Burkholderia Contaminans Research Articles

P148 Burkholderia contaminans in cystic fibrosis over a 15-year period in a reference centre in Argentina

P148 Burkholderia contaminans in cystic fibrosis over a 15-year period in a reference centre in Argentina

Multilocus sequence analysis, biofilm production, antibiotic susceptibility and synergy tests of Burkholderia species in patients with and without cystic fibrosis

Burkholderia spp. emerged as important pathogens in the airways of immunocompromised humans, especially those with cystic fibrosis (CF). Failure of identification with conventional techniques, high intrinsic resistance to most antibiotics and biofilm formation can cause difficulties in the treatment of these infections. The aim of this study was to identify Burkholderia spp. strains isolated from CF and non-CF patients with with routine microbiological methods, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multilocus sequence analysis (MLSA), to determine of the antibiotic susceptibility and synergies, and to evaluate biofilm formation of these isolates. A total of 38 Burkholderia spp. (25 CF, 13 non-CF) from 26 patients were identified by biochemical, phenotypical and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and sequence types were revealed by multilocus sequence analysis (MLSA). Sequence types of isolates were identified using the PubMLST database. Characteristics of biofilm formation of clinical isolates were evaluated by microplate method. Antibiotic susceptibilities of ceftazidime, meropenem, trimethoprim-sulfamethoxazole (TMP-SXT) and levofloxacin were determined by broth microdilution method according to CLSI (2017) guidelines. Synergy tests were performed by checkerboard method. Clinical isolates were identified as Burkholderia cenocepacia (n= 16), Burkholderia contaminans (n= 11), Burkholderia gladioli (n= 4), Burkholderia dolosa (n= 4), Burkholderia multivorans (n= 2) and Burkholderia seminalis (n= 1). Sequence types of these isolates were determined as ST19, ST72, ST102, ST180, ST482, ST602, ST629, ST740, ST839 and ST1392. The correct identification at the species-level with MALDI-TOF MS was 94-100% for all isolates except B.contaminans. Biofilm formation among the identified species in the study was determined as 53% (n= 20). There was no statistical difference when the biofilm production was evaluated separately among Burkholderia species and biofilm production rates between CF (56%, 14/25) and non-CF (46%, 6/13) Burkholderia isolates (p> 0.05). Overall rates of resistance to ceftazidime, meropenem, TMP-SXT, and levofloxacin of the isolates were 35%, 66%, 50% and 40%, respectively. The antibiotic resistance against Burkholderia spp., isolates obtained from CF patients were more susceptible to ceftazidime, but no significant difference was found for other antibiotics. Synergy was determined between meropenem and TMP-SXT in two isolates. Antagonism was detected in 15 isolates, 12 of them were between meropenem and ceftazidime, three of them were between ceftazidime and TMP-SXT. Numerous resistance mechanisms may lead to higher resistance in this bacteria, whereas the antagonism between meropenem and ceftazidime in this study might be attributed to the expression of beta-lactamases. In this study, the distinctness of sequence types between Burkholderia spp. isolated from CF and non-CF patient, provided a better understanding about the importance of biofilm formation for the infections with these bacteria and emphasized that the management of therapy should be driven by the antibiotic test results.

A novel enzymatic method for the synthesis of methyl 6-O-acetyl-α-d-glucopyranoside using a fermented solid containing lipases produced by Burkholderia contaminans LTEB11

Abstract Chemical methods for the regioselective modification of carbohydrates require several steps that involve the addition and removal of protective groups. Enzymatic methods are promising since they can give high selectivity in a single step. We developed a new method for acetylation of methyl-α- d -glucopyranoside with vinyl acetate, the reaction being catalyzed by a fermented solid containing lipases from Burkholderia contaminans LTEB11. The main product of the synthesis, methyl 6-O-acetyl-α- d -glucopyranoside (6-OAc-α-MetGlc), was identified by NMR and quantified by HPLC. The conversion of methyl-α- d -glucopyranoside to 6-OAc-α-MetGlc was evaluated at laboratory scale (4 mL) in different solvent systems. The conversion increased with increasing hydrophobicity of the solvent, with the highest conversion, 65% in 72 h, being obtained in the solvent-free system. This system was scaled up to a 1-L reaction medium. A conversion of 76% in 72 h was obtained, with a calculated yield of 1.05 g (4.43 mmol) of 6-OAc-α-MetGlc. This is the largest amount of 6-OAc-α-MetGlc (in moles) produced by enzymatic catalysis that has been reported in the literature to date. These results suggest that the fermented solid produced by B. contaminans LTEB11, which is a low-cost biocatalyst, could potentially be used for the industrial production of esters of α- d -glucose.

Exploring the value of MALDI-TOF MS for the detection of clonal outbreaks of B. contaminans

Background: Microbial diagnostics aims to identify the causative bacterial, fungal and parasitic agents of infectious diseases. Usually this is a lengthy process relying biochemical profiling of metabolic traits. Although molecular genetics has risen in both output and affordability to become the gold standard in diagnosis, it is not yet available for most routine clinical microbiology due to cost and the level of skill it requires. MALDI-TOF MS approaches may be useful in bridging the gap between low resolution phenotypic methods and bulky genotypic methods in the goal of epidemiological source-typing of microbes. Burkholderia cepacia complex has been shown to be identifiable at the species level using MALDI-TOF MS by the optimization of the database by adding reference Mass Spectra Profiles (MSPs) for B. contaminans on its commercial database. There have not yet been studies assessing the ability of MALDI-TOF MS to source-type Burkholderia contaminans isolates into epidemiologically relevant outbreak clusters Therefore, the aim of the present study was to compare the performance of MALDI-TOF MS as a typing method with the gold standard method pulsed-field gel electrophoresis (PFGE) Methods & Materials: 55 well characterised B. contaminans isolates were used to create a panel for analysis of MALDI-TOF MS biomarker peaks and their relation to outbreak strains, location, diagnosis and typeability. Previously all isolates were subjected to PFGE of XbaI-digested genomic DNA.The macrorestriction profiles of strains were distinct and classified into different clades on the basis of epidemiological information available Classification models were generated using biostatistical analysis software ClinProTools 3.1. Three algorithms were available: Genetic Algorithm (GA), Supervised Neural Network (SNN) using 3-nearest neighbours and the QuickClassifier (QC). Results: B. contaminans spectra derived from MALDI-TOF MS were of sufficiently high resolution to identify 100% of isolates. Classification algorithms showed promise for discrimination, Genetic Algorithm models showing 100% recognition capability for all outbreak models and the typeable vs non-typeable model, and 96.63% recognition for the location model. Conclusion: MALDI-TOF MS shows promise in the discrimination of B. contaminans isolates into epidemiological clusters. Further work should investigate this capability, and include peptide studies and genomic sequencing to identify individual proteins or genes responsible for this.

Effectiveness of multi-trait Burkholderia contaminans KNU17BI1 in growth promotion and management of banded leaf and sheath blight in maize seedling

Plant growth promoting (PGP) bacteria enhance plant growth and are a green alternative to chemical fertilizers. In our study, an effective plant growth promoting rhizobacteria (PGPR) strain, KNU17BI1, was isolated from rhizospheric soil of maize, South Korea. The strain was tested in vitro for specific PGP and antifungal traits, such as phosphate solubilization, zinc solubilization, indole acetic acid (IAA) production, ammonia production, nitrogen fixation, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, siderophore hydrogen cyanide production (HCN) and hydrolytic enzyme activity. Furthermore, in viro antifungal activity was done in a laboratory and in vivo effect of KNU17BI1 on banded leaf and sheath blight intensity as well as plant growth promotion on maize seedling were conducted under greenhouse conditions. The strain was found to be highly effective toward all the parameters except HCN production. The strain KNU17BI1 was identified on the basis of 16S RNA and multilocus sequence analysis (MLSA) and confirmed as Burkholderia contaminans. This study for the first time demonstrated potent in vitro antifungal activity of B. contaminans against Rhizoctonia solani AG-1(IA), Pythium graminicola, Fusarium moniliforme, Alternaria alternata, Alternaria solani, Fusarium graminearum, Stemphylium botryosum Wallr, Colletotrichum dematium, Stemphylium lycopersici and Fusarium oxysporum f.sp. melonis. Furthermore, in this study, for the first time, the potential of B. contaminans stain KNU17BI1 in controlling banded leaf and sheath blight of maize caused by R. solani AG-1(IA) was reported. Therefore, further studies are warranted on the structural identification of actual compounds behind such activities that would be exploited further for biocontrol as well as plant growth promotion.

Polyhydroxybutyrate and polyhydroxydodecanoate produced by Burkholderia contaminans IPT553.

In this paper, we introduce a new Burkholderia contaminans capable of producing a newly characterized polymer. CG-MS and magnetic nuclear resonance 1 H and 13 C were used to determine the constitution of polymers produced in glucose, glucose with casein, sucrose and sucrose with casein. Three pairs of primers were used to find the polyhydroxyalkanoates (PHA) synthase class and sequence. The synthesized polymers were composed by short-chain length PHA (scl-PHA), especially polyhydroxybutyrate (PHB), and medium chain length PHA (mcl-PHA), especially polyhydroxydodecanoate (PHDd), and their concentration, constitution and molecular weight depend on carbon source used. The bacterium showed only class I synthase which could not explain the mcl-PHA production. Burkholderia contaminans has a class I PHA synthase and produces PHB combined to PHDd when cultivated in sucrose or glucose, and PHDd concentration is affected when casein is used. PHA are natural polymers produced by a wide range of bacteria. The presence of PHDd monomers confers to the polymer elastomeric properties. Previously, PHDd was only obtained when bacteria were cultivated in related carbon source. In this work, B. contaminansIPT553 produced PHB with PHDd using simple and low-cost carbon sources that can make possible the cheaper production of a more flexible biopolymer with crystallinity and elasticity different from the more common PHAs.

Ag(I) camphorimine complexes with antimicrobial activity towards clinically important bacteria and species of the Candida genus.

The present work follows a previous report describing the antibacterial activity of silver camphorimine complexes of general formula [Ag(NO3)L]. The synthesis and demonstration of the antifungal and antibacterial activity of three novel [Ag(NO3)L] complexes (named 1, 2 and 3) is herein demonstrated. This work also shows for the first time that the previously studied complexes (named 4 to 8) also exert antifungal activity. The antibacterial activity of complexes was evaluated against Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia contaminans and Escherichia coli strains, while antifungal activity was tested against the Candida species C. albicans, C. glabrata, C. parapsilosis and C. tropicalis. The antimicrobial activity of the complexes ranged from very high (complex 4) to moderate (complex 6) or low (complex 8), depending on the structural and electronic characteristics of the camphorimine ligands. Notably, the highest antibacterial and anti-Candida activities do not coincide in the same complex and in some cases they were even opposite, as is the case of complex 4 which exhibits a high anti-bacterial and low antifungal activity. These distinct results suggest that the complexes may have different mechanisms against prokaryotic and eukaryotic cells. The antifungal activity of the Ag(I) camphorimine complexes (in particular of complex 1) was found to be very high (MIC = 2 μg/mL) against C. parapsilosis, being also registered a prominent activity against C. tropicalis and C. glabrata. None of the tested compounds inhibited C. albicans growth, being this attributed to the ability of these yeast cells to mediate the formation of less toxic Ag nanoparticles, as confirmed by Scanning Electron Microscopy images. The high antibacterial and anti-Candida activities of the here studied camphorimine complexes, especially of complexes 1 and 7, suggests a potential therapeutic application for these compounds.

Bioconversion of AHX to AOH by resting cells of Burkholderia contaminans CH-1.

Fairy rings are zones of stimulated grass growth owing to the interaction between a fungus and a plant. We previously reported the discovery of two novel plant-growth regulating compounds related to forming fairy rings, 2-azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH). In this study, a bacterial strain CH-1 was isolated from an airborne-contaminated nutrient medium containing AHX. The strain converted AHX to AOH and identified as Burkholderia contaminans based on the gene sequence of its 16S rDNA. The quantitative production of AOH by resting cells of the strain was achieved. Among seven Burkholderia species, two bacteria and two yeasts tested, B. contaminans CH-1 showed the highest rate of conversion of AHX to AOH. By batch system, up to 10.6mmol AHX was converted to AOH using the resting cells. The yield of this process reached at 91%.

Polyclonal outbreak of bacteremia caused by Burkholderia cepacia complex and the presumptive role of ultrasound gel

A nosocomial polyclonal outbreak associated to bacteremia caused by different Burkholderia cepacia complex (BCC) species and clones is reported. Molecular characterization identified Burkholderia stabilis, Burkholderia contaminans, and Burkholderia ambifaria among BCC isolates obtained from patients in neonatal and adult intensive care units. BCC was also isolated from an intrinsically contaminated ultrasound gel, which constituted the presumptive BCC source. Prior BCC outbreak related to contaminated ultrasound gels have been described in the setting of transrectal prostate biopsy. Outbreak caused strains and/or clones of BCC have been reported, probably because BCC are commonly found in the natural environment; most BCC species are biofilm producers, and different species may contaminate an environmental source. The finding of multiple species or clones during the analysis of nosocomial BCC cases might not be enough to reject an outbreak from a common source.

Incidence of Burkholderia contaminans at a cystic fibrosis centre with an unusually high representation of Burkholderia cepacia during 15 years of epidemiological surveillance.

The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria comprising around 20 related species. These bacteria are important opportunistic pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon, from 1995 to 2006, was re-examined in the present work. These isolates, formerly classified as Burkholderia cepacia (taxon K), are here reclassified as Burkholderia contaminans, including the former B. cepacia IST408, which was the focus of previous studies regarding the biosynthesis of the exopolysaccharide 'cepacian'. The CF population examined has been previously described as having an exceptionally high representation of B. cepacia, presumably due to a contamination arising from saline solutions for nasal application. Twenty-one additional isolates, obtained from a chronically infected patient, from 2006 to 2010, were also identified as B. contaminans. This study also provides insight into the potential clinical impact of B. contaminans, a species that is rarely associated with CF infections. Isolates belonging to this species were shown to be involved in chronic and transient respiratory infections, and were associated with severe lung function deterioration and with a case of death with cepacia syndrome. However, since the patients were co-infected with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to prevail over B. cenocepacia isolates during co-infection of at least one chronically infected patient.

90 Burkholderia contaminans in cystic fibrosis: genetic and phenotypic diversity among isolates from long-term infections

89 Hypermutation in Burkholderia cepacia complex is highly prevalent in chronic respiratory infections of cystic fibrosis patients A. Bosch1, P. Martina2, S. Feliziani3, C. Juan4, M. Bettiol5, B. Gatti6, B. Valeiras7, O. Yantorno7, A. Smania8, A. Oliver4. 1CINDEFI-CONICET-CCT La Plata, Universidad Nacional de La Plata, Chemistry, La Plata, Argentina; 2CINDEFIUNLP-CONICET-CCT La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Chemistry, La Plata, Argentina; 3CIQUIBIC-CONICET Centro de Investigaciones en Quimica Biologica de Cordoba, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina; 4Servicio de Microbiologia y Unidad de Investigacion, Hospital Universitario Son Espases, Palma de Mallorca, Spain, Palma de Mallorca, Spain; 5Sala de Microbiologia, Hospital de Ninos de La Plata ‘Sor Maria Ludovica’, Microbiologia, La Plata, Argentina; 6Sala de Microbiologia, Hospital de Ninos de La Plata ‘Sor Maria Ludovica’, La Plata, Argentina; 7CINDEFI-UNLP-CONICET-CCT La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina; 8CIQUIBIC-CONICET Centro de Investigaciones en Quimica Biologica de Cordoba, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina

MLST@SNaP: user-friendly software for simplification of multilocus sequence typing and dissemination of microbial population analyses

Summary 1. Microbial multilocus sequence typing is a course-grained approach for classifying micro-organisms that involves intense gene sequencing, limiting its application in clinical and research laboratories. Multiplexes targeting single-nucleotide polymorphisms might replace this impractical method by providing the SNaP profile. We here describe MLST@SNAP, a fast, user-friendly and automated process to convert thousands of MLST sequences or sequence types into SNaP profiles and vice versa. The software largely increases the promptness of microbial population data analyses, being presently designed for the micro-organisms Aspergillus fumigatus, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia contaminans and Pseudomonas aeruginosa. 2. MLST@SNAP is freely available for MACINTOSH, UNIX and WINDOWS systems as supplementary material of this manuscript, at https://www.mediafire.com/folder/kc4fwabtb8lu1/setupMLST%40SNaP and http://www.portugene.com/MLSTSNaP.html accompanied by tutorial, example files and some data.

Outbreak of Bacteremia Due toBurkholderia contaminansLinked to Intravenous Fentanyl From an Institutional Compounding Pharmacy

IMPORTANCE Many health care facilities compound medications on site to fulfill local demands when customized formulations are needed, national supply is critically low, or costs for manufactured pharmaceuticals are excessive. Small, institutional compounding facilities may perform the same high-risk procedures as large distributors of compounded medications. OBJECTIVES To investigate an outbreak related to contamination of compounded sterile preparations and to determine processes to prevent future outbreaks. DESIGN, SETTING, AND PARTICIPANTS We performed an outbreak investigation of inpatients at Duke University Hospital from August 31 through September 6, 2012. The investigation included a case-control study, compounding facility inspection and environmental sampling, observation of a mock compounding demonstration, and microbiologic and molecular testing of sequestered medication. EXPOSURES Intravenous fentanyl prepared by an institutional compounding pharmacy. MAIN OUTCOMES AND MEASURES Microbiologic and molecular evidence of contamination of a compounded sterile preparation and failure of routine sterility testing. RESULTS Blood cultures of 7 patients during a 7-day period at Duke University Hospital yielded pan-susceptible Burkholderia cepacia complex bacteria. The risk factor common to all patients was receipt of continuous fentanyl infusion prepared by our institutional compounding pharmacy (odds ratio, 11.22; 95% CI, 2.09-∞; P = .01). The outbreak was terminated after sequestration of compounded fentanyl. An intensive evaluation of the compounding facility, its practice, and its procedures was completed. Investigators evaluated the clean room, collected targeted microbiologic samples within the compounding pharmacy environment, and observed a mock demonstration of compounding practice. The B cepacia complex was found in the anteroom sink drain and pH probe calibration fluid from the compounding clean room. Multiple microbiologic analyses of sequestered fentanyl initially failed. Ultimately, a batched, vacuum-assisted filtration method produced B cepacia complex from a single lot. Molecular analyses using repetitive element polymerase chain reaction and pulsed-field gel electrophoresis confirmed a clonal Burkholderia contaminans strain from patients, fentanyl, and environmental samples. CONCLUSIONS AND RELEVANCE An outbreak of B contaminans bacteremia was linked to contamination of locally compounded intravenous fentanyl. Health care facilities that house institutional compounding facilities must be vigilant in efforts to prevent, recognize, and terminate medication-related outbreaks.

Clonally related Burkholderia contaminans among ventilated patients without cystic fibrosis

We investigated a cluster of 10 Burkholderia cepacia complex-positive cultures among ventilated patients and those with a tracheostomy in an acute care hospital. Isolates from 5 patients had outbreak-strain-related Burkholderia contaminans. Isolates of B. cepacia complex unrelated to the outbreak strain were cultured from a sink drain. The investigation identified practices that might have led to contamination of patient respiratory care supplies with tap water, which might have contributed to the cluster.

Performance of MALDI-ToF MS for species identification of Burkholderia cepacia complex clinical isolates

We evaluated the performance of matrix-assisted laser desorption ionization–time of flight (MALDI-ToF) for identification of Bcc species compared with that of recA sequencing. MALDI-ToF was able of identifying 100% of Bcc isolates at the genus level, but 23.1% of Bcc isolates tested were not correctly identified at the species level. The misidentification occurred most frequently with Burkholderia contaminans (100%) and B. cepacia (33.3%).

Genetic Diversity of Burkholderia contaminans Isolates from Cystic Fibrosis Patients in Argentina

A total of 120 Burkholderia cepacia complex isolates collected during 2004-2010 from 66 patients in two cystic fibrosis reference centers in Argentina were analyzed. Burkholderia contaminans was the species most frequently recovered (57.6%), followed by Burkholderia cenocepacia (15%), a species distribution not reported so far. The recA-PCR-based techniques applied to the B. contaminans isolates revealed that 85% of the population carried the recA-ST-71 allele. Our results showed the utility of BOX-PCR genotyping in analyzing B. contaminans diversity. This approach allowed us to address clonal transmission during an outbreak and the genetic changes occurring in infecting bacteria over the course of chronic infection.

Biosynthesis of an antifungal oligopeptide in Burkholderia contaminans strain MS14

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. When compared with the wild type strain, antifungal activities of mutants MS14MT13 and MS14MT18 against Geotrichum candidum were eliminated, and the activity of mutant MS14MT15 was reduced by approximately 80%. Sequence analysis of a 22.7-kb DNA segment revealed the presence of six open-reading frames (ORFs), including the genes disrupted in the mutants. The wild type level of antifungal activity was observed for mutant MS14MT15 ( ambR2::Tn 5) complemented in trans with the intact ambR2 gene, which encodes a LuxR type regulator. AmbR2 positively regulates the transcription of the other five ORFs identified, which including two nonribosomal peptide synthetase genes required for biosynthesis of antifungal compound. The compound consists of amino acid residues having a molecular weight of 1200.6 Da. These results show that strain MS14 possesses a novel set of genes dedicated to the biosynthesis of an antifungal oligopeptide.

Taxon K, a complex within the Burkholderia cepacia complex, comprises at least two novel species, Burkholderia contaminans sp. nov. and Burkholderia lata sp. nov.

The aim of the present study was to re-examine the taxonomic position and structure of taxon K (also known as group K) within the Burkholderia cepacia complex (Bcc). For this purpose, a representative set of strains was examined by a traditional polyphasic taxonomic approach, by multilocus sequence typing (MLST) analysis and by analysis of available whole-genome sequences. Analysis of the recA gene sequence revealed three different lineages, designated recA-I, recA-II and recA-III. DNA-DNA hybridization experiments demonstrated that recA-I and recA-II isolates each represented a single novel species. However, DNA-DNA hybridization values of recA-II strains towards recA-III strains and among recA-III strains were at the threshold level for species delineation. By MLST, recA-I isolates were clearly distinguished from the others and represented a distinct lineage referred to as MLST-I, whereas recA-II and recA-III isolates formed a second MLST lineage referred to as MLST-II. A divergence value of 3.5 % was obtained when MLST-I was compared with MLST-II. The internal level of concatenated sequence divergence within MLST-I and MLST-II was 1.4 and 2.7 %, respectively; by comparison with the level of concatenated sequence divergence in established Bcc species, these data demonstrate that the MLST-I and MLST-II lineages represent two distinct species within the Bcc. The latter conclusion was supported by comparison of the whole-genome average nucleotide identity (ANI) level of MLST-I and MLST-II strains with strains of established Bcc species and by a whole-genome-based phylogenetic analysis. We formally propose to classify taxon K bacteria from the MLST-I and MLST-II lineages as Burkholderia contaminans sp. nov. (with strain J2956T =LMG 23361T =CCUG 55526T as the type strain) and Burkholderia lata sp. nov. (with strain 383T =ATCC 17760T =LMG 22485T =CCUG 55525T as the type strain), respectively. The MLST approach was confirmed as a valuable instrument in polyphasic taxonomic studies; more importantly, the cumulative data for about 1000 Bcc isolates analysed demonstrate that the 3 % concatenated sequence divergence level correlates with the 70 % DNA-DNA hybridization or 95 % whole-genome ANI threshold levels for species delineation.