Molecular Epidemiology of Acinetobacter baumannii Bloodstream Isolates Obtained in the United States from 1995 to 2004 Using rep-PCR and Multilocus Sequence Typing

Higgins, Paul G.; Janßen, K.; Fresen, Maximilian M.; Wisplinghoff, Hilmar; Seifert, Harald

doi:10.1128/jcm.01759-12

Cited by 44 publications

(36 citation statements)

References 36 publications

(54 reference statements)

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“…In contrast, MLST reflects SNPs in conserved housekeeping genes and therefore more accurately represents population phylogeny. Although PFGE has improved with standardization, it remains subject to a number of factors that decrease reliability and reproducibility, such as DNA yield and purity, the type of enzyme used, and errors in gel resolution, intergel standardization, and band visualization and identification (25,26,49,92). These findings suggest that PFGE should not be used to determine relationships between distantly related bacterial isolates (93).…”

Section: Figmentioning

confidence: 89%

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Utility of Whole-Genome Sequencing in Characterizing Acinetobacter Epidemiology and Analyzing Hospital Outbreaks

2016

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bAcinetobacter baumannii frequently causes nosocomial infections and outbreaks. Whole-genome sequencing (WGS) is a promising technique for strain typing and outbreak investigations. We compared the performance of conventional methods with WGS for strain typing clinical Acinetobacter isolates and analyzing a carbapenem-resistant A. baumannii (CRAB) outbreak. We performed two band-based typing techniques (pulsed-field gel electrophoresis and repetitive extragenic palindromic-PCR), multilocus sequence type (MLST) analysis, and WGS on 148 Acinetobacter calcoaceticus-A. baumannii complex bloodstream isolates collected from a single hospital from 2005 to 2012. Phylogenetic trees inferred from core-genome single nucleotide polymorphisms (SNPs) confirmed three Acinetobacter species within this collection. Four major A. baumannii clonal lineages (as defined by MLST) circulated during the study, three of which are globally distributed and one of which is novel. WGS indicated that a threshold of 2,500 core SNPs accurately distinguished A. baumannii isolates from different clonal lineages. The bandbased techniques performed poorly in assigning isolates to clonal lineages and exhibited little agreement with sequence-based techniques. After applying WGS to a CRAB outbreak that occurred during the study, we identified a threshold of 2.5 core SNPs that distinguished nonoutbreak from outbreak strains. WGS was more discriminatory than the band-based techniques and was used to construct a more accurate transmission map that resolved many of the plausible transmission routes suggested by epidemiologic links. Our study demonstrates that WGS is superior to conventional techniques for A. baumannii strain typing and outbreak analysis. These findings support the incorporation of WGS into health care infection prevention efforts.A n important role of clinical microbiology is to identify relationships between bacterial isolates. At a broad level, phenotypic and genotypic tests are used to categorize bacterial isolates into the same or different species. Within a bacterial species, techniques are used to group isolates into clonal lineages, which are groups of closely related bacteria that share a recent common ancestor but have spread regionally or globally. At a more local level, infection control practitioners must determine whether a group of isolates constitutes a hospital outbreak by ascertaining whether the isolates have a degree of similarity consistent with a common source within the hospital. Once isolates belonging to a hospital outbreak have been identified, similarities and differences between these isolates can be exploited to generate a transmission map to aid in finding the source of the outbreak and in disrupting ongoing pathways of transmission. For some groups of bacteria, such as Acinetobacter, discernment at each level has medically important consequences and therefore must be accomplished by hospital-associated clinical microbiology laboratories.Within the Acinetobacter genus, the Acinetobacter calcoaceticusAcinetobac...

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Section: Figmentioning

confidence: 89%

“…The Dice statistic was used, and band tolerance was set to 1%. Isolates clustering together at Ͼ90% similarity based on the UPGMA dendrogram were considered to be the same type (23,48,49). Whole-genome sequencing and assembly.…”

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confidence: 99%

Utility of Whole-Genome Sequencing in Characterizing Acinetobacter Epidemiology and Analyzing Hospital Outbreaks

2016

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“…This picture appears to be the most common in both the United States and Europe (8,35). Molecular epidemiology surveys conducted in the United States on CRAb isolates obtained from 1995 to 2008 showed that IC-II isolates had spread to many centers as early as 1996 and became the dominant lineage (35,36). Interestingly, strains assigned to IC-II are known to form biofilms and adhere to abiotic surfaces more efficiently than strains assigned to IC-I (mainly characterized by elevated resistance to desiccation).…”

Section: Discussionmentioning

confidence: 99%

Epidemic Diffusion of OXA-23-Producing Acinetobacter baumannii Isolates in Italy: Results of the First Cross-Sectional Countrywide Survey

et al. 2014

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e Carbapenem-resistant Acinetobacter baumannii (CRAb) is emerging worldwide as a public health problem in various settings. The aim of this study was to investigate the prevalence of CRAb isolates in Italy and to characterize their resistance mechanisms and genetic relatedness. A countrywide cross-sectional survey was carried out at 25 centers in mid-2011. CRAb isolates were reported from all participating centers, with overall proportions of 45.7% and 22.2% among consecutive nonreplicate clinical isolates of A. baumannii from inpatients (n ‫؍‬ 508) and outpatients (n ‫؍‬ 63), respectively. Most of them were resistant to multiple antibiotics, whereas all remained susceptible to colistin, with MIC 50 and MIC 90 values of <0.5 mg/liter. The genes coding for carbapenemase production were identified by PCR and sequencing. OXA-23 enzymes (found in all centers) were by far the most common carbapenemases (81.7%), followed by OXA-58 oxacillinases (4.5%), which were found in 7 of the 25 centers. In 6 cases, CRAb isolates carried both bla OXA-23-like and bla OXA-58-like genes. A repetitive extragenic palindromic (REP)-PCR technique, multiplex PCRs for group identification, and multilocus sequence typing (MLST) were used to determine the genetic relationships among representative isolates (n ‫؍‬ 55). Two different clonal lineages were identified, including a dominant clone of sequence type 2 (ST2) related to the international clone II (sequence group 1 [SG1], SG4, and SG5) and a clone of ST78 (SG6) previously described in Italy. Overall, our results demonstrate that OXA-23 enzymes have become the most prevalent carbapenemases and are now endemic in Italy. In addition, molecular typing profiles showed the presence of international and national clonal lineages in Italy.

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“…Nowadays, sequencebased methods available for typing of Acinetobacter spp., such as pulsed-field electrophoresis (8,23) and multilocus sequence typing (MLST) (17), are rather time-consuming, laborious, and expensive and require a long period of time before generating a typing result, useful for outbreak investigation and control (24,25). The use of DL rep-PCR has been intensively investigated for detecting outbreaks of nosocomial infections.…”

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confidence: 99%

“…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).…”

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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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Molecular Epidemiology of Acinetobacter baumannii Bloodstream Isolates Obtained in the United States from 1995 to 2004 Using rep-PCR and Multilocus Sequence Typing

Cited by 44 publications

References 36 publications

Utility of Whole-Genome Sequencing in Characterizing Acinetobacter Epidemiology and Analyzing Hospital Outbreaks

Utility of Whole-Genome Sequencing in Characterizing Acinetobacter Epidemiology and Analyzing Hospital Outbreaks

Epidemic Diffusion of OXA-23-Producing Acinetobacter baumannii Isolates in Italy: Results of the First Cross-Sectional Countrywide Survey

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

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