Pedro Albuquerque

DNA signature-based approaches for bacterial detection and identification.

During the late eighties, environmental microbiologists realized the potential of the polymerase chain reaction (PCR) for the design of innovative approaches to study microbial communities or to detect and identify microorganisms in diverse and complex environments. In contrast to long-established methods of cultivation-based microbial identification, PCR-based techniques allow for the identification of microorganisms regardless of their culturability. A large number of reports have been published that describe PCR-inspired methods, frequently complemented by sequencing or hybridization profiling, to infer taxonomic and clonal microbial diversity or to detect and identify microorganisms using taxa-specific genomic markers. Typing methods have been particularly useful for microbial ecology-driven studies; however, they are not suitable for diagnostic purposes, such as the detection of specific species, strains or clones. Recently, comprehensive reviews have been written describing the panoply of typing methods available and describing their advantages and limitations; however, molecular approaches for bacterial detection and identification were either not considered or only vaguely discussed. This review focuses on DNA-based methods for bacterial detection and identification, highlighting strategies for selecting taxa-specific loci and emphasizing the molecular techniques and emerging technological solutions for increasing the detection specificity and sensitivity. The massive and increasing number of available bacterial sequences in databases, together with already employed bioinformatics tools, hold promise of more reliable, fast and cost-effective methods for bacterial identification in a wide range of samples in coming years. This tendency will foster the validation and certification of these methods and their routine implementation by certified diagnostic laboratories.

A novel approach for the identification of bacterial taxa-specific molecular markers

Aims:  To develop and establish a methodology for an oriented and fast identification of species taxa‐specific molecular markers useful for the identification of micro‐organisms.

Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples

Background Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. Methodology In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. Significance This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.

Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with Novel Markers and Using a Dot Blot Platform Coupled with Automatic Data Analysis

ABSTRACT Phytosanitary regulations and the provision of plant health certificates still rely mainly on long and laborious culture-based methods of diagnosis, which are frequently inconclusive. DNA-based methods of detection can circumvent many of the limitations of currently used screening methods, allowing a fast and accurate monitoring of samples. The genus Xanthomonas includes 13 phytopathogenic quarantine organisms for which improved methods of diagnosis are needed. In this work, we propose 21 new Xanthomonas-specific molecular markers, within loci coding for Xanthomonas-specific protein domains, useful for DNA-based methods of identification of xanthomonads. The specificity of these markers was assessed by a dot blot hybridization array using 23 non-Xanthomonas species, mostly soil dwelling and/or phytopathogens for the same host plants. In addition, the validation of these markers on 15 Xanthomonas spp. suggested species-specific hybridization patterns, which allowed discrimination among the different Xanthomonas species. Having in mind that DNA-based methods of diagnosis are particularly hampered for unsequenced species, namely, Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans, for which comparative genomics tools to search for DNA signatures are not yet applicable, emphasis was given to the selection of informative markers able to identify X. fragariae, X. axonopodis pv. phaseoli, and X. fuscans subsp. fuscans strains. In order to avoid inconsistencies due to operator-dependent interpretation of dot blot data, an image-processing algorithm was developed to analyze automatically the dot blot patterns. Ultimately, the proposed markers and the dot blot platform, coupled with automatic data analyses, have the potential to foster a thorough monitoring of phytopathogenic xanthomonads.

Detection and discrimination of common bovine mastitis-causing streptococci.

Detection and typing of bovine mastitis pathogens are currently limited by time-consuming and culture-based techniques. In this work, a novel genus-specific DNA marker for Streptococcus and species-specific DNA markers for the prevalent mastitis pathogens Streptococcus agalactiae and Streptococcus uberis were designed and assessed. In order to enable further discrimination of these mastitis-causing streptococci, metabolic and pathogenicity-related genes were used to infer additional functional markers. A total of 12 DNA markers were validated with a set of 50 reference strains and isolates, representative of the Streptococcus genus, of closely related species and of microorganisms with matching habitats. The experimental validation, using dot blot hybridization under high stringency conditions, confirmed the specificity of the selected markers. The broad-spectrum taxonomic marker (ST1) was specific to the Streptococcus genus and the markers selected for S. agalactiae (A1 and A2) and S. uberis (U1 and U2) were shown to be species-specific. The functional markers revealed strain-specific patterns of S. agalactiae and S. uberis. Markers derived from the fructose operon (FO1 and FO3) were specific to bovine isolates of S. agalactiae, and the nisin operon markers (NU1 and NU3) were able to discriminate isolates belonging to S. agalactiae and S. uberis. The virulence-associated markers (V1, V2 and V3) allowed the detection of S. uberis and of closely related species. This work suggests that the combined use of these novel taxa-specific markers coupled with discriminatory functional markers presents a promising approach for the rapid and cost-effective detection and discrimination of common bovine mastitis-causing pathogens, which will contribute to an improved treatment and control of this disease.

Persistence of a dominant bovine lineage of group B Streptococcus reveals genomic signatures of host adaptation.

Group B Streptococcus (GBS) is a host-generalist species, most notably causing disease in humans and cattle. However, the differential adaptation of GBS to its two main hosts, and the risk of animal to human infection remain poorly understood. Despite improvements in control measures across Europe, GBS is still one of the main causative agents of bovine mastitis in Portugal. Here, by whole-genome analysis of 150 bovine GBS isolates we discovered that a single CC61 clone is spreading throughout Portuguese herds since at least the early 1990s, having virtually replaced the previous GBS population. Mutations within an iron/manganese transporter were independently acquired by all of the CC61 isolates, underlining a key adaptive strategy to persist in the bovine host. Lateral transfer of bacteriocin production and antibiotic resistance genes also underscored the contribution of the microbial ecology and genetic pool within the bovine udder environment to the success of this clone. Compared to strains of human origin, GBS evolves twice as fast in bovines and undergoes recurrent pseudogenizations of human-adapted traits. Our work provides new insights into the potentially irreversible adaptation of GBS to the bovine environment.

Automatic Analysis of Dot Blot Images

This paper presents a method for the automatic analysis of macroarray (dot blot) images. The system developed receives as input a dot blot image, corrects it for grid rotation, identifies the visible markers and provides an evaluation of the status of each marker (ON/OFF). Two experiments were carried out to evaluate the detection and classification stages. A total of 222 test images were produced from 6 original dot blot images, with various rotations, translations, contrast and noise level. Over 7500 markers were identified automatically and compared to manual reference. The RMS error in positioning the molecular marker center was between 1.1 and 3.8 pixels and the marker radius error less than 4%. The automatic classification of markers (ON/OFF) was compared to the classification by 3 human experts, using 10 test images. The overall accuracy evaluated on 5118 markers was 94.0%. For those markers that had the same evaluation by all 3 experts, the classification accuracies were 96.6% (ON) and 95.9% (OFF).

Multiple DNA Markers for Identification of Xanthomonas arboricola pv. juglandis Isolates and its Direct Detection in Plant Samples

Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut (Juglans regia L.) bacterial blight (WBB), and has been associated to other walnut emerging diseases, namely brown apical necrosis (BAN) and vertical oozing canker (VOC), altogether severely affecting the walnut production worldwide. Despite the research efforts carried out to disclose Xaj genetic diversity, reliable molecular methods for rapid identification of Xaj isolates and culture-independent detection of Xaj in infected plant samples are still missing. In this work, we propose nine novel specific DNA markers (XAJ1 to XAJ9) selected by dedicated in silico approaches to identify Xaj isolates and detect these bacteria in infected plant material. To confirm the efficacy and specificity of these markers, dot blot hybridization was carried out across a large set of xanthomonads. This analysis, which confirmed the pathovar specificity of these markers, allowed to identify four broad-range markers (XAJ1, XAJ4, XAJ6, and XAJ8) and five narrow-range markers (XAJ2, XAJ3, XAJ5, XAJ7, and XAJ9), originating 12 hybridization patterns (HP1 to HP12). No evident relatedness was observed between these hybridization patterns and the geographic origin from which the isolates were obtained. Interestingly, four isolates that clustered together according the gyrB phylogenetic analysis (CPBF 1507, 1508, 1514, and 1522) presented the same hybridization pattern (HP11), suggesting that these nine markers might be informative to rapidly discriminate and identify different Xaj lineages. Taking into account that a culture-independent detection of Xaj in plant material has never been described, a multiplex PCR was optimized using markers XAJ1, XAJ6, and XAJ8. This triplex PCR, besides confirming the dot blot data for each of the 52 Xaj, was able to detect Xaj in field infected walnut leaves and fruits. Altogether, these nine Xaj-specific markers allow conciliating the specificity of DNA-detection assays with typing resolution, contributing to rapid detection and identification of potential emergent and acutely virulent Xaj genotypes, infer their distribution, disclose the presence of this phytopathogen on potential alternative host species and improve phytosanitary control.

Genotyping and epidemiological metadata provides new insights into population structure of Xanthomonas isolated from walnut trees

Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut diseases affecting leaves, fruits, branches and trunks. Although this phytopathogen is widely spread in walnut producing regions and has a considerable genetic diversity, there is still a poor understanding of its epidemic behaviour. To shed some light on the epidemiology of these bacteria, 131 Xanthomonas isolates obtained from 64 walnut trees were included in this study considering epidemiological metadata such as year of isolation, bioclimatic regions, walnut cultivars, production regimes, host walnut specimen and plant organs. Genetic diversity was assessed by multilocus sequence analysis (MLSA) and dot blot hybridization patterns obtained with nine Xaj-specific DNA markers (XAJ1 – XAJ9). The results showed that Xanthomonas isolates grouped in ten distinct MLSA clusters and in 18 hybridization patterns (HP). The majority of isolates (112 out of 131) were closely related with X. arboricola strains of pathovar juglandis as revealed by MLSA (clusters I to VI) and hybridize with more than five Xaj-specific markers. Nineteen isolates clustered in four MLSA groups (clusters VII to X) which do not include Xaj strains, and hybridize to less than five markers. Taking this data together, was possible to distinguish 17 lineages of Xaj, three lineages of X. arboricola and 11 lineages of Xanthomonas sp. Some Xaj lineages appeared to be widely distributed and prevalent across the different bioclimatic regions and apparently not constrained by the other features considered. Assessment of type III effector genes and pathogenicity tests revealed that representative lineages of MLSA clusters VII to X were nonpathogenic on walnut, with exception for strain CPBF 424, making this bacterium particularly appealing to address Xanthomonas pathoadaptations to walnut. IMPORTANCE Xanthomonas arboricola pv. juglandis is one of the most serious threats of walnut trees. New disease epidemics caused by this phytopathogen has been a big concern causing high economic losses on walnut production worldwide. Using a comprehensive sampling methodology to disclose the diversity of walnut infective Xanthomonas, we were able to identify a genetic diversity higher than previously reported and generally independent of bioclimatic regions and the other epidemiological features studied. Furthermore, co-colonization of the same plant sample by distinct Xanthomonas strains were frequent and suggested a sympatric lifestyle. The extensive sampling carried out resulted in a set of non-arboricola Xanthomonas sp. strains, including a pathogenic strain, therefore diverging from the nonpathogenic phenotype that have been associated to these atypical strains, generally considered to be commensal. This new strain might be particularly informative to elucidate novel pathogenicity traits and unveil pathogenesis evolution within walnut infective xanthomonads. Beyond extending the present knowledge about walnut infective xanthomonads, this study might contribute to provide a methodological framework for phytopathogen epidemiological studies, still largely disregarded.