[Implications of prehospital estimation of trauma patients for the treatment pathway-An evaluation of the TraumaRegister DGU®]. / Implikationen der prähospitalen Einschätzung des Traumapatienten auf den Behandlungsverlauf ­ Eine Auswertung aus dem TraumaRegister DGU®.

BACKGROUND: In the prehospital acute treatment phase of severely injured patients, the stabilization of the vital parameters is paramount. The rapid and precise assessment of the injuries by the emergency physician is crucial for the initial treatment and the selection of the receiving hospital. OBJECTIVE: The aim of this study was to determine whether the prehospital emergency medical assessment has an influence on prehospital and emergency room treatment. MATERIAL AND METHODS: Data from the TraumaRegister DGU® between 2015 and 2019 in Germany were evaluated. The prehospital emergency medical assessment of the injury pattern and severity was recorded using the emergency physician protocol and compared with the in-hospital documented diagnoses using the abbreviated injury scale. RESULTS: A total of 47,838 patients with an average injury severity score (ISS) of 18,7 points (SD 12.3) were included. In summary, 127,739 injured body regions were documented in the hospitals. Of these, a total of 87,921 were correctly suspected by the emergency physician Thus, 39,818 injured body regions were not properly documented. In 42,530 cases a region of the body was suspected to be injured without the suspicion being confirmed in the hospital. Traumatic brain injuries and facial injuries were mostly overdiagnosed (13.5% and 14.7%, respectively documented by an emergency physician while the diagnosis was not confirmed in-hospital). Chest injuries were underdocumented (17.3% missed by an emergency physician while the diagnosis was finally confirmed in-hospital). The total mortality of all groups was very close to the expected mortality calculated with the revised injury severity classification II(RISC II)-score (12.0% vs. 11.3%). CONCLUSION: In the prehospital care of severely injured patients, the overall injury severity is often correctly recorded by the emergency physician and correlates well with the derived treatment, the selection of the receiving hospital as well as the clinical course and the patient outcome; however, the assessment of injuries of individual body regions seems to be challenging in the prehospital setting.

Emergency laparoscopic cholecystectomy in an unselected cohort: a safe and viable option in a specialist centre.

INTRODUCTION: Patients presenting acutely with symptomatic gallstone-related disease have historically had their laparoscopic cholecystectomy (LC) deferred due to perceived increased operative risks in the acute setting, particularly conversion to open surgery. The aim of this study was to compare morbidity and mortality between unselected cohorts of patients undergoing elective and 'emergency' LC in a District General Hospital. METHODS: All gallstone-related elective and emergency admissions under the care of two specialist laparoscopic surgeons during a two-year period were included. Patients admitted acutely with a diagnosis of biliary colic, acute cholecystitis or gallstone pancreatitis underwent 'emergency' LC during the same admission. Data were collected prospectively on patient demographics, inpatient stay, post-operative course and POSSUM scores. RESULTS: 423 patients underwent LC, of which 301 (71.1%) were elective and 122 (28.9%) were 'emergency' procedures. ASA grades and POSSUM physiologic scores were similar between the two groups. The overall morbidity rates were similar in the emergency and elective groups (13.1% vs. 7.3%, p = 0.088), and there was no significant difference in the rates of major complications including conversion to open surgery (0% vs. 0.3%, NS), bile leak or re-operation between the two groups. 30-day mortality rates were similar in the two groups (0.8% vs. 0%, NS). CONCLUSION: When performed by specialist laparoscopic surgeons, LC in the acute setting is safe with mortality and morbidity rates, including conversion to open surgery, comparable to elective LC.

Screening of Bacillus thuringiensis serotypes by polymerase chain reaction (PCR) for insecticidal crystal genes toxic against coffee berry borer.

Using PCR,257 isolates of Bacillus thuringiensis(Bt) were screened for cry-type genes. Of 257 isolates/strains, 60 isolates were identified as cry7/8, 10 isolates as cry3 and 36 isolates as cry 1I. One specific strain of B. thuringiensis (sumiyoshiensis; T03B 001) was investigated for the presence of cry7 and cry8 genes. Genes Cry7 and cry8 were first detected in this strain using family primers prior to analysis by exclusion polymerase chain reaction (E-PCR) using specific type primers. E-PCR conducted with the above said primers led to the identification by agarose gel electrophoresis of a remaining 1.5 Kb family band indicating a potentially novel gene. This PCR product, (1.5 Kb), was purified from the gel and cloned in pGEM-T Easy vector. Twenty recombinant colonies bearing 1.5 Kb insert were identified and three randomly selected representatives of the group, clones 7, 8 and 10, were sequenced and compared to all cry7 and cry8 sequences available from Gene Bank. Alignments with available DNA and protein sequences showed that all these clones contained a gene related to cry8Aa1. Analysis using protein sequence alignment showed that the sequence from clone 7 differed from the closest relative, known under the new nomenclature as cry 8Aa1, by 44%. The crystal proteins from B. thuringiensis sumiyoshiensis (T03B 001) was toxic to coffee berry borer larvae.

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac.

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.

Lipid-induced pore formation of the Bacillus thuringiensis Cry1Aa insecticidal toxin.

After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death. Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear. Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions. Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the beta-sheet domains. Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression.

Exchange of domain I from Bacillus thuringiensis Cry1 Toxins Influences protoxin stability and crystal formation.

Influence of domain I exchange on the stability and production of Bacillus thuringiensis Cry1 protoxins as well as on the shape of inclusion and toxicity to Spodoptera exigua and Plutella xylostella larvae was investigated. Chimeric genes were prepared by exchanging the regions coding for domain I between Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The AcCC chimera accumulated into bipyramidal inclusion bodies, whereas CEE produced round-shaped inclusion bodies, and ECC and AaEE protoxins produced small granules. AbEE and EAaAa did not produce any inclusion body and were visualized by immunodetection only. AcCC, CEE, ECC, and AaEE were stable to trypsin, whereas AbEE and EAaAa were not. Bioassays showed that the chimeras were not toxic in vivo. However, S. exigua larvae fed with the activated AcCC toxin displayed a lower growth rate.

Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin.

The operon containing the genes encoding the subunits of the binary crystal toxin of Bacillus sphaericus strain LP1-G, BinA and BinB (41.9 kDa and 51.4 kDa, respectively), was cloned and sequenced. Purified crystals were not toxic to Culex pipiens larvae. Comparison of the amino-acid sequences of this strain (Bin4) with those of the three other known toxin types (Bin1, Bin2 and Bin3) revealed mutations at six positions, including a serine at position 93 of BinA4, whereas all other types of BinA toxin from B. sphaericus had a leucine at this position. Reciprocal site-directed mutagenesis was performed to replace this serine in BinA4 from LP1-G with a leucine and the leucine in the BinA2 protein from strain 1593 with a serine. Native and mutated genes were cloned and overexpressed. Inclusion bodies were tested on C. pipiens larvae. Unlike the native Bin4 toxin, the mutated protein was toxic, and the reciprocal mutation in Bin2 led to a significant loss of toxicity. In vitro receptor-binding studies showed similar binding behaviour for native and mutated toxins. In the absence of any experimental data on the 3D structure of these proteins, sequence analysis and secondary-structure predictions were performed. Amino acid 93 of the BinA polypeptide probably belongs to an alpha helix that is sensitive to amino-acid modifications. Position 93 may be a key element in the formation of the BinA-BinB complex responsible for the toxicity and stability of B. sphaericus Bin toxins.

Chemostat modeling of Escherichia coli persistence in conventionalized mono-associated and streptomycin-treated mice.

We have previously shown that Escherichia coli BJ4 has similar doubling time in mice that are mono-associated (having only the inoculated E. coli BJ4) or streptomycin-treated (having mainly gram-positive bacteria plus the inoculated E. coli BJ4). We also showed that when the mice were conventionalized (fed cecum homogenate from conventional mice or ones with a complete microbial flora), the introduction of complete flora in both cases increased the in vivo doubling time, while decreasing the colony counts in fecal samples. To determine whether the increase in doubling time could explain the decrease in colony counts, we analyzed our previous results by a chemostat model. The analysis shows that the increasing doubling time alone is sufficient to explain the decrease in colony counts in mono-associated mice, but not in the streptomycin-treated mice. The observed decreasing rate in colony counts in streptomycin-treated mice is slower than predicted. Furthermore, whereas the model predicted a decrease to extinction in both mice, the E. coli persist at a frequency 10-80 times higher in streptomycin-treated mice than in mono-associated mice. Thus, while a chemostat model is able to explain some of the population dynamics of intestinal bacteria in mice, additional factors not included in the model are stabilizing the system. Because we find that E. coli declines more slowly and to a higher stabilization frequency in streptomycin-treated mice, which have a more diverse flora before conventionalization, we take these results to suggest that the persistence of E. coli populations is promoted by species diversity. We propose that a mechanism for the persistence may be the presence of new E. coli niches created by keystone species in the more diverse flora.

The crystal proteins from Bacillus thuringiensis subsp. thompsoni display a synergistic activity against the codling moth, Cydia pomonella.

Crystal proteins from Bacillus thuringiensis subsp. thompsoni strain HnC are active against the codling moth, Cydia pomonella, a major pest of orchards. Inclusion bodies purified from strain HnC displayed an LC(50) of 3.34 x 10(-3) microgram/microliter. HnC-purified crystals were tenfold more active than Cry2Aa and Cry1Aa toxins, and 100-fold more toxic than Cry1Ab. The 34-kDa and 40-kDa proteins contained in HnC inclusion bodies were shown to act synergistically. The toxicity of crystal proteins produced by the recombinant B. thuringiensis strain BT-OP expressing the full-length native operon was about tenfold higher than that of the 34-kDa protein. When the gene encoding the non-insecticidal 40-kDa protein, which is not active, was introduced into the recombinant strain producing only the 34-kDa protein, the toxicity was raised tenfold and was similar to that of the strain BT-OP.

Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.

Expression of orf1 from the Bacillus thuringiensis NRD-12 cry2Aa1 operon.

The 5' untranslated region and the orf1 sequence from the cry2Aa1 operon from Bacillus thuringiensis subsp. kurstaki NRD-12 were sequenced and compared to that from strain HD-1. The start codon described in HD-1 does not yield in NRD-12 a protein of the expected size of 20 kDa, but a 10-amino acid peptide. A second, highly conserved start codon is located 25 bp downstream from the first one and corresponds to an open reading frame of the same size in all known orf1-related sequences. Expression of lacZ gene fusions created at the level of the first ATG, second ATG, and stop codon of the NRD-12 orf1 sequence showed that orf1 is translated from the second ATG. The expected protein is 19 kDa in size. The expression starts at t2, which is in agreement with the presence of a BtI promoter in the cry2Aa1 operon.

Estimation of growth rates of Escherichia coli BJ4 in streptomycin-treated and previously germfree mice by in situ rRNA hybridization.

The growth physiology of Escherichia coli during colonization of the intestinal tract was studied with four animal models: the streptomycin-treated mouse carrying a reduced microflora, the monoassociated mouse with no other microflora than the introduced strain, the conventionalized streptomycin-treated mouse, and the conventionalized monoassociated mouse harboring a full microflora. A 23S rRNA fluorescent oligonucleotide probe was used for hybridization to whole E. coli cells fixed directly after being taken from the animals, and the respective growth rates of E. coli BJ4 in the four animal models were estimated by correlating the cellular concentrations of ribosomes with the growth rate of the strain. The growth rates thus estimated from the ribosomal content of E. coli BJ4 in vivo did not differ in the streptomycin-treated and the monoassociated mice. After conventionalization there was a slight decrease of the bacterial growth rates in both animal models.

Ribosomal efficiency and growth rates of freshly isolated Escherichia coli strains originating from the gastrointestinal tract.

It has been previously reported that for natural Escherichia coli isolates from the ECOR collection, there were differences in the ribosomal efficiencies and there was a direct correlation between growth rate and the ribosome efficiency (R-factor). The aim of this study was to determine whether strains freshly isolated (i.e. subcultured < 5 times) from the gastrointestinal tract ecosystem also exhibited this correlation. Eleven E. coli and two Enterobacter spp. isolates from either humans, pigs, rats or a mammoth were investigated. Considerable variability in the R-factor was noted using an in vitro translation assay, however no consistent correlation between the R-factor and growth rate was noted.

Simultaneous production of the 34-kDa and 40-kDa proteins from Bacillus thuringiensis subsp. thompsoni is required for the formation of inclusion bodies.

Cooperation of two crystal proteins from Bacillus thuringiensis subsp. thompsoni. strain HnC was shown to be essential for the formation of inclusion bodies. Expression of the operon containing the 34-kDa and 40-kDa protein genes from HnC in a B. thuringiensis crystal minus strain resulted in the formation of inclusion bodies identical to those from strain HnC. Interruption of one of the genes in the operon led to the lack of inclusion body and to low production of the remaining protein. Absence of inclusion body and low rate of protein production were also observed when both genes were simultaneously expressed but on different vectors. To show a cooperative effect in the formation of the inclusion body, both proteins must be produced from the same transcript.

Influence of the 20-kDa protein from Bacillus thuringiensis ssp. israelensis on the rate of production of truncated Cry1C proteins.

The potential role of a molecular chaperone on the rate of production of extensively altered Bacillus thuringiensis Cry1C proteins was investigated. Analysis of the proteins produced by the recombinant B. thuringiensis strains showed that the truncated proteins were produced at a low rate. Expression of the 20-kDa protein gene from B. thuringiensis ssp. israelensis in tandem with the truncated-cry1C genes led to the production of a greater amount of proteins. The formation of inclusion bodies, however, did not occur even when the 20-kDa protein gene was expressed.

Physiological state of Escherichia coli BJ4 growing in the large intestines of streptomycin-treated mice.

Growth rates of Escherichia coli BJ4 colonizing the large intestine of streptomycin-treated mice were estimated by quantitative hybridization with rRNA target probes and by epifluorescence microscopy. The ribosomal contents in bacteria isolated from the cecal mucus, cecal contents, and feces were measured and correlated with the ribosomal contents of bacteria growing in vitro at defined rates. The data suggest that E. coli BJ4 grows at an overall high rate in the intestine. However, when taking into account the total intestinal volume and numbers of bacteria present in cecal mucus, cecal contents, and feces, we suggest that E. coli BJ4 in the intestine consists of two populations, one in the mucus which has an apparent generation time of 40 to 80 min and one in the luminal contents which is static.

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae).

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.