Archaea in the foregut of macropod marsupials: PCR and amplicon sequence-based observations.

AIMS: To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. METHODS AND RESULTS: DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). CONCLUSIONS: Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. SIGNIFICANCE AND IMPACT OF THE STUDY: Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future.

Characterization of culturable anaerobic bacteria from the forestomach of an eastern grey kangaroo, Macropus giganteus.

AIM: To determine the culturable biodiversity of anaerobic bacteria isolated from the forestomach contents of an eastern grey kangaroo, Macropus giganteus, using phenotypic characterization and 16S rDNA sequence analysis. METHODS AND RESULTS: Bacteria from forestomach contents of an eastern grey kangaroo were isolated using anaerobic media containing milled curly Mitchell grass (Astrebla lappacea). DNA was extracted and the 16S rDNA sequenced for phylogenetic analysis. Forty bacterial isolates were obtained and placed in 17 groups based on phenotypic characteristics and restriction enzyme digestion of 16S rDNA PCR products. DNA sequencing revealed that the 17 groups comprised five known species (Clostridium butyricum, Streptococcus bovis, Clostridium sporogenes, Clostridium paraputrificum and Enterococcus avium) and 12 groups apparently representing new species, all within the phylum Firmicutes. CONCLUSIONS: Foregut contents from Australian macropod marsupials contain a microbial ecosystem with a novel bacterial biodiversity comprising a high percentage of previously unrecognized species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study adds to knowledge of Australia's unique biodiversity, which may provide a future bioresource of genetic information and bacterial species of benefit to agriculture.

Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology.

A screening technique for integrons in members of the family Enterobacteriaceae and nonfermenting gram-negative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety of clinical specimens were screened for class 1 integrons by real-time PCR performed on a LightCycler instrument. This technique used a primer pair specific for a 300-bp conserved region at the 5' ends of class 1 integrons. The screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class 1 integrons, and the real-time PCR technique was thus shown to be both sensitive and specific. DNA from 50 of 226 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 of 50 (68%) of these isolates. In an attempt to study the molecular epidemiology of antimicrobial resistance genes carried within integrons, a comparison of the types of gene cassettes carried by isolates from different patients was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the study. Resistance to trimethoprim was also frequently found to be encoded within integrons. Furthermore, multiple bacterial isolates obtained from one patient over a 5-month period were all shown to carry an integron containing the same single adenyltransferase gene cassette, suggesting that these elements were relatively stable in this case.

Helicobacter pylori and tonsillectomy.

Tonsillar tissue is a component of mucosa-associated lymphoid tissue (MALT), which has evolved to protect vulnerable mucosal surfaces. Helicobacter pylori, implicated as an aetiological factor in duodenal ulcers and gastritis, induces the appearance of lymphoid aggregates (MALT) in the stomach. This organism is cytotoxic via a nitric oxide synthase cascade. The possibility that tonsillar tissue processes Helicobacter pylori or that Helicobacter pylori can colonize the palatine tonsils is explored. The study design was that of a prospective study. We determined if Helicobacter pylori (i) forms part of the normal microenvironment of the tonsil, (ii) plays a role in the pathogenesis of tonsillitis and (iii) is associated with increased expression of inducible nitric oxide synthase (iNOS) in macrophages of the tonsil. Serology for Helicobacter pylori was performed on 50 patients undergoing tonsillectomy. Tonsillar specimens were monitored for urease activity by CLO test (a sealed plastic slide holding an agar gel, which contains urea and detects the urease enzyme of Helicobacter pylori), and immunocytochemically probed for Helicobacter pylori and iNOS expression. The mean age of this patient group was 17.2 years (3-36 years). Fourteen (28%) were sero-positive for Helicobacter pylori but no evidence of this pathogen was found in any tonsillar specimen. The number of macrophages staining for iNOS, per field, under a magnification of x40, was increased in sero-positive patients (13.3 +/- 1.3 versus 9.9 +/- 0.7; P = 0.01). Helicobacter pylori does not appear to colonize the tonsil. We believe that Helicobacter pylori primes the tonsils by inducing macrophage iNOS expression. The higher expression in sero-positive patients is a reflection of a pro-inflammatory reaction to Helicobacter pylori that is both local and systemic.

Molecular epidemiology of outbreaks of gastroenteritis associated with small round-structured viruses in East Anglia, United Kingdom, during the 1996-1997 season.

During the winter season from November 1996 to May 1997, 550 fecal specimens were submitted from 94 outbreaks of gastroenteritis occurring in East Anglia, United Kingdom. These specimens were tested for the presence of small round-structured viruses (SRSVs) by electron microscopy, reverse transcriptase PCR, or both methods. SRSVs were shown to be associated with 64 of 94 (68%) of these outbreaks, of which 16 (25%) outbreaks occurred at a single location (Southend) within the region. Twenty-four specimens from 13 of the 16 SRSV-positive outbreaks occurring in Southend were available for genomic analysis, in which divergence within the RNA polymerase region of the SRSV genome was investigated. A further 27 specimens from 17 other SRSV-associated outbreaks, occurring at different locations within East Anglia but at the same time as those at Southend, were also studied. Fifty of the total of 51 (98%) specimens studied were shown to belong to genogroup II, and within this genogroup, 49 of 50 (98%) specimens were shown to be Grimsby-like viruses, with only one Mexico-like strain. Furthermore, phylogenetic analysis of the Grimsby-like viruses indicated clusterings according to the geographical location of the outbreak. One specimen contained a virus belonging to genogroup I, and this had the greatest sequence identity (83%) with Southampton virus.

An analysis of the origins of a cooperative binding energy of dimerization.

The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface. These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.

Semiquantitation of cooperativity in binding of vancomycin-group antibiotics to vancomycin-susceptible and -resistant organisms.

The association of vancomycin group antibiotics with the growing bacterial cell wall was investigated by using the cell wall precursor analog di-N-acetyl-Lys-D-Ala-D-Ala in competition binding experiments. The affinities of the antibiotics for the -D-Ala-D-Ala-containing cell wall precursors of Bacillus subtilis ATCC 6633 (a model for vancomycin-susceptible gram-positive bacteria) and for the -D-Ala-D-Lac-containing cell wall precursors of Leuconostoc mesenteroides (a model for vancomycin-resistant strains of Enterococcus faecium and Enterococcus faecalis) were determined by a whole-cell assay. The binding of strongly dimerizing antibiotics such as eremomycin to the bacterial surface was thus shown to be enhanced by up to 2 orders of magnitude (relative to the binding in free solution) by the chelate effect, whereas weakly dimerizing antibiotics like vancomycin and antibiotics carrying lipid tails (teicoplanin) benefited less (ca. 1 order of magnitude). The affinity measured in this way correlates well with the MIC of the antibiotic, and a consequence of this is that future design of semisynthetic vancomycin-group antibiotics should attempt to incorporate chelate effect-enhancing structural features.

A limitation of two-state analysis for transitions between disordered and weakly ordered states.

BACKGROUND: The stability of the secondary structure of particular peptide regions is often used to investigate the involvement of the region in protein folding. When analysing the relatively small populations of associated states that are formed by weak interactions (i.e. those interactions that are comparable to thermal energies), it is common practice to characterise the associated state by a parameter that is measured when this state is highly occupied. The accuracy of this method, however, has not yet been determined. RESULTS: Using as a model the vancomycin group of antibiotics, either forming dimers or binding to cell wall precursors, we have investigated the dependence of the limiting (i.e. fully associated) chemical shifts of two protons on the equilibrium constants for the formation of the fully associated states. The chemical shift shows a large variation with the equilibrium constant for the formation of the fully associated state. CONCLUSIONS: The results demonstrate, in two systems, that a parameter representing a fully associated state (chemical shift) varies greatly with the equilibrium constant for the formation of that associated state. The results have implications for two-state analyses of populations of protein fragments in which a parameter representing the fully associated state is taken to be independent of the equilibrium constant for its formation. Using two-state analysis to determine the population of associated states of protein fragments could result in an underestimation of the population of these associated states.

A twenty year (1971-1990) review of tracheostomies in a major paediatric hospital.

Changing trends in the indications for paediatric tracheostomies, with decreasing numbers of tracheostomies being performed, have been reported in the literature. In a retrospective analysis of the period 1971 to 1990 the experience of tracheostomies in children under the age of 15 at Our Lady's Hospital (Dublin) is reviewed. Only 29 tracheostomies were performed during this time with an increase in numbers (90%) performed during the second 10 year period. The major underlying indication for tracheostomy in both 10 year periods was for the management of an airway problem secondary to congenital abnormalities (65%). In 14 children the operation was performed during the first year of life. However, while 90% of the children were under the age of one in the period 1971-1980 this fell to 26% during 1981-1990. Complications occurred in 41% overall, however, in the under 1 year old group 64% developed complications. There were no deaths as a direct result of the tracheostomy or its complications, but six children died because of the severity of the underlying disease. The average length of time before decannulation was 2.1 years, with decannulation difficulties occurring infrequently (11%).

Cooperativity and anti-cooperativity between ligand binding and the dimerization of ristocetin A: asymmetry of a homodimer complex and implications for signal transduction.

BACKGROUND: Recent work has indicated that dimerization is important in the mode of action of the vancomycin group of glycopeptide antibiotics. NMR studies have shown that one member of this group, ristocetin A, forms an asymmetric dimer with two physically different binding sites for cell wall peptides. Ligand binding by ristocetin A and dimerization are slightly anti-cooperative. In contrast, for the other glycopeptide antibiotics of the vancomycin group that have been examined so far, binding of cell wall peptides and dimerization are cooperative. RESULTS: Here we show that the two halves of the asymmetric homodimer formed by ristocetin A have different affinities for ligand binding. One of these sites is preferentially filled before the other, and binding to this site is cooperative with dimerization. Ligand binding to the other, less favored half of the dimer, is anti-cooperative with dimerization. CONCLUSIONS: In dimer complexes, anti-cooperativity of dimerization upon ligand binding can be a result of asymmetry, in which two binding sites have different affinities for ligands. Such a system, in which one binding site is filled preferentially, may be a mechanism by which the cooperativity between ligand binding and dimerization is fine tuned and may thus have relevance to the control of signal transduction in biological systems.

Analysis of peptidoglycan precursors in vancomycin-resistant Enterococcus gallinarum BM4174.

Vancomycin resistance in enterococci is an increasing clinical problem, and several phenotypes have been identified. We demonstrate here that the resistance mechanism in the constitutively vancomycin-resistant Enterococcus gallinarum BM4174 involves an altered pathway of peptidoglycan synthesis and hydrolysis of the normal precursors in the vancomycin-sensitive pathway. A ligase encoded by the vanC gene catalyses synthesis of D-Ala-D-Ser and substitutes this dipeptide for D-Ala-D-Ala in peptidoglycan precursors. It is presumed that this substitution lowers the affinity of vancomycin for its target site. Destruction of D-Ala-D-Ala (D,D-peptidase activity) and of UDP-MurNAc-L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala by removal of the terminal D-Ala residue (D,D-carboxypeptidase activity) ensures that the normal vancomycin-sensitive pathway of peptidoglycan synthesis cannot function in the resistant strain.

Bezold's abscess.

Since the introduction of antibiotics for the treatment of suppurative otitis media the incidence of complications from this disease has been greatly diminished. Acute mastoiditis, resulting in the deep neck abscess known as Bezold's abscess, has become very rare. A case of Bezold's abscess is presented with special reference to the clinical presentation and pathogenesis of this now uncommon condition. The variations in the routes of spread of the abscess in the fascial planes of the neck are described in detail. The difference between what is known today as a Bezold's abscess and the abscess that Bezold described in the early part of this century are presented.