A comparative study of different PCR-based DNA fingerprinting techniques for typing of the Acinetobacter calcoaceticus-A. baumannii complex

Vila, Jordi; Marcos, Ma Ángeles; Anta, M. T. Jiménez de

doi:10.1099/00222615-44-6-482

Cited by 131 publications

(86 citation statements)

References 32 publications

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“…A dendrogram created using the Rep-PCR results demonstrated only one distinct cluster with Ͼ90% similarity (see Fig. S4 in the supplemental material), a cutoff level above which isolates can be considered genetically related (21,46,47). This cluster included eight A. baumannii isolates involved in a prolonged multistate outbreak from 2005 to 2006.…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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

2016

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“…The Mg 2ϩ concentration was 3 mM, and the primers were used at 0.5 M each. The primer pair REP1 (5Ј-IIIGCGCCGICATCAGGC-3Ј) and REP2 (5Ј-ACGTCTTATCAGGCCTAC-3Ј) was used to amplify putative REP-like elements in the genomic bacterial DNA (23). A total of 500 ng of chromosomal DNA was added to the reaction mixture.…”

Section: Selection Of Clinical Isolates and Patientsmentioning

confidence: 99%

Identification and Broad Dissemination of the CTX-M-14 β-Lactamase in Different Escherichia coli Strains in the Northwest Area of Spain

et al. 2002

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During the course of a molecular epidemiology study of mechanisms of antibiotic resistance in the area served by our hospital (516,000 inhabitants), we isolated the gene encoding CTX-M-14 ␤-lactamase. Thirty clinical strains (27 Escherichia coli and 3 Klebsiella pneumoniae isolates) with a phenotype of extended-spectrum ␤-lactamase were collected from January to October 2001 and studied for the presence of the CTX-M-14 ␤-lactamase gene. By isoelectric point determination, PCR, and nucleotide sequencing, we detected the presence of this gene in 17 E. coli strains belonging to 15 different genotypes (REP-PCR) causing infections in 17 different patients. Epidemiological studies based on medical records did not suggest any relationship between the patients infected with these E. coli strains and, interestingly, 7 of 30 patients harboring strains with extended-spectrum ␤-lactamases never had contact with the hospital environment before the clinical E. coli isolation. Conjugation experiments revealed that this gene was plasmid mediated in the 17 E. coli strains, and plasmid restriction fragment length polymorphisms showed 9 different patterns in the 17 E. coli strains. By PCR, the sequence of the tnpA transposase gene of the insert sequence ISEcp-1 was detected in all the plasmids harboring the CTX-M-14 gene. These results strongly suggest that plasmid dissemination between different E. coli strains in addition to a mobile element (transposon) around the ␤-lactamase gene may be involved in the spreading of the CTX-M-14 gene. This study reinforces the hypothesis that the epidemiology of the prevalence of the ␤-lactamase genes is changing and should alert the medical community to the increase in the emergence of the CTX-M ␤-lactamases worldwide.

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“…Contrary to this method, several authors (3,4) have shown that repetitive extragenic palindromic PCR (REP-PCR) is a simple, rapid, and relatively low-cost method, useful in the characterization of nosocomial outbreaks of A. baumannii infections, being comparable to pulsed-field gel electrophoresis in regard to discriminatory power and reproducibility. Moreover, REP-PCR has a higher power of discrimination and reproducibility than do other PCR-based fingerprinting systems (3,11,17,23,25).…”

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confidence: 99%

Comparison of a Repetitive Extragenic Palindromic Sequence-Based PCR Method and Clinical and Microbiological Methods for Determining Strain Sources in Cases of Nosocomial Acinetobacter baumannii Bacteremia

et al. 2002

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Using a repetitive extragenic palindromic PCR (REP-PCR), we genotypically characterized strains causing nosocomial Acinetobacter baumannii infections and analyzed the source of bacteremia in 67 patients from an institution in which infections by this bacterium were endemic. Six different genotypes were found, including 21, 27, 3, 9, 3, and 4 strains. The probable source of bacteremia, according to clinical and/or microbiological criteria, was known in 42 patients (63%): respiratory tract (n ‫؍‬ 19), surgical sites (n ‫؍‬ 12), intravascular catheters (n ‫؍‬ 5), burns (n ‫؍‬ 3), and urinary tract (n ‫؍‬ 3). The definite source of bacteremia, according to REP-PCR, could be established in 30 (71%) out of the 42 patients with strains from blood and other sites; in these cases clinical and microbiological criteria for the source of bacteremia were thus confirmed. In the remaining 12 patients (29%) the probable source was refuted by the REP-PCR method. The definite sources of bacteremia according to genotype were as follows: respiratory tract in 13 patients (31%), surgical sites in 8 (19%), intravascular catheters in 4 (9%), burns in 3 (7%), and urinary tract in 2 (5%). A comparison of strains from blood cultures and other sites with regard to their REP-PCR and antimicrobial resistance profiles was also made. Taking the REP-PCR as the "gold standard," the positive predictive value of antibiotype was 77% and the negative predictive value was 42%. In summary, the utility of the diagnosis of the source of nosocomial A. baumannii bacteremia using clinical and/or microbiological criteria, including antibiotyping, is limited, as demonstrated by REP-PCR.

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A comparative study of different PCR-based DNA fingerprinting techniques for typing of the Acinetobacter calcoaceticus-A. baumannii complex

Cited by 131 publications

References 32 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

Identification and Broad Dissemination of the CTX-M-14 β-Lactamase in Different Escherichia coli Strains in the Northwest Area of Spain

Comparison of a Repetitive Extragenic Palindromic Sequence-Based PCR Method and Clinical and Microbiological Methods for Determining Strain Sources in Cases of Nosocomial Acinetobacter baumannii Bacteremia

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