Jon Iredell

Horizontal Gene Transfer in a Polyclonal Outbreak of Carbapenem-Resistant Acinetobacter baumannii

ABSTRACT 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 blaOXA-23 gene. In all resistant strains examined, the blaOXA-23 gene was adjacent to the insertion sequence ISAba1 in a structure that has been found in Acinetobacter baumannii strains of a similar phenotype from around the world; blaOXA-51-like genes were also found in all A. baumannii strains but were not consistently associated with ISAba1, 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 ISAba1-blaOXA-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.

Antibiotic resistance in Enterobacteriaceae: mechanisms and clinical implications

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.

Unusual trafficking pattern of Bartonella henselae ‐containing vacuoles in macrophages and endothelial cells

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.

Meropenem versus piperacillin-tazobactam for definitive treatment of bloodstream infections due to ceftriaxone non-susceptible Escherichia coli and Klebsiella spp (the MERINO trial): study protocol for a randomised controlled trial

BackgroundGram-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/DesignThe 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 registrationThe 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).

Failure of the BD GeneOhm StaphS/R Assay for Identification of Australian Methicillin-Resistant Staphylococcus aureus Strains: Duplex Assays as the “Gold Standard” in Settings of Unknown SCCmec Epidemiology

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

Integron gene cassettes in Acinetobacter spp. strains from South China

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 vitro interactions of tobramycin with various nonantibiotics against Pseudomonas aeruginosa and Burkholderia cenocepacia

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.

Culture-Negative Endocarditis Due to Houston Complex Bartonella henselae Acquired in Noumea, New Caledonia

ABSTRACT 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.

Non-susceptibility to ceftaroline in healthcare-associated multiresistant MRSA in Eastern Australia

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

Antigenic and Genotypic Relationships between Bartonella henselae Strains

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