In vitro volatile organic compound profiling using GC×GC-TOFMS to differentiate bacteria associated with lung infections: a proof-of-concept study.

Chronic pulmonary infections are the principal cause of morbidity and mortality in individuals with cystic fibrosis (CF). Due to the polymicrobial nature of these infections, the identification of the particular bacterial species responsible is an essential step in diagnosis and treatment. Current diagnostic procedures are time-consuming, and can also be expensive, invasive and unpleasant in the absence of spontaneously expectorated sputum. The development of a rapid, non-invasive methodology capable of diagnosing and monitoring early bacterial infection is desired. Future visions of real-time, in situ diagnosis via exhaled breath testing rely on the differentiation of bacteria based on their volatile metabolites. The objective of this proof-of-concept study was to investigate whether a range of CF-associated bacterial species (i.e. Pseudomonas aeruginosa, Burkholderia cenocepacia, Haemophilus influenzae, Stenotrophomonas maltophilia, Streptococcus pneumoniae and Streptococcus milleri) could be differentiated based on their in vitro volatile metabolomic profiles. Headspace samples were collected using solid phase microextraction (SPME), analyzed using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and evaluated using principal component analysis (PCA) in order to assess the multivariate structure of the data. Although it was not possible to effectively differentiate all six bacteria using this method, the results revealed that the presence of a particular pattern of VOCs (rather than a single VOC biomarker) is necessary for bacterial species identification. The particular pattern of VOCs was found to be dependent upon the bacterial growth phase (e.g. logarithmic versus stationary) and sample storage conditions (e.g. short-term versus long-term storage at -18 °C). Future studies of CF-associated bacteria and exhaled breath condensate will benefit from the approaches presented in this study and further facilitate the production of diagnostic tools for the early detection of bacterial lung infections.

Delusional infestation: an Australian multicentre study of 23 consecutive cases.

Delusional infestation remains a debilitating condition that is therapeutically challenging for clinicians. This case series identifies 23 patients with delusional infestation in an Australian setting. The majority of patients are women and unlikely to have a psychiatric comorbid background. The use of unnecessary anti-parasitic medication is prevalent.

Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection.

We describe the success of adjunctive bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection in the context of bilateral ureteric stents and bladder ulceration, after repeated failure of antibiotics alone. No bacteriophage-resistant bacteria arose, and the kinetics of bacteriophage and bacteria in urine suggest self-sustaining and self-limiting infection.

Carbapenem-resistant Acinetobacter baumannii in intensive care unit patients: risk factors for acquisition, infection and their consequences.

A retrospective case-control study was performed to assess risk factors and the clinical and economic consequences associated with acquisition of carbapenem-resistant Acinetobacter baumannii (CR-AB) in an intensive care unit (ICU) over a 24-month period. CR-AB was acquired by 64 of 1431 ICU admissions; each was matched with two controls. Risk factors associated with CR-AB acquisition included ICU-wide variables, such as 'colonization pressure' (the prevalence of ICU colonized patients) and ICU antibiotic use over the preceding three months, as well as patient-related variables. Among colonized patients, risk factors for CR-AB infection included transfusion and 'colonization density' (the proportion of body sites colonized with CR-AB). CR-AB infection was independently associated with increased hospital mortality [mortality difference: 20%; 95% confidence interval (CI): 1-40%], prolonged ICU stay (median length of stay difference: 15 days; 95% CI: 9-21 days) and prolonged hospital stay (30 days, 11-38 days) compared with matched controls.

What do physicians think about evidence-based antibiotic use in critical care? A survey of Australian intensivists and infectious disease practitioners.

BACKGROUND: The analysis of factors that influence prescribing decisions is increasingly important. Antibiotic use is often based on limited evidence and lack of information about clinical decision-making processes is an important obstacle to improving antibiotic utilization. AIMS: To compare the attitudes of intensive care unit practitioners (ICUP) and infectious disease practitioners (IDP) to antibiotic use and to the evidence-based information support. METHOD: A postal survey conducted between March and July 2000 of ICUP and IDP representing all States and Territories in Australia. RESULTS: One hundred and fifty-three of 224 clinicians returned the questionnaire (68.3% response rate). In choosing an antibiotic, IDP placed significantly more weight than ICUP on the in vitro susceptibility of the pathogen (P = 0.001), antibiotic cost (P = 0.05) and possible development of antibiotic resistance (P = 0.007). More than 95% of both groups believed that unit-specific antibiotic susceptibility of endemic pathogens was an essential factor in rational prescribing, but only 68.5% of IDP and 38.7% of ICUP use microbiology laboratory databases. When in doubt about appropriate antibiotic use, 63.8% of ICUP seek and 76.3% usually follow the advice of IDP. Both groups agree that published antibiotic guidelines are useful, but IDP were more likely to consult them. ICUP were more likely to believe that guidelines are used to control clinicians rather than to improve quality of care (P = 0.001). A greater proportion of IDP (71.2%) than ICUP (52.5%) believed that antibiotic prescribing in their intensive care unit (ICU) was evidence based but most (91.8% and 86.9%, respectively) agreed that it should be. CONCLUSIONS: Australian clinicians have positive views about evidence-based prescribing and antibiotic guidelines. However, there are clinically significant differences in prescribing behaviour between ICUP and IDP. These may be explained by different disease spectra managed by each group or different cultures, training and/or cognitive styles. Improvements in the understanding of physicians' information and decision support needs are required to strengthen evidence-based prescribing.

Antibiotic therapy of ventilator-associated pneumonia--a reappraisal of rationale in the era of bacterial resistance.

Ventilator-associated pneumonia (VAP) is the most frequent nosocomial infection in intensive care units (ICU). Resistance patterns seen in ICUs suggest that prescribing recommendations should be reappraised to limit practices engendering resistance to large families of antibiotics. Despite concern surrounding the use of antibiotics in the management of VAP, there is limited evidence to assist the clinician in making decisions about the indications for such therapy, the selection of the correct antibiotic(s), the timing of initiation of therapy and its duration. The high amount of antibiotic use, in combination with the low grade colonisation of patients with multi-resistant pathogens at the time of admission, turns the ICU into an environment where antibiotic policy is likely to have an effect on the resistance problem. Opinions are changing as to the validity of invasive techniques in guiding prescribing decisions. Invasive and semi-invasive diagnostic testing increases physician confidence in the diagnosis and management of VAP and helps to limit or discontinue antibiotic treatment.

Failure to detect Bartonella henselae infection in synovial fluid from sufferers of chronic arthritis.

Bartonella henselae causes granulomatous and indolent infection in the immune competent human, and angioproliferation in the context of persistent infection and impaired immunity. This bacterium is found in up to 40% of household cats, from which humans acquire it by either a cat scratch or a bite (hence the name, cat-scratch disease). Approximately 5% of Australian and US blood donors have serological evidence of past infection, but most associated illnesses are mild or subclinical. A number of lines of evidence prompted us to consider a relationship between rheumatoid arthritis (RA) and Bartonella infection. These include epidemiological associations with household pet exposure; apparent responsiveness of some RA cases to tetracycline therapy; the granulomatous and angioproliferative nature of Bartonella lesions; the insidiousness and high seroprevalence of this infection in the community; and even reported Bartonella infection mimicking juvenile RA. In a small group of patients with chronic arthritides, we found no direct evidence of humoral antibodies to, nor of persistent infection with, Bartonella henselae in synovial fluid. While larger and more invasive studies are likely to provide more confident exclusions of this hypothesis, this suggests that persistent Bartonella infection is unlikely to play a major role in RA.

Lipopolysaccharide O-antigen expression and the effect of its absence on virulence in rfb mutants of Vibrio cholerae O1.

Using defined rfb mutants, defective in the biosynthesis of the O-antigen of the lipopolysaccharide (LPS), and monoclonal antibodies (MAbs) to the A, B and C LPS antigens, we have examined the distribution of the antigens and the effects of their loss. By immunogold electron microscopy, it has been possible to determine the relative amounts of the A, B and C antigens on Inaba and Ogawa cells, confirming previous studies based upon bacterial agglutination and hemagglutination inhibitions. These antigens are absent from rfb::Tn mutants selected as resistant to phages which have been shown to use the O-antigen as their receptor. These mutants were severely attenuated as measured by both LD50 and their ability to compete with the wild-type parents when analyzed in the infant mouse cholera model. These mutants were unchanged in the export of cholera toxin or other secreted proteins but revealed an altered outer membrane protein profile. The competition defect suggested an effect on TCP (toxin-coregulated pilus). An analysis of the rfb::Tn mutants revealed that they were unable to assemble TCP on their surface, but the major subunit, TcpA, could be found as an intracellular pool. These mutants could be complemented back to wild-type using the cloned rfb region, implying that functional TCP assembly is dependent upon an intact LPS.

Translocation failure in a type-4 pilin operon: rfb and tcpT mutants in Vibrio cholerae.

Defined chromosomal mutations that lead to assembly failure of the toxin coregulated pilus (TCP) of Vibrio cholerae provide useful insights into the biogenesis of a type-4 pilus. Mutants in rfb affecting LPS O-antigen biosynthesis, and strains depleted of the cytoplasmic membrane-associated ATP-binding protein TcpT, provide contrasting TCP export-defective phenotypes acting at different locations. Mutants in the perosamine biosynthesis pathway of V. cholerae 569B result in an rfb phenotype with an LPS consisting only of core oligosaccharide and lipid A. Such strains are unable to assemble TCP, and TcpA subunits are found in the periplasm and membrane fractions. In both rfb and tcpT mutants, the export defect is specific and complete. TcpT is a member of a large family of cytoplasmic membrane-associated ATP-binding proteins which are essential in type-4 pilin systems and in many non-pilin outer membrane transporters in Gram-negative bacteria. The behaviour of translocation-arrested TcpA in rfb and tcpT mutants is indistinguishable from that within assembled pilus under a range of conditions including flotation in density gradients, chemical cross-linking, and detergent extraction experiments. From the data presently available, it would appear that TcpA requires TcpT-mediated translocation from the cytoplasmic membrane and that TcpT stabilizes the subunit at or immediately beyond this stage, before crossing the outer membrane.

Outer membrane translocation arrest of the TcpA pilin subunit in rfb mutants of Vibrio cholerae O1 strain 569B.

The toxin-coregulated pilus (TCP) of Vibrio cholerae is a type 4-related fimbrial adhesin and a useful model for the study of type 4 pilus biogenesis and related bacterial macromolecular transport pathways. Transposon mutagenesis of the putative perosamine biosynthesis genes in the rfb operon of V. cholerae 569B eliminates lipopolysaccharide (LPS) O-antigen biosynthesis but also leads to a specific defect in TCP export. Localization of TcpA is made difficult by the hydrophobic nature of this bundle-forming pilin, which floats anomalously in sucrose density gradients, but the processed form of TcpA can be found in membrane and periplasmic fractions prepared from these strains. While TcpA cannot be detected by surface immunogold labelling in transmission electron microscope preparations, EDTA pretreatment facilitates immunofluorescent antibody labelling of whole cells, and ultrathin cryosectioning techniques confirm membrane and periplasmic accumulation of TcpA. Salt and detergent extraction, protease accessibility, and chemical cross-linking experiments suggest that although TcpA has not been assembled on the cell surface, subunit interactions are otherwise identical to those within TCP. In addition, TcpA-mediated fucose-resistant hemagglutination of murine erythrocytes is preserved in whole-cell lysates, suggesting that TcpA has obtained its mature conformation. These data localize a stage of type 4 pilin translocation to the outer membrane, at which stage export failure leads to the accumulation of pilin subunits in a configuration similar to that within the mature fiber. Possible candidates for the outer membrane defect are discussed.

Biotype-specific tcpA genes in Vibrio cholerae.

The tcpA gene, encoding the structural subunit of the toxin-coregulated pilus, has been isolated from a variety of clinical isolates of Vibrio cholerae, and the nucleotide sequence determined. Strict biotype-specific conservation within both the coding and putative regulatory regions was observed, with important differences between the El Tor and classical biotypes. V. cholerae O139 Bengal strains appear to have El Tor-type tcpA genes. Environmental O1 and non-O1 isolates have sequences that bind an El Tor-specific tcpA DNA probe and that are weakly and variably amplified by tcpA-specific polymerase chain reaction primers, under conditions of reduced stringency. The data presented allow the selection of primer pairs to help distinguish between clinical and environmental isolates, and to distinguish El Tor (and Bengal) biotypes from classical biotypes of V. cholerae. While the role of TcpA in cholera vaccine preparations remains unclear, the data strongly suggest that TcpA-containing vaccines directed at O1 strains need include only the two forms of TcpA, and that such vaccines directed at (O139) Bengal strains should include the TcpA of El Tor biotype.

The toxin-co-regulated pilus of Vibrio cholerae O1: a model for type 4 pilus biogenesis?

The toxin-co-regulated pilus (TCP), an important colonization factor of Vibrio cholerae, is similar to the type 4 pilus produced by a variety of pathogenic Gram-negative bacteria. The putative translocation and assembly machinery of TCP has broad similarities with known pilin and nonpilin export mechanisms.

Relieving medical officers in Queensland country hospitals.

OBJECTIVE: Evaluation of training and preparedness of Queensland country hospital relieving staff to perform their duties. DESIGN: A postal survey of 208 medical graduates who graduated in 1983 and were first enrolled on the Queensland Medical Register in 1985. RESULTS: Among the problems described was a lack of practical training in obstetrics, anaesthetics, paediatrics and emergency services. Most respondents (83%) stated that their total experience had been inadequate to provide the services required of them as relievers. Unsupervised services were provided by inexperienced junior doctors. Seventy-two per cent of all obstetric relievers, and 57% of all anaesthetic relievers, had completed three months or less of supervised postgraduate training at the time of the survey. This was reflected in poor levels of confidence to perform basic procedures, despite the fact that a high percentage of respondents had been called upon to do this. Both undergraduate and postgraduate training was felt to be inadequate. Suggestions made by respondents focused primarily on the provision of supervised "hands-on" clinical experience, with longer and more practically oriented training at both levels, decentralisation of teaching, and more readily available formal non-specialist qualifications in obstetrics and anaesthetics. CONCLUSIONS: Most respondents claimed that they were inadequately prepared for the relieving experience. The provision of supervised hands-on basic clinical training and that of competent country relievers might both be achieved by longer rotations to provincial centres on a regular basis in the early postgraduate years.