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BackgroundRobust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness.MethodsWe conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 (IFI27) in COVID-19 patients.FindingsWe show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression is associated with the presence of a high viral load. We further demonstrate that systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 severity. For clinical outcome prediction (e.g. respiratory failure), IFI27 expression displays a high positive (0.83) and negative (0.95) predictive value, outperforming all other known predictors of COVID-19 severity. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 swine influenza virus infection, IFI27-like genes were highly upregulated in the blood samples of severely infected patients.InterpretationThese data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus.Research in contextEvidence before this studyWe searched the scientific literature using PubMed to identify studies that used the IFI27 biomarker to predict outcomes in COVID-19 patients. We used the search terms “IFI27”, “COVID-19, “gene expression” and “outcome prediction”. We did not identify any study that investigated the role of IFI27 biomarker in outcome prediction. Although ten studies were identified using the general terms of “gene expression” and “COVID-19”, IFI27 was only mentioned in passing as one of the identified genes. All these studies addressed the broader question of the host response to COVID-19; none focused solely on using IFI27 to improve the risk stratification of infected patients in a pandemic.Added value of this studyHere, we present the findings of a multi-cohort study of the IFI27 biomarker in COVID-19 patients. Our findings show that the host response, as reflected by blood IFI27 gene expression, accurately predicts COVID-19 disease progression (positive and negative predictive values; 0.83 and 0.95, respectively), outperforming age, comorbidity, C-reactive protein and all other known risk factors. The strong association of IFI27 with disease severity occurs not only in SARS-CoV-2 infection, but also in other respiratory viruses with pandemic potential, such as the influenza virus. These findings suggest that host response biomarkers, such as IFI27, could help identify high-risk COVID-19 patients - those who are more likely to develop infection complications - and therefore may help improve patient triage in a pandemic.Implications of all the available evidenceThis is the first systemic study of the clinical role of IFI27 in the current COVID-19 pandemic and its possible future application in other respiratory virus pandemics. The findings not only could help improve the current management of COVID-19 patients but may also improve future pandemic preparedness.

ImportanceWhether selective decontamination of the digestive tract (SDD) reduces mortality in critically ill patients remains uncertain.ObjectiveTo determine whether SDD reduces in-hospital mortality in critically ill adults.Design, Setting, and ParticipantsA cluster, crossover, randomized clinical trial that recruited 5982 mechanically ventilated adults from 19 intensive care units (ICUs) in Australia between April 2018 and May 2021 (final follow-up, August 2021). A contemporaneous ecological assessment recruited 8599 patients from participating ICUs between May 2017 and August 2021.InterventionsICUs were randomly assigned to adopt or not adopt a SDD strategy for 2 alternating 12-month periods, separated by a 3-month interperiod gap. Patients in the SDD group (n = 2791) received a 6-hourly application of an oral paste and administration of a gastric suspension containing colistin, tobramycin, and nystatin for the duration of mechanical ventilation, plus a 4-day course of an intravenous antibiotic with a suitable antimicrobial spectrum. Patients in the control group (n = 3191) received standard care.Main Outcomes and MeasuresThe primary outcome was in-hospital mortality within 90 days. There were 8 secondary outcomes, including the proportion of patients with new positive blood cultures, antibiotic-resistant organisms (AROs), and Clostridioides difficile infections. For the ecological assessment, a noninferiority margin of 2% was prespecified for 3 outcomes including new cultures of AROs.ResultsOf 5982 patients (mean age, 58.3 years; 36.8% women) enrolled from 19 ICUs, all patients completed the trial. There were 753/2791 (27.0%) and 928/3191 (29.1%) in-hospital deaths in the SDD and standard care groups, respectively (mean difference, −1.7% [95% CI, −4.8% to 1.3%]; odds ratio, 0.91 [95% CI, 0.82-1.02]; P = .12). Of 8 prespecified secondary outcomes, 6 showed no significant differences. In the SDD vs standard care groups, 23.1% vs 34.6% had new ARO cultures (absolute difference, −11.0%; 95% CI, −14.7% to −7.3%), 5.6% vs 8.1% had new positive blood cultures (absolute difference, −1.95%; 95% CI, −3.5% to −0.4%), and 0.5% vs 0.9% had new C difficile infections (absolute difference, −0.24%; 95% CI, −0.6% to 0.1%). In 8599 patients enrolled in the ecological assessment, use of SDD was not shown to be noninferior with regard to the change in the proportion of patients who developed new AROs (−3.3% vs −1.59%; mean difference, −1.71% [1-sided 97.5% CI, −∞ to 4.31%] and 0.88% vs 0.55%; mean difference, −0.32% [1-sided 97.5% CI, −∞ to 5.47%]) in the first and second periods, respectively.Conclusions and RelevanceAmong critically ill patients receiving mechanical ventilation, SDD, compared with standard care without SDD, did not significantly reduce in-hospital mortality. However, the confidence interval around the effect estimate includes a clinically important benefit.Trial RegistrationClinicalTrials.gov Identifier: NCT02389036

Introduction This study aims to assess the association of piperacillin/tazobactam and meropenem minimum inhibitory concentration (MIC) and beta-lactam resistance genes with mortality in the MERINO trial. Methods Blood culture isolates from enrolled patients were tested by broth microdilution and whole genome sequencing at a central laboratory. Multivariate logistic regression was performed to account for confounders. Absolute risk increase for 30-day mortality between treatment groups was calculated for the primary analysis (PA) and the microbiologic assessable (MA) populations. Results In total, 320 isolates from 379 enrolled patients were available with susceptibility to piperacillin/tazobactam 94% and meropenem 100%. The piperacillin/tazobactam nonsusceptible breakpoint (MIC >16 mg/L) best predicted 30-day mortality after accounting for confounders (odds ratio 14.9, 95% confidence interval [CI] 2.8–87.2). The absolute risk increase for 30-day mortality for patients treated with piperacillin/tazobactam compared with meropenem was 9% (95% CI 3%–15%) and 8% (95% CI 2%–15%) for the original PA population and the post hoc MA populations, which reduced to 5% (95% CI −1% to 10%) after excluding strains with piperacillin/tazobactam MIC values >16 mg/L. Isolates coharboring extended spectrum β-lactamase (ESBL) and OXA-1 genes were associated with elevated piperacillin/tazobactam MICs and the highest risk increase in 30-day mortality of 14% (95% CI 2%–28%). Conclusions After excluding nonsusceptible strains, the 30-day mortality difference from the MERINO trial was less pronounced for piperacillin/tazobactam. Poor reliability in susceptibility testing performance for piperacillin/tazobactam and the high prevalence of OXA coharboring ESBLs suggests that meropenem remains the preferred choice for definitive treatment of ceftriaxone nonsusceptible Escherichia coli and Klebsiella.

Multidrug- and colistin-resistant Salmonella enterica serotype 4,[5],12:i:- sequence type 34 is present in Europe and Asia. Using genomic surveillance, we determined that this sequence type is also endemic to Australia. Our findings highlight the public health benefits of genome sequencing-guided surveillance for monitoring the spread of multidrug-resistant mobile genes and isolates.

Multidrug resistant carbapenemase-producingKlebsiella pneumoniaecapable of causing severe disease in humans is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as potential therapeutics against these multidrug resistant pathogens. We are currently developing bacteriophage therapy against carbepenem-resistantK. pneumoniaebelonging to the genetically diverse, globally disseminated clonal group CG258. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the bacterial genomes of 18 target strains, tested them against novel lytic bacteriophages, and generated phage-susceptibility profiles. The genomes ofK. pneumoniaeisolates carryingblaNDMandblaKPCwere sequenced and isolates belonging to CG258 were selected for susceptibility testing using a panel of lytic bacteriophages (n=65). The localK. pneumoniaeCG258 population was dominated by isolates belonging to sequence type ST258 clade 1 (86%). The primary differences between ST258 genomes were variations in the capsular locus (cps) and in prophage content. We showed that CG258-specific lytic phages primarily target the capsule, and that successful infection is blocked in many, post-adsorption, by immunity conferred by existing prophages. Five bacteriophages specifically active againstK. pneumoniaeST258 clade 1 (n=5) belonging to the Caudovirales order were selected for further characterization. Our findings show that effective control ofK. pneumoniaeCG258 with phage will require mixes of diverse lytic viruses targeting all relevantcpsvariants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic phage candidates against this serious pathogen.ImportanceBacteriophages are natural agents that exclusively and selectively kill bacteria and have the potential to be useful in the treatment of multidrug resistant infections.K. pneumoniaeCG258 is a main agent of life-threatening sepsis that is often resistant to last-line antibiotics. Our work highlights some key requirements for developing bacteriophage preparations targeting this pathogen. By defining the genomic profile of our clinicalK. pneumoniaeCG258 population and matching it with bacteriophage susceptibility patterns, we found that bacteriophage ability to lyse each strain correlates well withK. pneumoniaeCG258 structural subtypes (capsule variants). This indicates that preparation of bacteriophage therapeutics targeting this pathogen should aim at including phages against each bacterial capsular subtype. This necessitates a detailed understanding of the diversity of circulating isolates in different geographical areas in order to make rational therapeutic choices.

WOS: 000444341400012 PubMed ID: 30208454 IMPORTANCE Extended-spectrum beta-lactamases mediate resistance to third-generation cephalosporins (eg, ceftriaxone) in Escherichia coli and Klebsiella pneumoniae. Significant infections caused by these strains are usually treated with carbapenems, potentially selecting for carbapenem resistance. Piperacillin-tazobactam may be an effective "carbapenem-sparing" option to treat extended-spectrum beta-lactamase producers. OBJECTIVES To determine whether definitive therapy with piperacillin-tazobactam is noninferior to meropenem (a carbapenem) in patients with bloodstream infection caused by ceftriaxone-nonsusceptible E coli or K pneumoniae. DESIGN, SETTING, AND PARTICIPANTS Noninferiority, parallel group, randomized clinical trial included hospitalized patients enrolled from 26 sites in 9 countries from February 2014 to July 2017. Adult patients were eligible if they had at least 1 positive blood culture with E coli or Klebsiella spp testing nonsusceptible to ceftriaxone but susceptible to piperacillin-tazobactam. Of 1646 patients screened, 391 were included in the study. INTERVENTIONS Patients were randomly assigned 1: 1 to intravenous piperacillin-tazobactam, 4.5 g, every 6 hours (n = 188 participants) or meropenem, 1 g, every 8 hours (n = 191 participants) for a minimum of 4 days, up to a maximum of 14 days, with the total duration determined by the treating clinician. MAIN OUTCOMES AND MEASURES The primary outcome was all-cause mortality at 30 days after randomization. A noninferiority margin of 5% was used. RESULTS Among 379 patients (mean age, 66.5 years; 47.8% women) who were randomized appropriately, received at least 1 dose of study drug, and were included in the primary analysis population, 378 (99.7%) completed the trial and were assessed for the primary outcome. A total of 23 of 187 patients (12.3%) randomized to piperacillin-tazobactam met the primary outcome of mortality at 30 days compared with 7 of 191 (3.7%) randomized to meropenem (risk difference, 8.6%[1-sided 97.5% CI, -infinity to 14.5%]; P = .90 for noninferiority). Effects were consistent in an analysis of the per-protocol population. Nonfatal serious adverse events occurred in 5 of 188 patients (2.7%) in the piperacillin-tazobactam group and 3 of 191 (1.6%) in the meropenem group. CONCLUSIONS AND RELEVANCE Among patients with E coli or K pneumoniae bloodstream infection and ceftriaxone resistance, definitive treatment with piperacillin-tazobactam compared with meropenem did not result in a noninferior 30-day mortality. These findings do not support use of piperacillin-tazobactam in this setting. University of Queensland; Australian Society for Antimicrobials (ASA); International Society for Chemotherapy (ISC); National University Hospital Singapore Clinician Researcher Grant [NUHSRO/2014/121/CRG/07]; Australian Infectious Disease Centre; Australian Genome Research Facility; Royal College of Pathologists of Australasia (RCPA) Foundation; Study, Education, and Research Committee (SERC) of Pathology Queensland; National Health and Medical Research Council (NHMRC) Career Development Fellowship; Australian Postgraduate Award from the University of Queensland; NHMRC Career Development and Practitioner Fellowship; NHMRC Practitioner Fellowship The study was sponsored by the University of Queensland. This study was funded by grants from the Australian Society for Antimicrobials (ASA), International Society for Chemotherapy (ISC), National University Hospital Singapore Clinician Researcher Grant NUHSRO/2014/121/CRG/07. Whole-genome sequencing was funded by grants from the Australian Infectious Disease Centre and Australian Genome Research Facility; the Royal College of Pathologists of Australasia (RCPA) Foundation; and the Study, Education, and Research Committee (SERC) of Pathology Queensland. Dr Beatson was supported by a National Health and Medical Research Council (NHMRC) Career Development Fellowship during the course of the trial. Dr Harris was supported by an Australian Postgraduate Award from the University of Queensland. Dr Peleg was supported by a NHMRC Career Development and Practitioner Fellowship during the course of the trial. Dr Paterson was supported by a NHMRC Practitioner Fellowship during the course of the trial.

Objective To elicit the views of well-informed community members on the acceptability of proposed policy interventions designed to improve community use of antibiotics in Australia. Design Two community juries held in 2016. Setting and participants Western Sydney and Dubbo communities in NSW, Australia. Twenty-nine participants of diverse social and cultural backgrounds, mixed genders and ages recruited via public advertising: one jury was drawn from a large metropolitan setting; the other from a regional/rural setting. Main outcome measure Jury verdict and rationale in response to a prioritization task and structured questions. Results Both juries concluded that potential policy interventions to curb antibiotic misuse in the community should be directed towards: (i) ensuring that the public and prescribers were better educated about the dangers of antibiotic resistance; (ii) making community-based human and animal health-care practitioners accountable for their prescribing decisions. Patient-centred approaches such as delayed prescribing were seen as less acceptable than prescriber-centred approaches; both juries completely rejected any proposal to decrease consumer demand by increasing antibiotic prices. Conclusion These informed citizens acknowledged the importance of raising public awareness of the risks, impacts and costs of antibiotic resistance and placed a high priority on increasing social and professional accountability through restrictive measures. Their overarching aim was that policy interventions should be directed towards creating collective actions and broad social support for changing antibiotic use through establishing and explaining the need for mechanisms to control and support better prescribing by practitioners, while not transferring the burdens, costs and risks of interventions to consumers.

The Gram-Negative ‘Superbugs’ Conference of the Australasian Society for Infectious Diseases, Gold Coast, Queensland, Australia, 2–3 August 2013 The Australian Society for Infectious Diseases hosted a national conference focused specifically on raising awareness of and contemplating solutions to the rise in antimicrobial resistance, especially in Gram-negative bacteria. Presentations were primarily informative, although some lively interactive sessions were held, particularly to debate contentious areas and to discuss options for policy makers and practitioners in infection control and antimicrobial stewardship. The conference brought together a diversity of backgrounds and interests, and was the first national meeting focused on this area.

Resistance of the Enterobacteriaceae to antibiotics, especially of the β lactam type, is increasingly dominated by the mobilization of continuously expressed single genes that encode efficient drug modifying enzymes. Strong and ubiquitous selection pressure has seemingly been accompanied by a shift from "natural" resistance, such as inducible chromosomal enzymes, membrane impermeability, and drug efflux, to the modern paradigm of mobile gene pools that largely determine the epidemiology of modern antibiotic resistance. In this way, antibiotic resistance is more available than ever before to organisms such as Escherichia coli and Klebsiella pneumoniae that are important causes of major sepsis. Modulation of the phenotype by host bacteria makes gene transmission less obvious and may in part explain why tracking and control of carbapenem resistance has been particularly problematic in the Enterobacteriaceae. This review discusses the underlying principles and clinical implications of the mobility and fixation of resistance genes and the exploitable opportunities and potential threats arising from apparent limitations on diversity in these mobile gene pools. It also provides some illustrative paradoxes and clinical corollaries, as well as a summary of future options.

Motivation: Larger than gene structures (LGS) are DNA segments that include at least one gene and often other segments such as inverted repeats and gene promoters. Mobile genetic elements (MGE) such as integrons are LGS that play an important role in horizontal gene transfer, primarily in Gram-negative organisms. Known LGS have a profound effect on organism virulence, antibiotic resistance and other properties of the organism due to the number of genes involved. Expert-compiled grammars have been shown to be an effective computational representation of LGS, well suited to automating annotation, and supporting de novo gene discovery. However, development of LGS grammars by experts is labour intensive and restricted to known LGS. Objectives: This study uses computational grammar inference methods to automate LGS discovery. We compare the ability of six algorithms to infer LGS grammars from DNA sequences annotated with genes and other short sequences. We compared the predictive power of learned grammars against an expert-developed grammar for gene cassette arrays found in Class 1, 2 and 3 integrons, which are modular LGS containing up to 9 of about 240 cassette types. Results: Using a Bayesian generalization algorithm our inferred grammar was able to predict > 95% of MGE structures in a corpus of 1760 sequences obtained from Genbank (F-score 75%). Even with 100% noise added to the training and test sets, we obtained an F-score of 68%, indicating that the method is robust and has the potential to predict de novo LGS structures when the underlying gene features are known. Availability: http://www2.chi.unsw.edu.au/attacca. Contact: guyt@unsw.edu.au

The epidemiology of emerging antibiotic resistance genes in Asia is inadequately defined and studies within the major pools of transmissible genes such as integron gene cassettes are important. One hundred and twenty-two non-repetitive Acinetobacter spp. isolates were obtained from inpatients of a major hospital in South China. Fifty-three of these isolates contained class 1 integrons, and there is evidence of horizontal gene transfer between unrelated clones. The common pool of gene cassettes was dominated by four cassette arrays: arr3-aacA4 (24 isolates of several unrelated strains); aacC1-orfP-orfQ-aadA1a (11 isolates, probably all the same strain); aacA4-catB8-aadA1 (2 isolates); and dfrVII (1 isolate). We developed a simple restriction fragment length polymorphism (RFLP)-based identification of these and other cassettes reported in China, using readily available enzymes, to facilitate further studies of this type.

In the last few years, phenotypically carbapenem resistant Acinetobacter strains have been identified throughout the world, including in many of the hospitals and intensive care units (ICUs) of Australia. Genotyping of Australian ICU outbreak-associated isolates by pulsed-field gel electrophoresis of whole genomic DNA indicated that different strains were cocirculating within one hospital. The carbapenem-resistant phenotype of these and other Australian isolates was found to be due to carbapenem-hydrolyzing activity associated with the presence of the bla OXA-23 gene. In all resistant strains examined, the bla OXA-23 gene was adjacent to the insertion sequence ISAba 1 in a structure that has been found in Acinetobacter baumannii strains of a similar phenotype from around the world; bla OXA-51 -like genes were also found in all A. baumannii strains but were not consistently associated with ISAba 1 , which is believed to provide the promoter required for expression of linked antibiotic resistance genes. Most isolates were also found to contain additional antibiotic resistance genes within the cassette arrays of class 1 integrons. The same cassette arrays, in addition to the ISAba 1-bla OXA-23 structure, were found within unrelated strains, but no common plasmid carrying these accessory genetic elements could be identified. It therefore appears that antibiotic resistance genes are readily exchanged between cocirculating strains in epidemics of phenotypically indistinguishable organisms. Epidemiological investigation of major outbreaks should include whole-genome typing as well as analysis of potentially transmissible resistance genes and their vehicles.

Background Gram-negative bacteria such as Escherichia coli or Klebsiella spp. frequently cause bloodstream infections. There has been a worldwide increase in resistance in these species to antibiotics such as third generation cephalosporins, largely driven by the acquisition of extended-spectrum beta-lactamase or plasmid-mediated AmpC enzymes. Carbapenems have been considered the most effective therapy for serious infections caused by such resistant bacteria; however, increased use creates selection pressure for carbapenem resistance, an emerging threat arising predominantly from the dissemination of genes encoding carbapenemases. Recent retrospective data suggest that beta-lactam/beta-lactamase inhibitor combinations, such as piperacillin-tazobactam, may be non-inferior to carbapenems for the treatment of bloodstream infection caused by extended-spectrum beta-lactamase-producers, if susceptible in vitro. This study aims to test this hypothesis in an effort to define carbapenem-sparing alternatives for these infections. Methods/Design The study will use a multicentre randomised controlled open-label non-inferiority trial design comparing two treatments, meropenem (standard arm) and piperacillin-tazobactam (carbapenem-sparing arm) in adult patients with bacteraemia caused by E. coli or Klebsiella spp. demonstrating non-susceptibility to third generation cephalosporins. Recruitment is planned to occur in sites across three countries (Australia, New Zealand and Singapore). A total sample size of 454 patients will be required to achieve 80% power to determine non-inferiority with a margin of 5%. Once randomised, definitive treatment will be for a minimum of 4 days, but up to 14 days with total duration determined by treating clinicians. Data describing demographic information, antibiotic use, co-morbid conditions, illness severity, source of infection and other risk factors will be collected. Vital signs, white cell count, use of vasopressors and days to bacteraemia clearance will be recorded up to day 7. The primary outcome measure will be mortality at 30 days, with secondary outcomes including days to clinical and microbiological resolution, microbiological failure or relapse, isolation of a multi-resistant organism or Clostridium difficile infection. Trial registration The MERINO trial is registered under the Australian New Zealand Clinical Trials Register (ANZCTR), reference number: ACTRN12613000532707 (registered 13 May 2013) and the US National Institute of Health ClinicalTrials.gov register, reference number: NCT02176122 (registered 24 June 2014). Electronic supplementary material The online version of this article (doi:10.1186/s13063-014-0541-9) contains supplementary material, which is available to authorized users.

Sir,Malakoplakia and Whipple's disease are generally regarded as two discrete disease entities. Their occurrence in the same patient has not previously been reported. We report the case of 54‐year‐...

Bartonella henselae, the agent of cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen, whose interaction with macrophages and endothelial cells (ECs) is crucial in the pathogenesis of these diseases. However, little is known about the subcellular compartment in which B. henselae resides. Two hours after infection of murine macrophages and human ECs, the majority of B. henselae-containing vacuoles (BCVs) lack typical endocytic marker proteins, fail to acidify, and do not fuse with lysosomes, suggesting that B. henselae resides in a non-endocytic compartment. In contrast to human umbilical vein endothelial cells, bacterial death and lysosomal fusion with BCVs is apparent in J774A.1 macrophages at 24 h. This phenomenon of delayed lysosomal fusion requires bacterial viability, and is confined to the BCV itself. Using magnetic selection, we enriched for transposon-mutagenized B. henselae trapped in lysosomes of macrophages 2 h after infection. Genes affected appear to be relevant to the intracellular lifestyle in macrophages and ECs and include some previously implicated in Bartonella pathogenicity. We conclude that B. henselae has a specific capacity to actively avoid the host endocytic pathway after entry of macrophages and ECs, from within a specialized non-endocytic membrane-bound vacuole.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

Department of Microbiology, St Vincent’s Hospital, Darlinghurst, Sydney, Australia; School of Biological Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia; Centre for Infectious Diseases and Microbiology, Institute for Clinical Pathology and Medical Research, Westmead, Sydney, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, Australia; Department of HIV, Immunology and Infectious Diseases, St Vincent’s Hospital, Darlinghurst, Sydney, Australia; Sydney Clinical School, Faculty of Medicine, University of Sydney, Sydney, Australia

A 44-year-old man with a bioprosthetic aortic valve suffered destructive endocarditis with severe embolic disease due to Bartonella henselae infection. Multilocus sequence typing was successfully performed with crude preparations of operative tissue as templates, and the infecting organism was determined to be typical of the Houston clonal group, although it was never cultured from blood or tissue. This is the first report of B. henselae infection in the South Pacific, and it reminds one that B. henselae is a cause of potentially lethal culture-negative endocarditis which may respond poorly to conventional empirical therapy. Nothing is known of the epidemiology of the infection in this region, but it is likely to be common and to contain representatives of both major clonal complexes. This study emphasizes the ease with which multilocus sequence typing can be used directly with tissue, which is important because of suggestions of strain-dependent clinical outcomes.

Pseudomonas aeruginosa is a significant pathogen of patients with cystic fibrosis (CF), and renewed concerns over antibiotic resistance have been raised with reports of potential patient-to-patient transmission of antibiotic resistant clonal strains. A recent study conducted in two CF centres in Brisbane, Australia, identified a dominant clonal P. aeruginosa strain (P2). This P2 clone displayed increased resistance to tobramycin, one of the most commonly used antibiotics in patients with CF. Horizontal gene transfer mechanisms such as plasmids, transposons and integrons containing resistance genes can enhance the spread and development of antibiotic resistance in bacteria. We investigated whether integrons were present in mobile elements present in P. aeruginosa isolates harboured by patients with CF in two Brisbane CF centres, and if these integrons carried associated resistance cassettes.Overall, our results indicate a high occurrence of class 1 integron carriage and class 1 associated aadB gene cassette in P. aeruginosa isolates in Brisbane CF patients. Longitudinal studies are required to determine the true impact of this resistance element, if rates of resistance gene detection change over time, and whether horizontal transfer to other Gram negative bacteria occurs in patients with CF.© 2008 Royal College of Pathologists of Australasia

Pseudomonas aeruginosa and Burkholderia cepacia are the major pathogens that colonize the airway surface and cause progressive respiratory failure and high mortality, especially in cystic fibrosis (CF) patients. Tobramycin is the treatment of choice, but persistent usage enables the infectious organisms to activate defence mechanisms, making eradication rarely successful. Combinations of antibiotic and nonantibiotic compounds have been tested in vitro against P. aeruginosa and B. cepacia, but with mixed results. Sodium ions interfere with the bacterial tobramycin uptake system, but amiloride partially reverses this antagonism. In this pilot study, we extend previous findings of the effectiveness of tobramycin in combination with amiloride and other nonantibiotics against a P. aeruginosa type strain, and against four P. aeruginosa strains and one Burkholderia cenocepacia strain isolated from CF patients. Significantly, the four clinical P. aeruginosa strains were tobramycin resistant. We also find that Na+ and K+, but not Cl(-), are the chief antagonists of tobramycin efficacy. These results suggest that chemotherapy for CF patients might not only be compromised by antibiotic-resistant pathogens alone, but by a lack of penetration of antibiotics caused either by bacterial biofilms or the high sodium flux in the CF lung, or by antagonistic effects of some drug combinations, any of which could allow the persistence of drug-susceptible bacteria.

Twenty-two non-repetitive carbapenem-resistant Acinetobacter baumannii isolates were obtained from Intensive Care Unit patients. All of the isolates carried bla(OXA-23), bla(OXA-66), a novel cephalosporinase-encoding gene (bla(ADC-25)) and a class 1 integron with an aacC1-orfP-orfQ-aadA1a cassette array and had identical enterobacterial repetitive intergenic consensus (ERIC) profiles. ISAba1 was found upstream of bla(OXA-23), but was not associated with bla(OXA-66) or bla(ADC-25).

The greatest impact of microbiology data on clinical care is in the critically ill. Unfortunately, this is also the area in which microbiology laboratories are most often non-contributive. Attempts to move to rapid, culture-independent diagnostics are driven by the need to expedite urgent results. This is difficult in Gram-negative infection because of the complexity of the antibiotic resistance phenotype. Here, we discuss resistance to modern beta-lactams as a case in point. Recent outbreaks of transmissible carbapenem resistance among Gram-negative enteric pathogens in Sydney and Melbourne serve to illustrate the pitfalls of traditional phenotypical approaches. A better understanding of the epidemiology and mosaic nature of antibiotic resistance elements in the microflora is needed for us to move forward.

Aims Aztreonam/clavulanic acid combination therapy is a potential treatment for multi-resistant Enterobacteriaceae containing MBLs and ESBLs. We describe an aztreonam/clavulanic acid based phenotypic detection and susceptibility test for MBL and ESBL-containing multi-resistant Enterobacteriaceae derived from initial genotypic characterisation. Methods Three phenotypically pan-resistant (by Etest and automated broth microdilution) K. pneumoniae isolates from a clinical colonisation mini-outbreak were shown by PCR to contain MBL and ESBL β-lactamases. These isolates and thirteen other Enterobacteriaceae containing MBLs and ESBLs (with three also containing AmpC β-lactamases) were assessed for susceptibility to aztreonam/clavulanic acid by either broth microdilution or Etest, and our phenotypic disc-diffusion assay. β-lactamases were confirmed by multiplex PCR/reverse line blot. Results Clavulanic acid restored susceptibility to aztreonam in all ‘pan-resistant’ outbreak isolates but not strains also carrying an AmpC β-lactamase. Our disc-diffusion assay predicted 16/16 MBLs, 15/16 ESBLs and 3/3 AmpC β-lactamases and showed susceptibility to aztreonam/clavulanic acid in all non-AmpC isolates. Discussion Pan-resistant Gram-negative isolates are likely to become increasingly common. We have described a potential treatment for an initially phenotypically pan-resistant organism that was predicted from a genotype and developed a simple combined phenotypic detection and susceptibility test. Both potential treatment and test could be employed in resource poor settings.

Lyn Gilbert , Jon Iredell and John Merlino Marie Bashir Institute for Emerging Infections and Biosecurity, Centre for Values, Ethics and the Law in Medicine, University of Sydney, and Level 3, Institute of Clinical Pathology & Medical Research, Westmead Hospital, PO Box 533, Wentworthville, NSW 2145, Australia. Tel: +61 2 9845 6252, Email: lyn.gilbert@sydney.edu.au WestmeadMillennium Institute forMedical Research, C24 –WestmeadHospital, TheUniversity of Sydney, NSW2006, Australia. Tel: +61 2 9845 6255, Email: jonathan.iredell@sydney.edu.au Department of Microbiology and Infectious Diseases, Concord Hospital, and Department of Infectious Diseases and Immunology, Sydney Medical School, University of Sydney, Hospital Road, Concord, NSW 2139, Australia. Tel: +61 2 9767 6658, Email: JMerlino@med.usyd.edu.au

The advent of molecular diagnostics has allowed us to move into the clinically relevant timeframe for diagnosis for the first time. Bacterial load testing, real-time antibiotic resistance testing and infection control screening are discussed. The common platforms and their potential are discussed. The phenotype-genotype relationship is organism-specific to some extent and this is contrasted for the four major pathogens of septic shock, S. aureus, E. coli, K. pneumoniae and Streptococcus spp., including the natural minimum inhibitory concentration (MIC) distribution of the major transmissible resistance genes.

Objective Evaluation of the clinical and epidemiological features of Mycobacterium marinum infection in Queensland. Design Laboratory identification and in-vitro susceptibility tests of 29 isolates from the Queensland Health Department Tuberculosis Reference Laboratory were retrospectively gathered and followed up by contacting referring practitioners and obtaining clinical details of patients involved. Subjects 29 patients from whom M. marinum was isolated, with a male:female ratio of 3.1:1, and a mean age of 47.4 years. Results Of 26 patients for whom adequate information was available, 12 had evidence of involvement of deep tissues (including two cases of arthritis) and five suffered sporotrichoid spread of infection. The delay between onset of symptoms and consultation with a medical practitioner was five months (range, two weeks to two years), with a further mean delay to definitive diagnosis of 4.4 weeks. Cure was apparent in 22 of 23 cases. Chemotherapy alone was adequate in 11 cases, as was surgical intervention in three, while a combination approach was successful in eight cases. Trimethoprim/sulfamethoxazole was successful in seven of nine cases and combination rifampicin and ethambutol in six of seven. Tetracyclines were employed as single-agent therapy in nine patients and were effective in seven. Conclusions Synovitis was a common presenting feature of M. marinum infection in Queensland patients. Occupational and recreational exposure to salt or fresh water was common, and although this history was available to practitioners a mean delay to definitive diagnosis of 4.4 weeks still occurred. The data suggest that chemotherapy alone is often adequate, even with deep tissue involvement. Combinations of conventional antimycobacterial drugs may be the therapy of choice, especially for serious infections, although success was recorded with trimethoprim/sulfamethoxazole alone.

Identification and susceptibility testing of presumed staphylococci in blood cultures require >24 h by routine phenotypic methods. Delayed appropriate antibiotic therapy results in increased mortality in critically ill patients, while unnecessary vancomycin use may result in suboptimal therapy and

The ubiquitous feline intraerythrocytic pathogen Bartonella henselae causes a range of important human infections, and seroprevalence in blood donors in Australia is around 5% (6). Two antigenically distinct subtypes have been described, named for the type strain (Houston-1) and the more recently recognized Marseille strain (5), which have characteristic DNA sequences in the 16S rRNA gene (rDNA) and the gltA (citrate synthase) gene (2, 3, 5). Culture of this fastidious, gram-negative bacterium is difficult, and serology is a primary diagnostic approach. A recent report that Houston-like strains (including Houston-1, 90-615, and SA-2) may be distinguished from serologically distinct Marseille-like strains (including URBHLLY-8, URBHLIE-9, Fizz, and CAL-1) by the faster migration of a characteristic band (ca. 29 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting of whole-cell lysates is therefore particularly noteworthy (7). However, the SDS-PAGE data appear to contrast with previous publications from the same laboratory (8, 9) and others (11). We therefore compared local human isolates with the Houston-1 and Marseille strains and also found two closely related genotypes with corresponding patterns on SDS-PAGE of whole-cell lysates (Fig. ​(Fig.1).1). All human strains appear to have either the 30-kDa band (Houston) or the 29-kDa band (Marseille), in agreement with earlier studies (10, 11). The agar-pitting colony phenotype and number of in vitro passages have no apparent influence (Fig. ​(Fig.11 and Table ​Table1).1). However, a local feline strain of the type II (Marseille-like) 16S rDNA genotype has the faster-migrating (29-kDa) band and a gltA gene sequence typical of Houston strains, consistent with the considerable diversity known to exist in feline strains (1, 10) and illustrating a dissociation of the accepted genotype-phenotype relationships. The strains in the recent study (7) were all human isolates and were assigned to genotypes by individual sequencing of several genes, including groEL (12) and the 16S rDNA (3). While the 29- to 30-kDa antigen(s) may yet prove useful for serotyping of human isolates, it thus seems unlikely that reported inconsistencies in the data can be easily explained by phase variation or genotype. In addition, we have no reason to assume that the more diverse feline strains are all nonpathogenic in humans. For both of thesereasons, we therefore urge caution in adopting this promising target as a basis for serotype distinction until the picture is clearer. FIG. 1. Marseille and Houston types. Coomassie-stained SDS-PAGE of whole-cell lysates harvested from chocolate agar plates (grown at 35°C in 5% CO2). High-molecular-weight (lane 1 and left border) and low-molecular-weight (right border) markers are shown, ... TABLE 1. B. henselae isolates used in this study

Among the agents of community-acquired septic shock in countries such as Australia and the US, the major risk of antibiotic failure not easily identified by a simple gene target resides with the Enterobacteriaceae , particularly Escherichia coli and Klebsiella pneumoniae . Streptococcus pneumoniae and Staphylococcus aureus are easily managed with a small handful of markers but the potential causes of β-lactam and aminoglycoside resistance among E. coli and K. pneumoniae are legion. However, the local gene pool may be restricted and a ‘local profile’ may be small enough to use in ‘realtime’ diagnostic and screening tools. Here, we review the experience with the common phenotypes, the approach to screening, and the major questions we need to resolve before we move these tools into routine practice. The focus in this session is on the markers of β-lactam and aminoglycoside resistance in E. coli and K. pneumoniae , and on the opportunities and pitfalls in the assay systems available to detect them.

Strongyloides stercoralis is a parasitic nematode that can cause infections ranging in severity from the asymptomatic to fatal. Aim To investigate whether the loop mediated isothermal amplification (LAMP) of nucleic acid can be applied to the detection of S. stercoralis DNA in stool. Methods Primers were designed that were specific to the stron-gyloides 28S rRNA gene. The LAMP reaction was optimised using Strongyloides ratti, which was available through laboratory culture. LAMP products were characterised with restriction enzyme digestion and cloning with sequence analysis. Analytical sensitivity was determined using serial dilutions of a PCR product and human stool specimens spiked with S. ratti. Stool specimens with S. stercoralis were tested. Specificity was tested using a range of bacteria, fungi and parasites. Results S. stercoralis and spiked S. ratti were detected in human stool specimens. Tests showed a high degree of analytic sensitivity, between 11.75 and 2.35 copies of DNA, and good specificity. Discussion The most effective methods used to detect S. stercoralis in stool require live larvae. Nucleic acid detection may enable easier specimen transport and reduce the risk of laboratory acquired infections. This preliminary study indicates that LAMP may be a useful method to detect S. stercoralis in the stool.

Determining the origin, spread and characteristics of important resistance genes and plasmids in Gram-negative bacteria may be more informative than determining the epidemiology of bacteria themselves. Infection control resistance surveillance and containment efforts should focus on the transmission characteristics of the resistance rather than those of the index organism at the point of recognition.

The study of bacterial resistance to antimicrobial agents is a complex science that requires an understanding of the genetic, molecular and physiological mechanisms of resistance, and the effect of antibiotic selection pressures on bacterial populations in different environments.