Molecular genotyping of Acinetobacter spp. isolated in Arizona, USA, using multilocus PCR and mass spectrometry

Acinetobacter spp. are a diverse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter, but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has hampered understanding of disease incidence, transmission patterns and diversity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house amplified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis.

Molecular Identification of Japanese Barberry (Berberis thunbergii) Cultivars Using Amplified Fragment Length Polymorphism

Plants of the genus Berberis have been forbidden entry in Canada because common barberry (Berberis vulgaris) is an alternate host for black stem rust of wheat. Japanese barberry (Berberis thunbergii), an ornamental shrub appreciated for its hardiness and attractiveness, is generally considered immune to rust. Therefore, the Canadian government has established a program allowing importation of 11 approved Japanese barberry cultivars. The success of the program relies on the proper identification of the approved varieties, which may not always be possible when the plant is dormant or missing some essential morphologic criteria. Therefore, a cultivar verification method has been established using the amplified fragment length polymorphism (AFLP) genotyping technique. The method was validated using DNA extracted from 41 plants sampled across the country and representing 23 Japanese barberry cultivars and representative of the 11 approved cultivars. The method successfully differentiated and confirmed the identity of all approved cultivars. Furthermore, the method was able to detect and identify two of six samples submitted for cultivar verification as mislabeled by the exporter because the AFLP patterns generated did not correspond to their reference cultivar patterns.

AFLP data and the origins of domesticated crops

Amplified fragment length polymorphism (AFLP) datasets have been used to construct neighbor-joining trees from which monophyletic origins for crops such as einkorn wheat, barley, and emmer wheat have been inferred. We simulated several different multiple domestication scenarios for an imaginary cereal crop and examined the resulting domesticated populations. The simulations showed that the population biology aspects of the domestication process can result in independently domesticated populations merging in such a way that a monophyletic origin is erroneously inferred when the resulting population is examined by AFLP genotyping and neighbor-joining analysis. The results bring into question the use of this method to infer the origins of real crops.Key words: AFLPs, agriculture, neighbor-joining, plant domestication.

Antibiotic Resistance Is a Major Risk Factor for Epidemic Behavior ofAcinetobacter baumannii

Objective:To study the presence of bacterial factors in clinical isolates ofAcinetobacterspecies in order to identify markers of epidemic potential.Design:Case-control study.Methods:Forty-six isolates ofAcinetobacterspecies, including 23 epidemic and 23 sporadic strains from different outbreaks in nine European countries, were compared for the presence of the following factors: hemagglutination, presence of capsules and fimbriae, binding to salivary mucins, resistance to drying, and antibiogram typing. Genotyping of all strains was performed by amplified fragment-length polymorphism (AFLP).Results:All outbreak strains except two (91%) were identified asAcinetobacter baumannii. Binding to salivary mucins and resistance to antibiotics were significantly associated with epidemic behavior. Antibiogram typing showed clustering of predominantlyA baumanniistrains within one group, and these strains were significantly more resistant to antibiotics than sporadic strains. AFLP genotyping revealed a great heterogeneity among the different EuropeanAcinetobacterstrains. Cluster analysis of AFLP fingerprints showed several small clusters of differentA baumanniioutbreak strains. AFLP genotyping could not identify a common epidemic marker within the strains studied.Conclusions:Antibiogram typing can be used in routine clinical laboratories as a screening method to recognize potentially epidemicA baumanniistrains. Several other factors were found, both in different outbreaks as well as in sporadicAcinetobacterisolates. These characteristics were unable to predict epidemic behavior and therefore cannot be used as discriminative epidemic markers. AFLP genotyping demonstrated no common clonal origin of European epidemicA baumanniistrains. This indicates that any clinicalA baumanniiisolate with resistance to multiple antibiotics can be a potential nosocomial outbreak strain.