sugar-specific PTS components, such as the genes encoding the tagatose-6-P pathway and the 6-phospho-beta-glycosidases. These phosphoglycosidases belong to a subfamily of the beta-glycosidase family I among about 300 different glycosidases. The active site nucleophile was recently identified to be Glu 358 in Agrobacterium beta-glucosidase. This corresponds to Glu 375 in staphylococcal and lactococcal 6-phospho-beta-galactosidase. This enzyme is inactivated by mutating Glu 375 to Gln. Diffracting crystals of the lactococcal 6-P-beta-galactosidase allow the elucidation of its tertiary structure which helps to derive the structures for the entire glycosidase family 1. In addition, a fusion protein with 6-phospho-beta-galactosidase and staphylococcal protein A was constructed.

Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes.

Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing alpha and unprocessed beta subunits. A human homologue of the yeast proteasome maturation factor Ump1 was identified in 2D gels of 16 S precursor preparations and designated as POMP (proteasome maturation protein). We show that POMP is detected only in precursor fractions and not in fractions containing mature 20 S proteasome. Northern blot experiments revealed that expression of POMP is induced after treatment with interferon gamma. To analyse the role of the beta 5 propeptide for proper maturation and incorporation of the beta 5 subunit into the complex, human T2 cells, which highly express derivatives of the beta 5i subunit (LMP7), were studied. In contrast to yeast, the presence of the beta 5 propeptide is not essential for incorporation of LMP7 into the proteasome complex. Mutated LMP7 subunits either carrying the prosequence of beta 2i (LMP2) or containing a mutation in the active threonine site are incorporated like wild-type LMP7, while a LMP7 derivative lacking the prosequence completely is incorporated to a lesser extent. Although the absence of the prosequence does not affect incorporation of LMP7, its deletion leads to delayed proteasome maturation and thereby to an accumulation of precursor complexes. As a result of the precursor accumulation, an increased amount of the POMP protein can be detected in these cells.

Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light.

A comprehensive comparison of antioxidant defenses in the dermis and epidermis and their response to exposure to ultraviolet (UV) irradiation has not previously been attempted. In this study, enzymic and non-enzymic antioxidants in epidermis and dermis of hairless mice were compared. Enzyme activities are presented both as units/gram of skin and units/milligram of protein; arguments are presented for the superiority of skin wet weight as a reference base. Catalase, glutathione peroxidase, and glutathione reductase (units/gram of skin) were higher in epidermis than dermis by 49%, 86%, and 74%, respectively. Superoxide dismutase did not follow this pattern. Lipophilic antioxidants (alpha-tocopherol, ubiquinol 9, and ubiquinone 9) and hydrophilic antioxidants (ascorbic acid, dehydroascorbic acid, and glutathione) were 24-95% higher in epidermis than in dermis. In contrast, oxidized glutathione was 60% lower in epidermis than in dermis. Mice were irradiated with solar light to examine the response of these cutaneous layers to UV irradiation. After irradiation with 25 J/cm2 (UVA + UVB, from a solar simulator), 10 times the minimum erythemal dose, epidermal and dermal catalase and superoxide dismutase activities were greatly decreased. alpha-Tocopherol, ubiquinol 9, ubiquinone 9, ascorbic acid, dehydroascorbic acid, and reduced glutathione decreased in both epidermis and dermis by 26-93%. Oxidized glutathione showed a slight, non-significant increase. Because the reduction in total ascorbate and catalase was much more severe in epidermis than dermis, it can be concluded that UV light is more damaging to the antioxidant defenses in the epidermis than in the dermis.

Dose-response effects of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis.

There has not as yet been an integrated, comprehensive study of the responses of dermis and epidermis in vivo to a wide range of ultraviolet (UV) doses, encompassing all major antioxidants and a sensitive marker of oxidative damage. We have irradiated hairless mice with simulated solar light at doses of 2, 5, 12.5, and 25 J/cm2 combined UVA and UVB (0.8 to 10 MED) and measured enzymic and non-enzymic antioxidants as well as lipid hydroperoxides in both epidermis and dermis to elucidate the response of cutaneous antioxidant defense mechanisms to UV stress. Among the nonenzymic antioxidants two different dose-response patterns were seen. Ascorbate was rapidly depleted at doses between 0 and 5 J/cm2 but was less affected between 5 and 25 J/cm2. In contrast, glutathione, ubiquinol/one, and alpha-tocopherol levels remained approximately equal to control levels between 0 and 5 J/cm2, then decreased to varying degrees from 5 to 25 J/cm2; ubiquinol was almost completely depleted, whereas alpha-tocopherol dropped only 30%. The concentration of lipid hydroperoxides increased throughout the dose range. These results may be explained partly by direct destruction of some antioxidants by UV light, partly by the separate antioxidant functions of the compounds, and partly by recycling of some antioxidants (e.g., alpha-tocopherol) at the expense of others (e.g., ubiquinol). Even at the lowest dose (0.8 MED) lipid hydroperoxide formation was observed. Among the enzymic antioxidants, superoxide dismutase activity decreased significantly (to 63.6% of initial activity for epidermis and 51.5% for dermis at 25 J), whereas activities of glutathione peroxidase and glutathione reductase decreased slightly. Catalase activity decreased dramatically at doses above 5 J (to 11.8% of initial activity in epidermis and 27.7% in dermis at 25 J). The dramatic loss of catalase is almost entirely accounted for by direct destruction by the simulated solar light, but superoxide dismutase was unaffected by direct exposure; hence its destruction must be due to indirect effects, either mediated by free radicals or other harmful species formed upon irradiation. At low doses of UV light many components of the cutaneous antioxidant system were damaged, whereas at high doses all components were damaged and some were almost completely destroyed.

Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin.

We measured enzymic and non-enzymic antioxidants in human epidermis and dermis from six healthy volunteers undergoing surgical procedures. Epidermis was separated from dermis by curettage and antioxidants were measured by high-performance liquid chromatography (HPLC) or standard spectrophotometric methods. The concentration of every antioxidant (referenced to skin wet weight) was higher in the epidermis than in the dermis. Among the enzymic antioxidants, the activities of superoxide dismutase, glutathione peroxidase, and glutathione reductase were higher in the epidermis compared to the dermis by 126, 61 and 215%, respectively. Catalase activity in particular was much higher (720%) in the epidermis. Glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, which provide reduced nicotinamide adenine dinucleotide phosphate (NADPH), also showed higher activity in the epidermis than the dermis by 111% and 313%, respectively. Among the lipophilic antioxidants, the concentration of alpha-tocopherol was higher in the epidermis than the dermis by 90%. The concentration of ubiquinol 10 was especially higher in the epidermis, by 900%. Among the hydrophilic antioxidants, concentrations of ascorbic acid and uric acid were also higher in the epidermis than in the dermis by 425 and 488%, respectively. Reduced glutathione and total glutathione were higher in the epidermis than in the dermis by 513 and 471%. Thus the antioxidant capacity of the human epidermis is far greater than that of dermis. As the epidermis composes the outermost 10% of the skin and acts as the initial barrier to oxidant assault, it is perhaps not surprising that it has higher levels of antioxidants.

alpha-Lipoic acid as a biological antioxidant.

alpha-Lipoic acid, which plays an essential role in mitochondrial dehydrogenase reactions, has recently gained considerable attention as an antioxidant. Lipoate, or its reduced form, dihydrolipoate, reacts with reactive oxygen species such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also protects membranes by interacting with vitamin C and glutathione, which may in turn recycle vitamin E. In addition to its antioxidant activities, dihydrolipoate may exert prooxidant actions through reduction of iron. alpha-Lipoic acid administration has been shown to be beneficial in a number of oxidative stress models such as ischemia-reperfusion injury, diabetes (both alpha-lipoic acid and dihydrolipoic acid exhibit hydrophobic binding to proteins such as albumin, which can prevent glycation reactions), cataract formation, HIV activation, neurodegeneration, and radiation injury. Furthermore, lipoate can function as a redox regulator of proteins such as myoglobin, prolactin, thioredoxin and NF-kappa B transcription factor. We review the properties of lipoate in terms of (1) reactions with reactive oxygen species; (2) interactions with other antioxidants; (3) beneficial effects in oxidative stress models or clinical conditions.

The threshold of age in exercise and antioxidants action.

Physical activity and exercise are important factors in determining the quality of life in old animals and humans. With age there is a slow but significant reduction in muscle mass and ability to perform certain physical activities. This may be due to changes with the age of muscle composition and protein turnover, as well as decrease of trophic influence in neural control of muscles of old individuals. Exercise in general was shown to improve muscle performance even in old age. However a concept of threshold of age in exercise was advanced forward in the 1970s. Accordingly, the idea was that for a given exercise of a particular duration and intensity there is a certain age beyond which this exercise may not have a positive influence, but can become detrimental to the exercising animal or human. Recent studies on the effect of antioxidants such as Vitamins C and E and selenium have shown that these agents could decrease the free radical associated muscle damage caused by extensive exercise. Thus, administration of these antioxidants especially vitamins C and E may reduce the oxidative damage due to exercise, and may alter the threshold of age by delaying it to an older age.

Ocular signs in thiamine-deficient monkeys and in Wernicke's disease in humans.

Thiamine deficiency in the monkey is the animal counterpart of Wernicke's disease in humans. In the present study, thiamine deficiency was induced in 11 monkeys while three monkeys were given paired feedings supplemented by thiamine hydrochloride and three monkeys were maintained on regular chow. The typical clinical symptoms were apathy, inattention to peripheral stimuli, ataxia, ptosis, mydriasis progressing to pupillary areflexia, nystagmus, and ophthalmoparesis progressing to total ophthalmoplegia. With thiamine treatment, recovery was prompt and complete in mild to moderate cases but delayed and incomplete in severe cases. The animals were killed six or more months after discontinuance of the experiments to determine the chronic effects of treated thiamine deficiency. The significant abnormalities in the brain stem were symmetric gliosis and neuronal loss in the inferior colliculi, the regions of the third and sixth nerve nuclei, and the medial vestibular nuclei. White matter was characteristically spared. With the exception of the inferior colliculi, the target sites for neuropathologic changes were the centers for ocular motor control.

Sex differences in muscarinic receptor binding after chronic ethanol administration in the rat.

Male and female rats were administered ethanol (5% v/v) in a liquid diet for 18 weeks. Pair-fed control animals were fed the same diet except that dextrose was substituted isocalorically for ethanol. Normal controls received a commercial laboratory chow for the same duration. Results showed that, in females, chronic ingestion of an ethanol liquid diet significantly increased the number of muscarinic receptor binding sites compared to both control groups. In contrast, for males, there was no significant difference in the mean number of binding sites among the treatment groups. Furthermore, the mean maximum number of binding sites for males and females varied across brain areas. Males had a significantly greater number of receptor binding sites than females in the striatum, while females had a greater number in the cortex. It was suggested that the geuder differences observed in the present study could be mediated by hormonal effects on central muscarinic functioning.

Elevated muscle vitamin E does not attenuate eccentric exercise-induced muscle injury.

The purpose of this study was to evaluate the effect of elevated muscle vitamin E content on skeletal muscle damage from eccentric exercise. Sixty Sprague-Dawley rats were put on a normal (40 IU vitamin E/kg food) or supplemented (10,000 IU vitamin E/kg food) diet for 5 wk. Injury in soleus muscle was determined using several criteria: reductions in maximal tetanic force and number of intact fibers per square millimeter and elevations in muscle glucose 6-phosphate dehydrogenase activity and plasma creatine kinase activity, either immediately (0 h) or 2 days (48 h) after a downhill walking protocol. Sedentary animals were also tested but did not exercise. Muscle vitamin E levels were significantly elevated (approximately 3- to 4-fold), and susceptibility of the muscles to oxidant stress was decreased, after supplementation. However, vitamin E supplementation did not attenuate injury by any of the criteria employed. Maximal tetanic force decreased approximately 20% at 0 and 48 h after exercise in both groups. The number of intact fibers per square millimeter decreased approximately 30-35% in both groups at 0 and 48 h. Glucose 6-phosphate dehydrogenase activity increased approximately 50-100% in both groups at 48 h, and plasma creatine kinase activity was elevated approximately 2- to 2.5-fold at 0 h in both groups. These findings do not support a major role for free radical damage to muscle membranes in the initiation of injury from eccentric exercise, although they do not disprove free radical involvement in the etiology.

Comparison of Ca-DTPA and Zn-DTPA in removing 234Th from the rat.

The relative effectiveness of Ca-DTPA and Zn-DTPA as a function of the dose, number of doses and time of their administration in removing 234Th that had been administered as the citrate or nitrate from various rat organs was investigated. With a single early treatment (1.5 min after intravenous injection of 234Th), Zn-DTPA was clearly less effective than Ca-DTPA over the whole dose range (30 mumol/kg body weight-1000 mumol/kg body weight) tested. In the skeleton, 1000 mumol Zn-DTPA/kg removed as much 234Th (about 50%) as 30 mumol Ca DTPA/kg. Prompt treatment with 100 mumol Ca-DTPA/kg body weight diminished the skeletal 234Th content by about 70%; but when it was delayed by 6 h or 4 d, the 234Th content was reduced by only about 20% and 10%, respectively. The effect of DTPA in the bone was independent of 234Th compound, while in the soft tissues 234Th injected as nitrate was less affected than the citrate. The effect of DTPA was potentiated when treatment was repeated; the end effect was dependent on the time of the first DTPA injection and on the total number of doses. Thus, about 90% and 50% less 234Th was found in the bones after repeated treatment started at 1.5 min and 4 d after administration of 234Th, respectively. Under the conditions of a delayed repeated treatment, Ca-DTPA and Zn-DTPA were equally effective.

Effects of information-giving and communication during orthodontic consultation and treatment. Part 3: Optimized orthodontist-patient communication.

Effective, orthodontist-patient communication must meet certain minimum requirements whose importance might vary, depending on the individual case and stage of treatment. In the course of treatment the following tasks are of primary importance: 1. Before the start of treatment, during preliminary consultation, information tailored in extent and quality to the individual patient has to be provided, a professional assessment of treatment needs made, and a relationship of mutual trust established. 2. In the initial phase of treatment, the individual shaping and optimisation of treatment needs is a matter of priority if initial acceptance of the appliance is to be assured. The patient should be advised how to meet the treatment needs in his individual situation. 3. In the further course of treatment, the fulfillment of the demands has to be repeatedly clarified. Feedback with regard to patient cooperation should be handled with caution. A tight recall schedule, a time-keeping record and involvement of the parents are obvious interventions in cases of inadequate cooperation. The treatment may also have to be reevaluated.

Effectiveness of intra- and extraoral aids to the bionator. A controlled study within the scope of the "Wuerzburg concept".

A bimaxillary device is effective only if the therapeutic position of the mandible is maintained for as long a period as possible by day and night, i.e. the lower jaw cannot "drop" from the construction bite position. One simple means of preventing the mandible from dropping out of the bimaxillary appliance during sleep and thus of ensuring adaptation of the muscles and of the joint structures during the night without impairing active adaptation during daytime functions (speaking, swallowing) is being used at Wuerzburg University Department of Orthodontics: In connection with anterior traction, which anchors the bimaxillary appliance to the maxilla, attachments are fixed to the mandibular canines or first premolars, so that the mandible is kept in the therapeutic position during the night by means of elastics. Besides the description of this method, the results of a clinical study on its effectiveness are presented in this paper. In the experimental group (20 Class II/1 patients) treated with the combination of bionator with anterior extraoral traction and up-and-down elastics, the increase of the SNB angle and accordingly of the ANB angle was twice as great as in a control group treated only with bionator and extraoral traction without additional securing of the mandible.

Lüscher colour test in orthodontic patients. Practicability for compliance assessment?

The Lüscher colour test has been repeatedly recommended as a tool for the prediction of orthodontic treatment course and patient cooperation despite its scientific shortcomings. A series of studies involving patients aged 9 to 16 years undergoing treatment with removable appliances at various orthodontic practices and at one university clinic provided no evidence of systematic correlations between Lüscher test colour preferences and cooperation with appliance wear. There was only slight consistency in colour preferences in a re-test conducted a few months after baseline measurements. Statistical evaluation of specific hypotheses and exploratory analysis of the global potential of the test provided no support for generalizable and useful correlations between colour preferences and patient compliance. Therefore the Lüscher test cannot be generally recommended for the rating of patient compliance in the clinical orthodontic practice.

Assessment of the combined orthodontic-surgical treatment from the patients' point of view. A longitudinal study.

In a longitudinal study, 40 patients who underwent a combined orthodontic-surgical treatment, were interviewed 4 weeks before operation and 1 week and/or half a year after surgery in order to evaluate patients' expectations and appraisal of the operation and their psycho-social situation. The comparisons between the patients' pre- and postoperative statements yielded the following picture: the patients showed a high degree of satisfaction with the result of the operation. They judged their postoperative facial appearance, with the help of a "semantic differential', to be significantly more attractive. Satisfaction also was expressed in a higher degree of psychological well-being and thus an increase in self-confidence and motivation. Postoperatively, the "Giessen test' showed also a distinctly positive change of the patients' mood and improved experiences with social interaction. Before the operation almost half of the patients feared sensitivity loss or disturbance. One out of 5 patients did in fact suffer from this problem--but not from pain--half a year after the operation. Some patients felt they had received insufficient prior information about the procedure and the risk of the operation. In spite of all burdens for the patients, the assessment of the result of the operation is (generally) positive, not only from the medical point of view.

Parental reactions following the birth of a cleft child.

Parental experiences and behaviour following the birth of a cleft child were studied by comparing the results of 2 interview studies conducted at a 7-year interval. The parents of a total of 55 cleft children, most of whom had passed the primary treatment, were interviewed about their psychosocial adaptation, using an interview scheme with the focus on: a) emotional, cognitive and social reactions following birth, b) information-giving and support provided by the medical staff, c) interaction with the cleft child, and d) impact on family life. In both studies, more than 70% of the parents were very shocked at the birth of the cleft child. In the later study, parents more often expressed depression, social avoidance and feelings of guilt. They indicated better psychosocial care and more understanding provided by the medical staff compared to the earlier study. Twice as many parents were satisfied with the information provided by the cleft centre, however, the amount of information had little impact on coping with the shock. In conclusion, further sensitisation of the medical staff with respect to parental coping processes and needs for information and emotional support was called for, along with the inclusion of these topics in the respective curricula.

Effectiveness of bionator therapy for Class II malocclusions: a comparative long-term study.

OBJECTIVE: The goal of this retrospective study was to examine the effectiveness of isolated bionator therapy in Class II patients both longitudinally and over the long term. We aimed to determine whether skeletal and/or dentoalveolar reactions differ in relationship to the Angle Class (II, Division 1 vs. II, Division 2). PATIENTS AND METHODS: A total of 50 juvenile patients with Class II malocclusion (♀ n = 26, ♂ n = 24) were included. A total of 24 patients presented an Angle Class II, Division 1 and 26 an Angle Class II, Division 2. We compared the patients' lateral cephalograms taken at the beginning of treatment (t1: 10.1 years), after active therapy (t2: 13.8 years), and at the end of retention (t3: 16.4 years) analyzing the following cephalometric parameters: SNA, SNB, ANB, ANB(ind), SNPog, Wits appraisal, U1-SN, U1-SpP, L1-MeGo, interincisal angle. Mean and standard deviations of each of the variables were calculated. Differences between t1-t2 and t2-t3 were tested for statistical significance. Changes in the variables were then analyzed biometrically for specific differences in terms of Angle Class (II, Division 1 vs. II, Division 2). RESULTS: Between t1 and t2, SNB (p = 0.000) and SNPog (p = 0.000) increased significantly, as did ANB (p = 0.000), while the difference between ANB and ANB(ind) (p = 0.000) and Wits appraisal (p = 0.000) decreased significantly. The dentoalveolar variables U1-SN, U1-SpP, and the interincisal angle changed significantly in both groups. The inclination of the upper incisors was corrected by retrusion in the Class II, Division 1 and by protrusion in the Class II, Division 2 group. Only marginal changes in all variables between t2 and t3 were observed. CONCLUSION: A significant skeletal effect (even in long-time stability) through bionator treatment could be confirmed in this study of Class II, Divisions 1 and 2 patients. The desired effect on the upper front teeth was realized, and there was no appreciable dentoalveolar compensation in the mandible.