Use of different PCR-based DNA fingerprinting techniques and pulsed-field gel electrophoresis to investigate the epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii complex

Liu, Peter Yuk-Fong; Wu, Woa-Ling

doi:10.1016/s0732-8893(97)00080-1

Cited by 34 publications

(25 citation statements)

References 33 publications

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“…Acinetobacter baumannii hospital outbreaks have been assessed with various DNA typing methods (5)(6)(7)(8). Very few typing methods assess outbreaks in real time.…”

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confidence: 99%

Typing of Nosocomial Outbreaks of Acinetobacter baumannii by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

et al. 2013

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Acinetobacter baumannii has become a major nosocomial threat, causing infections with high rates of morbidity and mortality (1-4).Acinetobacter baumannii hospital outbreaks have been assessed with various DNA typing methods (5-8). Very few typing methods assess outbreaks in real time. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed for the rapid identification of pathogens (9). There are a growing number of reports showing the utility of MALDI-TOF MS to type different bacterial species for easy and early detection of nosocomial outbreaks (10-16).The aim of this study was to assess the potential utility of MALDI-TOF MS to detect the nosocomial spread of A. baumannii isolates. For this purpose, 35 multidrug-resistant strains of A. baumannii, isolated from colonized or infected patients hospitalized in a general hospital in Italy in 2010, were studied using the repetitive sequence-based PCR (rep-PCR) DiversiLab system (DL; bioMérieux, Marcy l'Etoile, France) and MALDI-TOF MS system (Bruker Daltonics, Bremen, Germany), and the results were compared. After storage at Ϫ70°C, the isolates were thawed out and cultured simultaneously on MacConkey agar.DiversiLab rep-PCR (bioMérieux) was performed according to the manufacturer's instructions (5, 17). Briefly, DNA was extracted from a bacterial culture, amplified using rep-PCR, loaded in LabChip, and run using an Agilent 2100 Bioanalyzer (Agilent Technologies). The results were analyzed by DiversiLab with the Pearson correlation (PC) coefficient to emphasize peak intensities more than peak presence or absence. The strains were considered indistinguishable, similar, or different if the similarity was Ն97.5% without differences in the fingerprinting pattern, Ͼ95% and Ͻ97.5%, or Յ95%, respectively.For MALDI-TOF MS, bacteria were cultured for 24 h at 37°C on MacConkey agar and extracts were prepared as described previously (18, 19). For isolate identification, the row spectra were compared with those in the Biotyper database and a log(score) of Ն2.3 was considered to represent a secure species identification. Spectra were acquired with a microflex LT mass spectrometer (Bruker Daltonics) and recorded in the positive linear mode at a laser frequency of 20 Hz, ion source 1 voltage of 20 kV, ion source 2 voltage of 8.5 kV, and mass range from 2,000 to 20,000 kDa, as described elsewhere (18). Reference spectra of newly created main spectra were added to the original Biotyper database. To evaluate the spectrum variation within each strain and to estimate the mass spectral variance of biological replicates, a principal component analysis (PCA) was performed; two biological replicates from six randomly selected Acinetobacter strains were tested, as described above, and spectra for each selected strain were acquired in the same experiment on the mass spectrometer. Technical mass-spectral variance was also evaluated, analyzing five technical replicates from each Acinetobacter independent culture on the same MALDI t...

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“…Acinetobacter baumannii hospital outbreaks have been assessed with various DNA typing methods (5)(6)(7)(8). Very few typing methods assess outbreaks in real time.…”

mentioning

confidence: 99%

Typing of Nosocomial Outbreaks of Acinetobacter baumannii by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

et al. 2013

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“…XDR A. baumannii isolates were defined as isolates that were resistant to all the tested antimicrobials except for tigecycline, rifampin, and polymyxin B (5, 6), while pandrug-resistant (PDR) A. baumannii was resistant to all tested antimicrobial agents (5,6). Clonal relatedness of study isolates was determined by multiplex PCR strain typing (11), with cluster analysis of banding data performed using the unweighted pair group method with arithmetic mean.…”

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confidence: 99%

In Vitro Antibiotic Synergy in Extensively Drug-Resistant Acinetobacter baumannii : the Effect of Testing by Time-Kill, Checkerboard, and Etest Methods

Tan

Lim

Lee

et al. 2011

Antimicrob Agents Chemother

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Treatment of extensively drug-resistant (XDR) Acinetobacter baumannii infections is challenging because of both the limited choice of antibiotic and the tendency of such infections to occur in critically ill hosts with limited physiologic reserves (17). Polymyxins demonstrate in vitro activity against A. baumannii, but resistance is also reported (8,17). In the absence of feasible alternatives, unconventional antibiotics, such as minocycline, rifampin, and tigecycline, have been used, both singly and in combination (19). In vitro combination susceptibility testing poses a significant challenge for clinical microbiology laboratories, with a lack of standardization and a variety of in vitro testing methods. This study evaluated the effects of various antibiotic combinations against a panel of XDR-Acinetobacter baumannii and compared in vitro synergy testing results obtained from time-kill, checkerboard, and Etest methods.(Some elements of this study were presented at the 20th European Congress of Clinical Microbiology and Infectious Diseases, 2010.) Clinical isolates of antibiotic-resistant A. baumannii were collected from four hospitals in Singapore over a 2-year period. Species identification was confirmed by a multiplex PCR assay (2). MICs to ampicillin-sulbactam, ciprofloxacin, gentamicin, imipenem, meropenem, aztreonam, piperacillin-tazobactam, polymyxin B, tigecycline, ceftazidime, amikacin, and cefepime were obtained by broth microdilution (Sensititre, Trek Diagnostics, United Kingdom), while MICs for rifampin were obtained by broth macrodilution (3). Categorical susceptibility was based on Clinical and Laboratory Standards Institute breakpoints (4). XDR A. baumannii isolates were defined as isolates that were resistant to all the tested antimicrobials except for tigecycline, rifampin, and polymyxin B (5, 6), while pandrug-resistant (PDR) A. baumannii was resistant to all tested antimicrobial agents (5, 6). Clonal relatedness of study isolates was determined by multiplex PCR strain typing (11), with cluster analysis of banding data performed using the unweighted pair group method with arithmetic mean.All strains were tested for the presence of in vitro synergy to polymyxin B-rifampin, polymyxin B-tigecycline, and tigecycline-rifampin combinations by three methods. These combinations were selected based on previously published data, which demonstrated a high likelihood of achieving in vitro synergy (10). Time-kill assays were performed following methods published by the Clinical and Laboratory Standards Institute (13), with colony enumeration performed at 0-and 24-h time points. The lower limit of detection was 400 CFU/ml. Antibiotic concentrations used for the time-kill assay were 2 mg/liter for each antibiotic, representing achievable serum concentrations for polymyxin B (9) and rifampin (7) and achievable tissue levels for tigecycline (18).Etest and checkerboard synergy testing were performed according to published methods (12,20). Fractional inhibitory concentrations (FIC) were calculated as follows: ...

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“…2), and S. warneri (22%) (Fig. 3) organisms isolated from the hands and environ- Although Acinetobacter colonization on human skin and fingertips and prolonged survival in the environment (27 days) has been reported (2,12,17), this is the first study to document same-strain sharing between hands and environmental surfaces within a community setting. Over the past two decades, Acinetobacter species have become virulent pathogens, responsible for nosocomial infections and outbreaks, particularly in intensive care units (36).…”

Section: Discussionmentioning

confidence: 77%

Molecular Characterization of Hand Flora and Environmental Isolates in a Community Setting

Pancholi

Healy²,

Bittner³

et al. 2005

J Clin Microbiol

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We analyzed 69 bacterial isolates, comprising seven species of gram-negative bacterial rods and three species of coagulase-negative staphylococci, recovered from both the hands of caretakers and their environment in households sampled in upper Manhattan. Repetitive sequence-based PCR and dendrogram analysis were used to determine strain similarity. Greater than 25% of individual species of Acinetobacter, Enterobacter, and coagulase-negative staphylococci recovered from the hands and immediate environment within each household shared the same genotype. This study is the first to demonstrate the frequency of bacteria shared within community households. These strains may serve as potential reservoirs for either community-or hospitalacquired infections.

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Use of different PCR-based DNA fingerprinting techniques and pulsed-field gel electrophoresis to investigate the epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii complex

Cited by 34 publications

References 33 publications

Typing of Nosocomial Outbreaks of Acinetobacter baumannii by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Typing of Nosocomial Outbreaks of Acinetobacter baumannii by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

In Vitro Antibiotic Synergy in Extensively Drug-Resistant Acinetobacter baumannii : the Effect of Testing by Time-Kill, Checkerboard, and Etest Methods

Molecular Characterization of Hand Flora and Environmental Isolates in a Community Setting

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