Xenorhabdus khoisanae SB10 produces Lys-rich PAX lipopeptides and a Xenocoumacin in its antimicrobial complex.

BACKGROUND: Xenorhabdus spp. live in close symbiosis with nematodes of the Steinernema genus. Steinernema nematodes infect an insect larva and release their symbionts into the haemocoel of the insect. Once released into the haemocoel, the bacteria produce bioactive compounds to create a semi-exclusive environment by inhibiting the growth of bacteria, yeasts and molds. The antimicrobial compounds thus far identified are xenocoumacins, xenortides, xenorhabdins, indole derivatives, xenoamicins, bicornutin and a number of antimicrobial peptides. The latter may be linear peptides such as the bacteriocins xenocin and xenorhabdicin, rhabdopeptides and cabanillasin, or cyclic, such as PAX lipopeptides, taxlllaids, xenobactin and szentiamide. Thus far, production of antimicrobial compounds have been reported for Xenorhabdus nematophila, Xenorhabdus budapestensis, Xenorhabdus cabanillasii, Xenorhabdus kozodoii, Xenorhabdus szentirmaii, Xenorhabdus doucetiae, Xenorhabdus mauleonii, Xenorhabdus indica and Xenorhabdus bovienii. Here we describe, for the first time, PAX lipopeptides and xenocoumacin 2 produced by Xenorhabdus khoisanae. These compounds were identified using ultraperformance liquid chromatography, linked to high resolution electrospray ionisation mass spectrometry and tandem mass spectrometry. RESULTS: Cell-free supernatants of X. khoisanae SB10 were heat stable and active against Bacillus subtilis subsp. subtilis, Escherichia coli and Candida albicans. Five lysine-rich lipopeptides from the PAX group were identified in HPLC fractions, with PAX1' and PAX7 present in the highest concentrations. Three novel PAX7 peptides with putative enoyl modifications and two linear analogues of PAX1' were also detected. A small antibiotic compound, yellow in colour and λmax of 314 nm, was recovered from the HPLC fractions and identified as xenocoumacin 2. The PAX lipopeptides and xenocoumacin 2 correlated with the genes and gene clusters in the genome of X. khoisanae SB10. CONCLUSION: With UPLC-MS and MSe analyses of compounds in the antimicrobial complex of X. khoisanae SB10, a number of PAX peptides and a xenocoumacin were identified. The combination of pure PAX1' peptide with xenocoumacin 2 resulted in high antimicrobial activity. Many of the fractions did, however, contain labile compounds and some fractions were difficult to resolve. It is thus possible that strain SB10 may produce more antimicrobial compounds than reported here, as suggested by the APE Ec biosynthetic complex. Further research is required to develop these broad-spectrum antimicrobial compounds into drugs that may be used in the fight against microbial infections.

Bacteriocin production and adhesion properties as mechanisms for the anti-listerial activity of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA.

Probiotics play an important role in maintaining a healthy and stable intestinal microbiota, primarily by preventing infection. Probiotic lactic acid bacteria (LAB) are known to be inhibitory to many bacterial enteric pathogens, including antibiotic-resistant strains. Whilst the positive role that probiotics have on human physiology, specifically in the treatment or prevention of specific infectious diseases of the gastro-intestinal tract (GIT) is known, the precise mechanistic basis of these effects remains a major research goal. In this study, molecular evidence to underpin the protective and anti-listerial effect of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA against orally administered Listeria monocytogenes EGDe in the GIT of mice is provided. Bacteriocins plantaricin 423 and mundticin ST4SA, produced by L. plantarum 423 and E. mundtii ST4SA, respectively, inhibited the growth of L. monocytogenes in vitro and in vivo. Bacteriocin-negative mutants of L. plantarum 423 and E. mundtii ST4SA failed to exclude L. monocytogenes EGDe from the gastrointestinal tract (GIT) of mice. Furthermore, L. plantarum 423 and E. mundtii ST4SA failed to inhibit recombinant strains of L. monocytogenes EGDe in vivo that expressed the immunity proteins of the two bacteriocins. These results confirmed that bacteriocins plantaricin 423 and mundticin ST4SA acted as anti-infective mediators in vivo. Compared to wild type strains, mutants of L. plantarum 423 and E. mundtii ST4SA, in which the adhesion genes were knocked out, were less effective in the exclusion of L. monocytogenes EGDe from the GIT of mice. This work demonstrates the importance of bacteriocin and adhesion genes as probiotic anti-infective mechanisms.

Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 excludes Listeria monocytogenes from the GIT, as shown by bioluminescent studies in mice.

Listeria monocytogenes is an opportunistic food-borne pathogen and is life-threatening to individuals with a weakened immune system. The aim of this study was to determine if Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA could prevent colonisation of L. monocytogenes in the gastro-intestinal tract (GIT). Mice were gavaged with L. plantarum 423, E. mundtii ST4SA, and a combination of the two strains, for 6 consecutive days and orally infected with a bioluminescent strain of L. monocytogenes (strain EGDe) on the last day of treatment. 30 min after infection, high cell numbers of L. plantarum 423, E. mundtii ST4SA and L. monocytogenes EGDe were isolated from faeces. L. monocytogenes EGDe cells were absent from the small intestine of L. plantarum 423-treated mice 4 h after infection and from the large intestine 2 h later. No bioluminescent, and thus metabolically active, cells of L. monocytogenes EGDe were recorded in the GIT of mice treated with E. mundtii ST4SA, suggesting that their growth was repressed. L. plantarum 423 and E. mundtii ST4SA colonised the colon the strongest. These strains may be considered for the competitive exclusion of L. monocytogenes from the GIT.

Two large, related, cryptic plasmids from geographically distinct isolates of Sulfobacillus thermotolerans.

Two large cryptic plasmids (59.2 and 65.9 kb) from isolates of Sulfobacillus thermotolerans from Yellowstone National Park (United States) and the Caribbean island of Montserrat were isolated and sequenced. This analysis revealed a common "backbone" region coding for a potential plasmid stability system plus a nonpheromone conjugation system containing homologues of both type IV and type II (tight adherence, or Tad-like) secretion systems.

Antibodies to protein tyrosine phosphatase receptor type O (PTPro) increase glomerular albumin permeability (P(alb)).

Glomerular capillary filtration barrier characteristics are determined in part by the slit-pore junctions of glomerular podocytes. Protein tyrosine phosphatase receptor-O (PTPro) is a transmembrane protein expressed on the apical surface of podocyte foot processes. Tyrosine phosphorylation of podocyte proteins including nephrin may control the filtration barrier. To determine whether PTPro activity is required to maintain glomerular macromolecular permeability, albumin permeability (P(alb)) was studied after incubation of glomeruli from normal animals with a series of monoclonal (mAb) and polyclonal antibodies. Reagents included mAbs to rabbit and rat PTPro and polyclonal rabbit immune IgG to rat PTPro. mAb 4C3, specific to the amino acid core of PTPro, decreased its phosphatase activity and increased P(alb) of rabbit glomeruli in a time- and concentration-dependent manner. In contrast, mAb P8E7 did not diminish phosphatase activity and did not alter P(alb). Preincubation of 4C3 with PTPro extracellular domain fusion protein blocked glomerular binding and abolished permeability activity. In parallel experiments, P(alb) of rat glomeruli was increased by two mAbs (1B4 and 1D1) or by polyclonal anti-rat PTPro. We conclude that PTPro interaction with specific antibodies acutely increases P(alb). The identity of the normal ligand for PTPro and of its substrate, as well as the mechanism by which phosphatase activity of this receptor affects the filtration barrier, remain to be determined.

Obesity and the pulmonologist.

Obese children have more respiratory symptoms than their normal weight peers and respiratory related pathology increases with increasing weight. Some will need specialist assessment (box 1). Obesity produces mechanical effects on respiratory system performance. Breathlessness, wheeze, and cough are not related to increased airway responsiveness and may respond more to weight loss than bronchodilator therapy. A significant number of obese children have signs and symptoms of obstructive sleep apnoea largely related to the effect of obesity on upper airway dimensions. It seems likely that unless action is taken soon, increasing numbers of children will experience preventable respiratory morbidity as a result of nutritional obesity.

Initial management of the poly-trauma patient: a practical approach in an Australian major trauma service.

The initial management of the poly-trauma patient is of vital importance to minimizing both patient morbidity and mortality. We present a practical approach to the early management of a severely injured patient as practiced at Liverpool Hospital in Sydney, Australia. Specific attention is paid to innovations in care and specific controversies in early management as well as local solutions to challenging problems.

Trauma training in Australia and New Zealand: results of a survey of advanced surgical trainees.

BACKGROUND: The surgical management of trauma within Australia and New Zealand has recently been undergoing major organizational changes. The aim of the present paper was to evaluate the attitudes and experience of Australian and New Zealand advanced surgical trainees in this changing climate and to identify problems with trauma training. METHODS: A survey assessing important areas of trauma management and training was sent to all advanced surgical trainees of the Royal Australasian College of Surgeons. RESULTS: Two hundred and seventy-two of 587 trainees responded (46%). Overall 85% of trainees believed they would be involved in trauma management in the future. The majority of trainees reported low rates of involvement and consultant supervision in trauma resuscitations. Only 32% of general surgical trainees believed that their exposure to major trauma operations was very adequate despite an average of 12.3 trauma operations per year. Seventy per cent of general surgical trainees reported a very adequate level of consultant supervision at trauma operations. In contrast 86% of orthopaedic trainees reported a very adequate exposure to trauma operations with an average of 221 orthopaedic trauma operations per year. Only 46% of orthopaedic trainees reported a very adequate level of consultant supervision at trauma operations. CONCLUSIONS: Regional rotations may need to be developed to even out trainees' experience in trauma management. The low level of supervision in trauma resuscitations and orthopaedic surgical training requires attention. This survey warrants repeating in a prospective manner.

Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon.

A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.

The chromosomal arsenic resistance genes of Thiobacillus ferrooxidans have an unusual arrangement and confer increased arsenic and antimony resistance to Escherichia coli.

The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC, arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) and arsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidans is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional trxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR and arsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.

Intra-abdominal hypertension is an independent cause of postoperative renal impairment.

HYPOTHESIS: Intra-abdominal hypertension exerts an effect on renal function independent of other confounding variables. DESIGN: A prospective study of all patients admitted to an intensive care unit following abdominal surgery was undertaken between September 1, 1994, and July 31, 1997, in a single university hospital. MAIN OUTCOME MEASURES: Intra-abdominal pressure (IAP) was measured every 8 hours (normal IAP, 0-17 mm Hg); 18 mm Hg or higher was considered increased. Forward stepwise logistic regression determined whether intra-abdominal hypertension is an independent cause of renal impairment. RESULTS: A total of 263 patients (174 after emergency surgery), whose mean +/- SD age was 61.0 +/- 18.7 years and Acute Physiology and Chronic Health Evaluation II score was 14.6 +/- 7.7, were studied. Intra-abdominal pressure was increased in 107 (40.7%) of the 263 patients. Renal impairment occurred in 35 (32.7%) of the 107 patients with intra-abdominal hypertension and in 22 (14.1%) of the 156 with a normal IAP (odds ratio, 1.62-5.42). Using the Wald maximizing model, renal impairment was independently associated with 4 antecedent factors: hypotension (P= .09), sepsis (P = .006), age older than 60 years (P = .03), and increased IAP (P = .004). CONCLUSIONS: To our knowledge, for the first time in a large clinical study, IAP has been shown to be an independent cause of renal impairment, and it ranks in importance after hypotension, sepsis, and age older than 60 years. Surgeons need to be aware of the importance of intra-abdominal hypertension in patients postoperatively.

Temporary abdominal closure: a prospective evaluation of its effects on renal and respiratory physiology.

This study prospectively analyzed outcomes in 49 consecutive patients undergoing temporary abdominal closure (TAC) between 1993 and 1996 at a single university hospital. There were 37 males and 12 females, mean age was 57 years (range, 25-79 years), mean Acute Physiology and Chronic Health Evaluation score was 27 (+7.8 SD), and mean Simplified Acute Physiology II score was 53.0 (+/-15.4). The reason for TAC was decompression in 22 patients, inability to close the abdomen in 10 patients, to facilitate reexploration for sepsis in 8 patients, and multifactorial in 9 patients. After TAC, there was a significant reduction in intra-abdominal pressure from 24.2+/-9.3 to 14.1+/-5.5 mm Hg and improvement in lung dynamic compliance from 24.1+/-7.9 to 27.6+/-9.4 mL/cm H2O (p < 0.05). Although 10 patients experienced brisk diuresis, there was no significant improvement in renal function; in fact, serum creatinine increased. The median length of stay was 35 days (range, 1-232 days). The mean number of abdominal operations after mesh insertion was 2.6+/-2.4. There were 21 deaths, for a standardized mortality rate of 0.80. Although it achieved significant reductions in abdominal pressures and improved lung dynamic compliance, TAC did not result in improved renal function or patient oxygenation.

Prospective evaluation of early missed injuries and the role of tertiary trauma survey.

BACKGROUND: This study prospectively evaluated the prevalence, clinical significance, and contributing factors to early missed injuries and the role of tertiary survey in minimizing frequency of missed injuries in admitted trauma patients. Missed injury, clinically significant missed injury, tertiary survey, and contributing factors were defined. Tertiary survey was conducted within 24 hours. RESULTS: Of 206 patients, 134 patients (65%) had 309 missed injuries composing 39% of all 798 injuries seen. Tertiary trauma survey detected 56% of early missed injuries and 90% of clinically significant missed injuries within 24 hours. Clinically significant missed injuries occurred in 30 patients with complications in 11 patients and death in two patients. Of 224 contributing errors, 123 errors were in clinical assessment, 83 errors were in radiology, 14 errors were patient related, and four errors were technical. The missed injury rate was significantly higher in patients with multiple injuries and in those involved in road crashes. CONCLUSIONS: Secondary trauma survey is not a definitive assessment and should be supplemented by tertiary trauma survey.

Long-term duct-occluded segmental pancreatic autografts: absence of microvascular diabetic complications.

BACKGROUND: Current insulin therapies for control of glucose metabolism in patients with type I diabetes mellitus prevent major metabolic consequences of insulin deficiency, but none prevents or arrests long-term complications. In experimental models of canine diabetes, retinopathy, neuropathy, and nephropathy have been shown to develop within 5 years. The aim of this study was to determine in a canine model whether glucose control provided by segmental duct-occluded pancreas autografts could prevent the long-term complications of diabetes. METHODS: Thirty-five outbred mongrel dogs underwent segmental pancreas autotransplantation with residual pancreatectomy. Follow-up over 5 years included endocrine, retinal fundus photography, fluorescein angiography, and nerve conduction studies. At endpoint, analysis of organ specific changes was undertaken. RESULTS: Long-term survival was achieved in 14 dogs for 4 to 5 years and in 3 dogs for 3 to 5 years. Glycosylated hemoglobin levels remained within normal limits, although response to glucose challenge was suboptimal. Fundus photography and fluorescein angiography demonstrated the absence of retinal vascular aneurisms, capillary leakage, and obliteration. Retinal digest showed no vascular changes and normal endothelial/pericyte ratios. Nerve conduction was normal, and histology of nerves revealed normal density of myelinated fibers and absence of intrafascicular vessels and glycogen deposits, with no change in spectrum of fiber diameters and ovoids. Renal histology revealed no evidence of nephropathy with normal glomerular basement membranes. CONCLUSIONS: We have demonstrated that duct-occluded segmental pancreatic autografts are capable of providing satisfactory metabolic control for up to 5 years, thereby preventing development of the long-term microvascular complications of diabetes.

Cloning and sequencing of the gene for the Thiobacillus ferrooxidans ATCC33020 glutamate synthase (GOGAT) small subunit and complementation of an Escherichia coli gltD mutant.

A recombinant plasmid which contains the gltD gene coding for the glutamate synthase (GOGAT) small subunit was isolated from a Thiobacillus ferrooxidans ATCC33020 gene bank by complementation of an Escherichia coli gltD mutant. The sequence of gltD was determined. The deduced amino acid sequence shows strong similarity to the two other prokaryote gltD sequences available, namely those of E. coli and A. brasilense (53% and 45% identity, respectively). A cosmid containing the gltBD region was isolated from a T. ferrooxidans cosmid gene bank, but was unable to complement an E. coli gltB mutant.

Intraabdominal pressure and gastric intramucosal pH: is there an association?

This study evaluated the potential association between increased intraabdominal pressure (IAP) and abnormally low gastric intramucosal pH (pHi) (/= 20 mmHg and a pHi of

The gene for gamma-glutamylcysteine synthetase from Thiobacillus ferrooxidans has low homology to its Escherichia coli equivalent and is linked to the gene for citrate synthase.

The gene for gamma-glutamylcysteine synthetase (gshA) from Thiobacillus ferrooxidans was isolated from a family of cosmids by its ability to complement an Escherichia coli gshA trxA double mutant which was unable to grow on minimal medium lacking glutathione. The predicted sequence of the gamma-glutamylcysteine synthetase was found to have only 18% amino acid sequence identity to the equivalent enzyme from E. coli. In spite of this low sequence homology, concentrations of GSH in a cell extract prepared from the E. coli gshA trxA mutant containing the cloned gene were almost as high as in a cell extract prepared from a wild-type E. coli strain. The gshA gene was found to be physically and transcriptionally linked to the T. ferrooxidans gene for citrate synthase (gltA). The T. ferrooxidans and E. coli citrate synthases shared 37% amino acid sequence identity and the cloned T. ferrooxidans citrate synthase gene was able to complement an E. coli gltA mutant.

Is penetrating injury on the increase in south-western Sydney?

BACKGROUND: Few Australian studies describe the epidemiology of penetrating trauma. This study describes the incidence and demographic features of penetrating injuries with emphasis on trends and severity analysis. METHODS: Case analysis was performed utilizing data from the Liverpool Hospital Trauma Registry (1989-94), NSW Department of Health Hospital Separations (1991-93), and the NSW Bureau of Crime Statistics (1991-93) with reference to the Liverpool and Fairfield Local Government Areas (LGA). RESULTS: The Trauma Registry revealed 251 of penetrating trauma. The median age was 26 years (interquartile range 21-33). Ninety-one per cent of the victims were male. Fourteen per cent of patients had an Injury Severity Score (ISS) > 15. Sixty-five per cent of cases were stab injuries and 20% gunshot injuries with the abdomen being the most commonly injured site. Twenty-one per cent of patients underwent laparotomy, 1.6% thoracotomy and 1.2% thoracotomy and laparatomy. There were 10 (4%) deaths. Trends in incidence of penetrating trauma and violent crime involving weapons were analysed. Static trends were observed for the annual incidence of penetrating trauma from the Liverpool Hospital Trauma Registry. Separations for penetrating trauma from Liverpool and Fairfield hospitals showed a slightly increasing trend. Violent crimes involving weapons in the Liverpool and Fairfield LGA showed a static trend. Nevertheless, separations for penetrating trauma and rates of violent crimes involving weapons were higher in south-western Sydney than metropolitan Sydney or NSW. Eight per cent of the LGA population are Vietnamese but this study identified 16% of victims as being Vietnamese. CONCLUSIONS: This study found no significant increase in penetrating trauma or violent crime predisposing to penetrating injury in south-western Sydney.

Trauma outcomes: a death analysis study.

Survival and mortality outcomes for trauma patients admitted to Liverpool Hospital, Sydney were analysed to determine the adequacy of trauma care. TRISS and ASCOT survival probabilities and peer review were utilised to determine if deaths were avoidable. Evaluation methods were compared for assessment of care. During the study period 2205 trauma patients were admitted, 518 of which fulfilled the study entry criteria. There were 38 deaths. The age and Injury Severity Score (ISS) of survivors was 34 +/- 18 years, 9.8 +/- 9 (mean +/- sd) compared to age and ISS for nonsurvivors 37 +/- 22 years and 45 +/- 22*, *p < 0.001. Peer review suggested that 32 deaths were non avoidable, 4 potentially avoidable and 2 were probably avoidable. TRISS and ASCOT survival probabilities were > 0.5 in 16 and 18 patients respectively. TRISS and ASCOT had low positive predictive value (25%) in identifying avoidable deaths. The Z Score was 1.79. The standardised mortality ratio (SMR) was 1.16. The Effectiveness (E) value for outcome was 0.91. Poor communication within the Area Trauma System was the greatest contributor to avoidable deaths. All trauma deaths need peer review rather than solely relying upon ASCOT and TRISS probabilities to identify "unexpected" deaths for detailed review.