[Malnutrition in Elderly Trauma Patients - Comparison of Two Assessment Tools]. / Die Erfassung des Ernährungsstatus alterstraumatologischer Patienten ­ ein Vergleich zweier etablierter Screeningmethoden.

Background: The prevalence of malnutrition in hospitalised patients is reported to be between 16 and 55 % across disciplines. Within hospital care, screening for malnutrition is required. However, in orthopaedics and trauma surgery, there is still no generally accepted recommendation for the methods for such a data survey. In the present study, the following aspects are to be investigated with the help of two established scores: (1) the prevalence of malnutrition in the patient population of geriatric trauma care, and (2) the correlation between methods of data survey. Material and Methods: Between June 2014 and June 2015, a consecutive series of hospitalised trauma patients were studied prospectively with two validated screening instruments to record nutritional status. The study was carried out at a municipal trauma surgery hospital, which is a first level interregional trauma centre as well as a university hospital. The Nutritional Risk Screening (NRS) and the Mini Nutritional Assessment (MNA Short and Long Form) were used. All patients were divided into three age groups: < 65 years, 65-80 years, and > 80 years. The prevalence of malnutrition in geriatric trauma patients and the correlation between the screening instruments were determined. For a better comparison, prescreening and main assessment were applied to all patients. For statistical evaluation, both quantitative and semi-quantitative parameters were used. Furthermore, the Kolmogorov-Smirnov test, Spearman's correlation analysis and the chi-square test were applied. These tests were two-sided and had a level of significance of 5 %. The present study was partially funded by the Oskar-Helene-Heim Foundation. Results: 521 patients (43.8 % women, 56.2 % men), with a mean age of 53.96 ± 18.13 years, were statistically evaluated within the present study. Depending on the method of the data survey, malnutrition (NRS≥3) in geriatric trauma patients varied from 31.3 % (65-80 years) to 60 % (> 80 years). With MNA, 28.8 and 54.3 % of patients were at risk of malnutrition (MNA 17-23.5), while the fractions of patients already suffering from malnutrition (MNA < 17) were 5.4 and 8.6 %, respectively. The correlation between the NRS and MNA total scores increases with the age of the patients. The correlation coefficient for patients under 65 years is r = - 0.380, while among patients aged between 65 and 80, it is r = - 0.481, and for patients over 80 years, there is a medium to strong correlation of r = - 0.638 (each with a Spearman correlation of p < 0.001). For the total population as well as the different age groups, statistically significant correlations were recorded between the categorised scores (chi-square test for linear trend, p < 0.001). Summary: The present study demonstrates high prevalence of malnutrition among the geriatric trauma patients. Because of its easy and rapid application, the NRS has an advantage in clinical use. It was shown that the two methods of data survey were highly correlated.

Analysis of transcriptional control mechanisms of capsule expression in Neisseria meningitidis.

The major virulence factor which contributes to the survival of Neisseria meningitidis in the blood stream and the cerebrospinal fluid is the capsular polysaccharide. Expression of the capsule genes of N. meningitidis serogroups B, C, W-135 and Y is controlled by an intergenic region separating the capsule biosynthesis operon (siaA-D) and the capsule transport operon (ctrA-D). To further investigate capsule expression in N. meningitidis we amplified and sequenced the intergenic region of 42 meningococcal isolates of different serogroups. Sequence variations were found mainly in a repeat region preceding the siaA start codon. Correlation between sequence variation and serogroup could not be observed. To measure the transcriptional and translational activity of the respective intergenic regions we performed transcriptional and translational fusions with the lacZ gene integrated into the chromosome of N. meningitidis. Sequence variations preceding the siaA start codon had no effect on beta-galactosidase activity. Different in vitro growth conditions such as temperature, glucose concentration, osmolarity, pH and iron concentration also did not influence beta-galactosidase activity. Sequential deletions of the intergenic region showed that an Up-like element adjacent to the predicted -35 box is necessary for full transcriptional activity. The deletion of the untranslated region preceding the siaA start codon led to a threefold higher beta-galactosidase activity compared with the full-length construct suggesting that the respective region may be involved in capsule regulation.

Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells.

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive intracellularly in eukaryotic cells such as monocytes, macrophages, and protozoan organisms. The Mip (macrophage infectivity potentiator) protein represents a factor of L. pneumophila necessary for optimal intracellular survival. Interestingly, Mip belongs to the substance class of FK 506-binding proteins and exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. In order to identify amino acids most likely to be involved in the enzymatic activity of Mip, site-directed mutagenized Mip proteins were constructed and characterized. It was shown that an Asp-142 to Leu-142 mutation and a Tyr-185 to Ala-185 substitution resulted in strongly reduced PPIase activity of the recombinant Mip proteins (5.3 and 0.6% of the activity of the wild-type Mip, respectively). Genes coding for the wild-type and for site-directed-mutagenized Mip proteins were used to complement three different Mip-negative mutants of the L. pneumophila Corby, Philadelphia I, and Wadsworth. While Mip protein expression could be restored in the corresponding complementants, significant Mip-specific PPIase activity could be detected only in Mip mutants complemented with wild-type mip genes. To investigate the influence of the PPIase activity of Mip on intracellular survival of L. pneumophila, invasion assays were performed using the macrophage-like cell line U937, human blood monocytes, and Acanthamoeba castellanii. The Mip-negative mutants were approximately 50- to 100-fold less infective for A. castellanii and for human mononuclear phagocytes in vitro compared with their isogenic Mip-positive parental strains. The wild-type invasion rate could be restored by introducing an intact copy of the mip gene into Mip-negative strains. In addition, no differences in intracellular survival were observed between the wild-type isolates and the Legionella strains exhibiting strongly reduced PPIase activity. These data indicated that the enzymatic activity of Mip does not contribute to intracellular survival of L. pneumophila.

The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis.

The lly locus (legiolysin) mediates the browning of the culture medium of Legionella pneumophila in the late stationary growth phase, presumably as a result of synthesis of homogentisic acid. Mutagenesis of the lly gene of the L. pneumophila Philadelphia I derivative JR32 did not affect intracellular replication in the natural host Hartmannella vermiformis. The Lly-negative mutant, however, showed a markedly decreased resistance to ordinary light. The cloned lly gene conferred an increased resistance to light in recombinant L. pneumophila and Escherichia coli K-12, indicating a contribution of the Lly protein to ecological adaptation of Legionella species.

Characterization of Mip proteins of Legionella pneumophila.

The Mip ('macrophage infectivity potentiator') protein of Legionella pneumophila has been shown to be an essential virulence factor, exhibiting peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. The cloning and sequencing of mip genes from three different L. pneumophila strains revealed a single amino acid substitution which did not affect the isomerase property of the enzyme. Mip proteins isolated from two wild-type L. pneumophila strains and from two corresponding Escherichia coli K-12 recombinant clones derived from these strains exhibited identical enzymatic properties and the precursor proteins are processed at identical cleavage sites. The mature Mip proteins exist in an oligomeric form. Site-directed mutagenesis demonstrated that a substitution of an Asp residue at position 142 by a Leu residue affects PPIase activity of Mip.

Sequence determination and mutational analysis of the lly locus of Legionella pneumophila.

The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus.

Analysis of virulence factors of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease is a facultative intracellular bacterium, which in the course of human infection multiplies in lung macrophages predominantly manifesting as pneumonia. The natural habitat of Legionella is found in sweet water reservoirs and man-made water systems. Virulent L. pneumophila spontaneously convert to an avirulent status at a high frequency. Genetic approaches have led to the identification of various L. pneumophila genes. The mip (macrophage infectivity potentiator) determinant remains at present the sole established virulence factor. The Mip protein exhibits activity of a peptidyl prolyl cis trans isomerase (PPIase), an enzyme which is able to bind the immunosuppressant FK506 and is involved in protein folding. The recently cloned major outer membrane protein (MOMP) could play a role in the uptake of legionellae by macrophages. Cellular models are useful in studying the intracellular replication of legionellae in eukaryotic cells. Human cell lines and protozoan models are appropriate for this purpose. By using U 937 macrophage-like cells and Acanthamoeba castellanii as hosts, we could discriminate virulent and avirulent L. pneumophila variants since only the virulent strain was capable of intracellular growth at 37 degrees C. By using these systems we further demonstrated that a hemolytic factor cloned and characterized in our laboratory, legiolysin (lly), had no influence on the intracellular growth of L. pneumophila.

Characterization of legiolysin (lly), responsible for haemolytic activity, colour production and fluorescence of Legionella pneumophila.

A genomic library of Legionella pneumophila, the causative agent of Legionnaires' disease in humans was constructed in Escherichia coli K12 and the recombinant clones were tested for haemolysis and other phenotypic properties. Seven clones were identified which were able to confer haemolysis of human, sheep, and canine erythrocytes but which were unable to mediate proteolytic activities or cytotoxic effects on CHO- or Vero cells. Clones that exhibited this haemolytic property were also able to produce a brown colour and a yellow-green fluorescence activity detected on M9 plates containing tyrosine. The genetic determinant encoding these properties, termed legiolysin (lly) was mapped by Tn1000 mutagenesis and by subcloning experiments. Southern hybridization with an lly-specific gene probe showed that this determinant is part of the genome of L. pneumophila but is not identical to a protease gene of L. pneumophila which also mediates haemolysis. Minicell analysis of lly-specific plasmids exhibited a protein of 39 kDa. Polyclonal antibodies generated against a LacZ-Lly hybrid protein also recognized a 39 kDa protein produced either by the recombinant legiolysin-positive E. coli K12 clones or by L. pneumophila wild-type strains.

Legiolysin, a new hemolysin from L. pneumophila.

Legionella pneumophila generates exotoxins, cytolysins, proteases or hemolysins that damage host cells like erythrocytes or tissue culture cells. The gene for a new L. pneumophila hemolysin without a proteolytic activity was identified, cloned in E. coli and sequenced. The gene product was analysed by SDS-Polyacrylamide-gel-electrophoresis.