Online-Only Abstracts: These articles are available online at wileyonlinelibrary.com

In recent years, matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has proved a rapid and reliable method for the identification of bacteria and yeasts that have already been isolated. The objective of this study was to evaluate this technology as a routine method for the identification of microorganisms directly from blood culture bottles (BCBs), before isolation, in a large collection of samples. For this purpose, 1000 positive BCBs containing 1085 microorganisms have been analysed by conventional phenotypic methods and by MALDI-TOF MS. Discrepancies have been resolved using molecular methods: the amplification and sequencing of the 16S rRNA gene or the Superoxide Dismutase gene (sodA) for streptococcal isolates. MALDI-TOF predicted a species- or genus-level identification of 81.4% of the analysed microorganisms. The analysis by episode yielded a complete identification of 814 out of 1000 analysed episodes (81.4%). MALDI-TOF identification is available for clinicians within hours of a working shift, as oppose to 18 h later when conventional identification methods are performed. Moreover, although further improvement of sample preparation for polymicrobial BCBs is required, the identification of more than one pathogen in the same BCB provides a valuable indication of unexpected pathogens when their presence may remain undetected in Gram staining. Implementation of MALDI-TOF identification directly from the BCB provides a rapid and reliable identification of the causal pathogen within hours.

A comparative study of clinical Aeromonas dhakensis and Aeromonas hydrophila isolates in southern Taiwan: A. dhakensis is more predominant and virulent

P.-L. Chen¹,², C.-J. Wu²,³, C.-S. Chen⁴, P.-J. Tsai⁵,⁶, H.-J. Tang⁷,⁸ and W.-C. Ko¹,⁹1) Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan, 2) Graduate Institute of Clinical Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan, 3) National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan, 4) Department of Biochemistry and Molecular Biology, National Cheng Kung University College of Medicine, Tainan, Taiwan, 5) Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University College of Medicine, Tainan, Taiwan, 6) Research Centre of Infectious Disease and Signalling, National Cheng Kung University, Tainan, Taiwan, 7) Department of Medicine, Chi Mei Medical Centre, Tainan, Taiwan, 8) Department of Health and Nutrition, Chia Nan University of Pharmacy and Science, Tainan, Taiwan and 9) Department of Medicine, National Cheng Kung University College of Medicine, Tainan, TaiwanOriginal Submission: 2 August 2013; Revised Submission: 9 November 2013; Accepted: 9 November 2013Article published online: 16 December 2013

Abstract

Aeromonas dhakensis, often phenotypically identified as Aeromonas hydrophila, is an important human pathogen. The present study aimed to compare the clinical and biological features of A. dhakensis and A. hydrophila isolates from human wounds. A total of 80 Aeromonas wound isolates collected between January 2004 and April 2011 were analysed. The species was identified by the DNA sequence matching of rpoD and gyrB (or rpoB if necessary). Most of the Aeromonas isolates were identified as A. dhakensis (37, 46.3%), and 13 (16.3%) as A. hydrophila. Both species alone can cause severe skin and soft-tissue infections. More A. dhakensis isolates were found in wounds exposed to environmental water (32.4% vs 0%, p 0.042). More biofilm formation was noted among A. dhakensis isolates (mean optical density at 570 nm, 1.23 ± 0.09 vs 0.78 ± 0.21, p 0.03). The MICs of ceftriaxone, imipenem and gentamicin for A. dhakensis isolates were higher (p <0.0001, <0.04, and <0.01, respectively). The survival rates of Caenorhabditis elegans co-incubated with A. dhakensis from day 1 to day 3 were lower than those of worms infected with A. hydrophila in liquid toxicity assays (all p values <0.01). Isolates of A. dhakensis exhibited more cytotoxicity, as measured by the released leucocyte lactate dehydrogenase levels in human normal skin fibroblast cell lines (29.6 ± 1.2% vs 20.6 ± 0.6%, p <0.0001). The cytotoxin gene ast was primarily present in A. hydrophila isolates (100% vs 2.7%, p <0.0001). In summary, A. dhakensis is the predominant species among Aeromonas wound isolates, and more virulent than A. hydrophila.

Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene

M. Mentasti¹, A. Underwood², C. Lück³, N. A. Kozak-Muiznieks⁴, T. G. Harrison¹ and N. K. Fry¹1) Respiratory and Vaccine Preventable Bacteria Reference Unit, 2) Applied Laboratory and Bioinformatics Unit, Public Health England, London, UK, 3) Institut für Medizinische Mikrobiologie und Hygiene, TU Dresden, Dresden, Germany and 4) Centers for Disease Control and Prevention, Atlanta, GA, USAOriginal Submission: 1 May 2013; Revised Submission: 12 November 2013; Accepted: 12 November 2013Article published online: 12 December 2013

Abstract

Sequence-based typing (SBT) combined with monoclonal antibody subgrouping of Legionella pneumophila isolates is at present considered to be the reference standard during epidemiological investigation of Legionnaires‘ disease outbreaks. In some isolates of L. pneumophila, the seventh allele of the standard SBT scheme, neuA, is not amplified, because a homologue that is refractory to amplification with the standard neuA primers is present. Consequently, a complete seven-allele profile, and hence a sequence type, cannot be obtained. Subsequently, primers were designed to amplify both neuA and the homologue, but these yielded suboptimal sequencing results. In this study, novel primers specific for the neuA homologue were designed and internationally validated by members of the ESCMID Study Group for Legionella Infections at national and regional Legionella reference laboratories with a modified version of the online L. pneumophila sequence quality tool. To date, the addition of the neuAh target to the SBT protocol has allowed full typing data to be obtained for 108 isolates of 11 different serogroups, namely 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, and 14, which could not previously be typed with the standard SBT neuA primers. Further studies are necessary to determine why it is still not possible to obtain either a neuA or a neuAh allele from three serogroup 11 isolates.