Chlamydiae in free-ranging and captive frogs in Switzerland.

A total of 210 frog samples originating either from a mass mortality (1991/1992) or from routine postmortem investigations of the years 1990 to 2004 were examined retrospectively for a possible involvement of Chlamydiae. For a prevalence study of Chlamydia in a selected Swiss amphibian population, 403 samples from free-ranging Rana temporaria were examined. Histopathology, immunohistochemistry using a monoclonal antibody against chlamydial lipopolysaccharide, and a 16S rRNA polymerase chain reaction (PCR) followed by DNA sequencing were performed on the formalin-fixed and paraffin-embedded tissues. Using PCR, 8 of 54 (14.8%) frog samples from the mass mortality (1991/1992) were positive for Chlamydia suis S45. A control group of healthy Xenopus laevis had 3 of 38 positive samples, sequenced as C suis S45 (2/3) and an endosymbiont of Acanthamoeba species UWE1 (1/3). Chlamydophila pneumoniae TW-183 was detected from exotic frogs kept in a zoo. Of the frogs collected for the prevalence study, 6 of 238 (2.5%) tested positive, 1 each for C suis S45, Cp pneumoniae TW-183, and uncultured Chlamydiales CRG22, and the remaining 3 revealed Chlamydophila abortus S26/3. In immunohistochemistry, there were 2 positive labeling reactions, 1 in intestine and the other in the epithelium coating the body cavity, both testing positive for Cp pneumoniae TW-183 in PCR. Histologically there were no lesions recorded being characteristic for Chlamydia. Although there is a prevalence of Chlamydia in Swiss frogs, no connection to a mass mortality (1991/1992) could be established. For the first time, C suis S45 and Cp abortus S26/3 were detected in frog material.

LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli.

The function of the LysR-type regulator LrhA of Escherichia coli was defined by comparing whole-genome mRNA profiles from wild-type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent K(D) approximately 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA. The expression of flhDC (encoding FlhD(2)C(2)) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD(2)C(2) is known as the master regulator for the expression of flagellar and chemotaxis genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified. The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies. It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD(2)C(2).

[Diagnostic imaging in vascular diagnosis]. / Bildgebende Gefässdiagnostik.

Previously, vascular imaging was done exclusively using angiography (X-ray projectional imaging of vessels opacified by iodinated contrast media). Recently angiography especially is used in combination with angioplasty (percutaneous enlargement of a closed or stenotic vessel). There has been a change from invasive to non invasive procedures. New developments in computed tomography (CT), magnetic resonance imaging (MR) and ultrasound have reduced the indications for the invasive digital subtraction angiography (DSA). Further improvement especially im MR-angiography and colour-coded dopplersonography will cover all primary diagnostic vascular examinations.

Multicopy suppression of a gacA mutation by the infC operon in Pseudomonas fluorescens CHA0: competition with the global translational regulator RsmA.

The gacA gene of the biocontrol strain Pseudomonas fluorescens CHA0 codes for a response regulator which, together with the sensor kinase GacS (=LemA), is required for the production of exoenzymes and secondary metabolites involved in biocontrol, including hydrogen cyanide (HCN). A gacA multicopy suppressor was isolated from a cosmid library of strain CHA0 and identified as the infC-rpmI-rplT operon, which encodes the translation initiation factor IF3 and the ribosomal proteins L35 and L20. The efficiency of suppression was about 30%, as determined by the use of a GacA-controlled reporter construct, i.e. a translational hcnA'-'lacZ fusion. Overexpression of the rsmA gene (coding for a global translational repressor) reversed the suppressive effect of the amplified infC operon. This finding suggests that some product(s) of the infC operon can compete with RsmA at the level of translation in P. fluorescens CHA0 and that important biocontrol traits can be regulated at this level.

Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation.

Root diseases caused by fungal pathogens can be suppressed by certain rhizobacteria that effectively colonize the roots and produce extracellular antifungal compounds. To be effective, biocontrol bacteria need to be present at sufficiently high cell densities. These conditions favor the operation of positive feedback mechanisms that control the production of antifungal compounds in biocontrol strains of fluorescent pseudomonads, via both transcriptional and post-transcriptional mechanisms.

Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis.

A few bacterial species are known to produce and excrete hydrogen cyanide (HCN), a potent inhibitor of cytochrome c oxidase and several other metalloenzymes. In the producer strains, HCN does not appear to have a role in primary metabolism and is generally considered a secondary metabolite. HCN synthase of proteobacteria (especially fluorescent pseudomonads) is a membrane-bound flavoenzyme that oxidizes glycine, producing HCN and CO2. The hcnABC structural genes of Pseudomonas fluorescens and P. aeruginosa have sequence similarities with genes encoding various amino acid dehydrogenases/oxidases, in particular with nopaline oxidase of Agrobacterium tumefaciens. Induction of the hcn genes of P. fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the -40 region of the hcn promoter, and nonlimiting amounts of iron. In addition, expression of the hcn genes depends on a regulatory cascade initiated by the GacS/GacA (global control) two-component system. This regulation, which is typical of secondary metabolism, manifests itself during the transition from exponential to stationary growth phase. Cyanide produced by P. fluorescens strain CHA0 has an ecological role in that this metabolite accounts for part of the biocontrol capacity of strain CHA0, which suppresses fungal diseases on plant roots. Cyanide can also be a ligand of hydrogenases in some anaerobic bacteria that have not been described as cyanogenic. However, in this case, as well as in other situations, the physiological function of cyanide is unknown.

Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Surveillance of traumatic spinal cord injury in Australia: the identification of information needs.

Monitoring the occurrence of disease through any surveillance program necessarily requires the expenditure of scarce resources. The type of information accessible through surveillance and how it may be obtained deserve careful consideration in order to justify these costs. Therefore before establishing a new system of surveillance it is advisable to ascertain the information needs of potential users and to determine the feasibility of developing a system to meet them. As part of the planning for a national traumatic spinal cord injury surveillance system in Australia these data were sought by conducting a survey of key informants in 1993. The planning and evaluation of health care services, a knowledge of spinal cord injury epidemiology and its sequelae, injury prevention, external demands for information, and facilitation of research, were identified as the most important needs for information. It has been shown that the prevalence of spinal cord injury in Australia is increasing. As this occurs the need for specialised health services will also rise. Therefore, to facilitate the rational planning of services, and to monitor the well-being of the Australian spinal cord injured population, accurate surveillance data are essential.

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.

[Cholelithiasis--diagnostic imaging today]. / Cholelithiasis--bildgebende Diagnostik heute.

The recent change in the therapeutic approach to gallstone disease (dissolution therapy, lithotripsy and especially laparoscopic cholecystectomy) has promoted an obvious transition in biliary imaging. In a short review the actually most relevant imaging modalities are presented and validated. In the light of the explosive growth of laparoscopic cholecystectomy, a main concern of this presentation is focused on biliary duct imaging.

Prevalence of spinal cord injury: an international comparison.

Information on the prevalence of spinal cord injury is becoming more important as the life expectancy of survivors is increasing, but few prevalence studies have been published. This paper summarises the internationally available findings on prevalence, including the methodologies and data sources used. Prevalence rates ranged from 11 to 112 per 100,000 population. Methodological difficulties in comparing findings over time, country, defined population and data source are discussed, and a more standardised methodology is recommended.

Legionnaires' disease outbreak, Fairfield 1992: public health aspects.

An investigation of an outbreak of Legionnaires' disease in 1992 in Fairfield, a municipality of Sydney, was carried out to determine the source of the outbreak. Cases of Legionnaires' disease with onset of symptoms between 11 and 20 April 1992 were included. Definite cases were individuals with a history consistent with Legionnaires' disease, confirmed by direct fluorescent antibody testing plus serology or culture. There were two control groups: patients admitted to the same hospital as the cases, matched for age and sex, and patients admitted to hospital with a presumptive diagnosis of legionnaires' disease, in whom the diagnosis was subsequently excluded. There were 26 definite cases with onset of symptoms between 11 and 20 April 1992. Six (23 per cent) died. Twenty-two cases (85 per cent) reported visiting the Fairfield business district during the ten days prior to the onset of symptoms. They were 20 times more likely to have visited Fairfield than were matched controls. Matching of Legionella pneumophila serogroup 1 from environmental and clinical samples was achieved by cytogenetic fingerprinting. Fourteen cases were linked to a single environmental sample. The epidemiological findings were consistent with a point source of Legionella in the Fairfield business district. It is most likely that the exposure occurred on 10 April 1992.

The pattern of diagnosed HIV infection in Australia, 1984-1992.

OBJECTIVE: To describe the pattern of newly diagnosed HIV infection in Australia, between 1984 and 1992. METHODS: State and Territory health authorities reported cases of newly diagnosed HIV infection to the national HIV surveillance centre. Information sought on each case included the State or Territory of diagnosis, the case identifying number, the sex, date of birth and postcode of residence of the person with newly diagnosed HIV infection, the source of exposure to HIV and the date of specimen collection for the diagnosis of infection. RESULTS: By the end of December 1992, a total of 16,765 cases of newly diagnosed HIV infection had been reported in Australia. The annual number of cases declined between 1985 and 1992. Most diagnoses were among males, and exposure to HIV was attributed to male homosexual contact for more than 80% of cases for which information on exposure to HIV was available. Cases of HIV infection attributed to heterosexual contact represented an increasing proportion of the annual number of diagnoses over the period 1985-1992, among both men and women. CONCLUSION: National surveillance for newly diagnosed HIV infection has complemented national surveillance for diagnoses of AIDS as a key mechanism for monitoring the course of the HIV epidemic in Australia. The pattern of newly diagnosed HIV infection was similar to the pattern of AIDS diagnoses, with the overwhelming majority of diagnoses of infection being in adult males whose exposure to HIV was attributed to homosexual contact. Limitations of HIV surveillance include the lack of information on HIV testing patterns, incomplete information on HIV exposure histories and duplication of reported diagnoses.

The acquired immunodeficiency syndrome in Australia: incidence 1982-1991.

OBJECTIVE: To describe the incidence of the acquired immunodeficiency syndrome (AIDS) in Australia between 1982 and 1991. DESIGN: State and Territory Health Departments notified new diagnoses of AIDS to the National AIDS Registry. Information reported for each case included sex, date of birth, date of AIDS diagnosis, presumed mode of exposure to the human immunodeficiency virus (HIV), and illness(es) on which the diagnosis of AIDS was based. RESULTS: To the end of March 1992, 3,160 cases of AIDS were reported as having been diagnosed between 1982 and the end of 1991. The cumulative incidence per head of population was about twice as high in New South Wales as in Australia as a whole. Over 97% of cases were in men, of whom 91% were adults or adolescents reporting homosexual contact. In women, 40% of cases were acquired through receipt of blood, blood products or tissue. The annual incidence of AIDS rose sharply until about 1988, but the annual rates of increase slowed in subsequent years. This trend was also apparent in cases acquired through sexual contact between men. In other exposure groups, numbers of cases were much smaller and trends less apparent. However, there was no indication of a similar levelling in AIDS incidence, except among blood transfusion recipients, in whom incidence may be declining. CONCLUSION: Transmission of HIV among people with AIDS in Australia has been overwhelmingly attributed to sexual contact between men. The annual incidence of cases attributed to sexual contact between men appears to be stabilising.

Reduction of the concentration and total amount of keratan sulphate in synovial fluid from patients with osteoarthritis during treatment with piroxicam.

To study the effects of piroxicam on cartilage metabolism in vivo, a three phase (placebo/piroxicam 20 mg/day by mouth/placebo) double blind controlled trial was conducted in patients with osteoarthritis of the knee joint. Twenty one patients were recruited, 19 of whom (11 women, eight men, median age 70 years) completed the treatment schedule. The knee joint under study was aspirated to dryness at four week intervals. Treatment with piroxicam was accompanied by a decrease in the pain score, an improvement in the functional index, and an increased range of movement. Reductions in the concentration (mean (SEM) 120 (6) to 110 (8) micrograms/ml) and the total amount (1.22 (0.34) to 0.99 (0.37) mg) of keratan sulphate, but not the effusion volume (9.4 (2.5) to 8.3 (2.6) ml) were observed during treatment with piroxicam. These findings are consistent with decreased proteoglycan catabolism during treatment with piroxicam. Neither depressed synthesis nor enhanced clearance of degraded proteoglycan fragments can be excluded, however.