Oligonucleotide array for identification and detection of pythium species

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.     

Oligonucleotide Array for Identification and Detection of Pythium Species

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.     

16S rRNA sequencing in routine bacterial identification: a 30-month experiment

Accurate identification of bacterial isolates is an essential task in clinical microbiology. Phenotypic methods are time-consuming and either fail to identify some bacteria such as Gram-positive rods entirely or at least fail to do so in some clinical situations. 16S rDNA sequencing is a recent method of identification which offers a useful alternative. In this study, we investigate the usefulness of this method for identifying a range of bacteria in a clinical laboratory under routine conditions. Over a period of 30 months, 683 isolates were obtained from clinical specimens, sequenced and analysed. For 568 of these isolates (83.1%), the sequence provided species level identification. For 108 isolates (15.8%), the identification was limited to the genus level, and for 7 isolates (1%), the sequence remained unidentifiable by 16S rDNA sequence analysis. For the isolates identified only to the genus level, the 16S rDNA approach failed to identify bacteria to the taxonomic level for 3 reasons: failure to differentiate between species in 72 isolates (66%), the lack of any closely related sequence in the database for 15 isolates (13.8%) and the presence of more than 1% of undetermined position in the sequence for 13 isolates (12%).     

Use of a novel multiplex probe array for rapid identification of <Emphasis Type="Italic">Mycobacterium</Emphasis> species from clinical isolates

Conventional identification of mycobacteria is based on the analysis of their phenotypic and biochemical characteristics after culture; thus this method is time-consuming, laborious, and is not always conclusive. Developing a fast and accurate method for rapid identification of Mycobacterium species is in urgent need for early diagnosis of mycobacteriosis and effective patient management. In this study, an efficient and affordable novel multiplex probe array which allows simultaneous identification of 15 medically important mycobacterial species was developed. A pair of genus-specific primers and a set of genus- and species-specific probes were designed according to the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer (ITS) sequence, and 23S rRNA gene of mycobacteria. This probe array was applied for the identification of 78 clinical mycobacterial isolates recovered from Henan, China. The results showed that the specificity and sensitivity of the probe array were 100% for both genus-specific probe and Mycobacterium tuberculosis complex-specific probe. Among 52 isolates of nontuberculous mycobacteria, 43 isolates (82.7%) can be rapidly identified to the species level. Genetic variability of 16S-23S rRNA gene ITS region in M. avium, M. intracellulare, M. chelonae, M. abscessus and M. fortuitum were analyzed. With the accumulation of the sequences of ITS identified and further optimization of probes, the multiplex probe array has the potential to be developed into a practical tool for rapid and accurate identification of mycobacterial species in clinical laboratory.     

Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Background

MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory.

Materials and Methods

First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification.

Results

The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides.

Conclusions

This work''s seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.     

Identification of Fungal Pathogens by Visible Microarray System in Combination with Isothermal Gene Amplification

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 10³ CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.     

粪便样品中大肠杆菌多态性分子研究

以粪便样品中分离到的大肠杆菌为研究对象,比较了3种不同方法在分离鉴定大肠杆菌过程中的应用。首先,通过传统方法从粪便样品中分离,筛选和确定了一批大肠杆菌疑似菌株,再用现代分子生物学方法对待鉴定的大肠杆菌疑似菌株,已知大肠杆菌MG1655以及几种其它细菌进行ARDRA(AmplifiedRibosomalDNARestrictionAnalysis)分析,最后利用ERIC-PCR技术在个体水平上分析菌株的多样性。结果表明,所有由传统方法确定的大肠杆菌疑似菌株和MG1655都属于同一ARDRA型,并与其它细菌的ARDRA条码型不同。这说明ARDRA分析得到的结果与传统分析方法的结果吻合,利用ARDRA分析可以区分大肠杆菌和其它肠道细菌。但是在本实验中ARDRA分析不能反映大肠杆菌中不同菌株之间的多样性,ERIC-PCR则可以区分它们。     

Anomalies in species identification of enterococci from veterinary sources using a commercial biochemical identification system

AIMS: A commercial biochemical panel ID kit was used to identify presumptive enterococci isolates of veterinary or agricultural origin obtained during different steps of culture. METHODS AND RESULTS: Fifty isolates identified as enterococci using a genus PCR assay were tested for genus and species identification using the BBL Crystal Identification Gram-Positive ID kit (Becton Dickinson, Sparks, MD, USA). Following sub-culture of the isolates three times, 59% agreement with the original panel ID was obtained. After four and six sub-cultures, percentage agreement increased to 61 and 64%, respectively. Nineteen of the 50 cultures were identified as both Enterococcus faecalis and E. faecium. CONCLUSIONS: As a result of the variability between speciation of isolates following re-culture, additional methods for speciation are warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the identification of the genus and species of non-human enterococcal isolates can vary greatly during successive passages when using this kit.     

Identification of aerobic mesophilic bacilli isolated from board and paper products containing recycled fibres

AIMS: To identify aerobic mesophilic bacteria isolated from coreboard, kitchen roll paper and food packaging boards containing recycled fibres and to create a rapid fingerprint-based database for their identification. METHODS AND RESULTS: A total of 197 isolates and 20 relevant type strains were characterized by automated ribotyping and as far as possible identified by the similarities of their riboprints to the relevant type strains. One strain from each unidentified ribotype, a total of 87 strains, was subjected to partial 16S rDNA sequencing and in most cases also to fatty acid analysis and physiological tests. From the isolates 113 and seven different ribotypes were generated belonging to the genera Bacillus and Paenibacillus, respectively. The dominating species, or closest related to them, were B. simplex (22.8% of isolates), B. licheniformis (18.3%) and B. amyloliquefaciens (12.7%); 5.1% of the isolates were identified as B. cereus, a potential food-borne pathogen. In particular, this species was present in one food packaging board (26.3% of isolates). Based on these results, 40.1% of the isolates and 45.0% of ribotypes were so different from the relevant type strains that they may represent novel species. CONCLUSIONS: All isolates were aerobic spore-formers, indicating that all non-spore-formers were eliminated during the drying stage of the processes. Although many isolates could be affiliated to described species of Bacillus or Paenibacillus, a significant proportion of the isolates could not be identified unambiguously as members of a described species. SIGNIFICANCE AND IMPACT OF THE STUDY: A RiboPrint identification database, composed of 120 composite patters, was established for bacteria originating from the pulp and paper industry. Considering the discrimination power of ribotyping, this database will be extremely useful in future for the reliable and rapid identification of bacteria isolated from pulp and paper industrial sources.     

Identification of Trueperella pyogenes Isolated from Bovine Mastitis by Fourier Transform Infrared Spectroscopy

The present study was designed to investigate the potential of Fourier transform infrared (FT-IR) spectroscopy to identify Trueperella (T.) pyogenes isolated from bovine clinical mastitis. FT-IR spectroscopy was applied to 57 isolates obtained from 55 cows in a period from 2009 to 2012. Prior to FT-IR spectroscopy these isolates were identified by phenotypic and genotypic properties, also including the determination of seven potential virulence factor encoding genes. The FT-IR analysis revealed a reliable identification of all 57 isolates as T. pyogenes and a clear separation of this species from the other species of genus Trueperella and from species of genus Arcanobacterium and Actinomyces. The results showed that all 57 isolates were assigned to the correct species indicating that FT-IR spectroscopy could also be efficiently used for identification of this bacterial pathogen.     

Reliable and rapid identification of Listeria monocytogenes and Listeria species by artificial neural network-based Fourier transform infrared spectroscopy

Differentiation of the species within the genus Listeria is important for the food industry but only a few reliable methods are available so far. While a number of studies have used Fourier transform infrared (FTIR) spectroscopy to identify bacteria, the extraction of complex pattern information from the infrared spectra remains difficult. Here, we apply artificial neural network technology (ANN), which is an advanced multivariate data-processing method of pattern analysis, to identify Listeria infrared spectra at the species level. A hierarchical classification system based on ANN analysis for Listeria FTIR spectra was created, based on a comprehensive reference spectral database including 243 well-defined reference strains of Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri. In parallel, a univariate FTIR identification model was developed. To evaluate the potentials of these models, a set of 277 isolates of diverse geographical origins, but not included in the reference database, were assembled and used as an independent external validation for species discrimination. Univariate FTIR analysis allowed the correct identification of 85.2% of all strains and of 93% of the L. monocytogenes strains. ANN-based analysis enhanced differentiation success to 96% for all Listeria species, including a success rate of 99.2% for correct L. monocytogenes identification. The identity of the 277-strain test set was also determined with the standard phenotypical API Listeria system. This kit was able to identify 88% of the test isolates and 93% of L. monocytogenes strains. These results demonstrate the high reliability and strong potential of ANN-based FTIR spectrum analysis for identification of the five Listeria species under investigation. Starting from a pure culture, this technique allows the cost-efficient and rapid identification of Listeria species within 25 h and is suitable for use in a routine food microbiological laboratory.     

Barcoding and microcoding using "identiprimers" with Leptographium species

Leptographium species provide an ideal model to test the applications of a PCR microcoding system for differentiating species of other genera of ascomycetes. Leptographium species are closely related and share similar gross morphology. Probes designed for a PhyloChip for Leptographium have been transferred and tested as primers for PCR diagnostic against Leptographium species. The primers were combined with complementary universal primers to identify known and suspected undescribed species of Leptographium. The primer set was optimized for 56 species, including the three varieties of L. wageneri, then blind-tested against 10 random DNA samples. The protocols established in this study successfully identified species from the blind test as well as eight previously undescribed isolates of Leptographium. The undescribed isolates were identified as new species of Leptographium with the aid of the microcoding PCR identification system established in this study. The primers that were positive for each undescribed isolate were used to determine close relatives of these species and some of their biological characteristics. The transfer of oligonucleotides from a micro-array platform to a PCR diagnostic was successful, and the identification system is robust for both known and unknown species of Leptographium.     

A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was developed to detect and identify typical Leuconostoc species. This method utilises a set of specific primers for amplification of the 16S rDNA region of typical Leuconostoc species. All Leuconostoc-type strains, all Leuconostoc isolates from kimchi, Korea's traditional, fermented vegetable product, and strains from closely related genera were examined to verify the identification by this method. The primers resulted in amplification only for nine typical Leuconostoc spp., but not for any other genera tested. The size of the amplified products was 976 bp and the amplicons of the different species could be differentiated from each other with MseI, HaeIII and Tsp509I endonucleases, except for the species Leuconostoc argentinum and Leuconostoc lactis, which were indistinguishable. A PCR-RFLP method for the typical Leuconostoc species was optimized to identify a large number of isolates from fermented vegetable product. This PCR-RFLP method enables the rapid and reliable identification of Leuconostoc species and to distinguish them from the other phylogenetically related lactic acid bacteria in food samples.     

BIOLOG细菌自动鉴定系统的应用与研究

利用ＢＩＯＬＯＧＭｉｃｒｏｓｔａｔｉｏｎ细菌自动鉴定系统对环境和临床来源的２０个属９０株菌进行了检测,同时以传统方法进行验证。２４ｈＢＩＯＬＯＧ系统鉴定结果：６２株革兰氏阴性菌中,５８株准确鉴定到属的水平（９３．５％）,５２株准确到种的水平（８３．９％）。２８株革兰氏阳性菌中,２５株准确到属的水平（８９．３％）,１１株准确到种的水平（３９．３％）,总计属的水平准确率９２．２％（８３／９０）,种的水平准确率７０．０％（６３／９０）,其中环境来源的菌株准确率高于临床样品。本文还对某些属遇到的问题     

Isolation of culturable yeasts from market wines and evaluation of the 5.8S-ITS rDNA sequence analysis for identification purposes

AIMS: To test the possibility that wines available in the marketplace may contain culturable yeasts and to evaluate the 5.8S-ITS rDNA sequence analysis as adequate means for the identification of isolates. METHODS AND RESULTS: As a case study, typical Greek wines were surveyed. Sequence analysis of the 5.8S-ITS rDNA was tested for its robustness in species or strain identification. Sixteen isolates could be assigned into the species Brettanomyces bruxellensis, Saccharomyces cerevisiae and Rhodotorula pinicola, whereas four isolates could not be safely identified. B. bruxellensis was the dominant species present in house wines, while non-Saccharomyces sp. were viable in aged wines of high alcohol content. CONCLUSIONS: Yeast population depends on postfermentation procedures or storage conditions. Although 5.8S-ITS rDNA sequence analysis is generally a rapid method to identify wine yeast isolates at the species level, or even below that, it may not be sufficient for some genera. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to show that commercial wines may possess diverse and potentially harmful yeast populations. The knowledge of yeasts able to reside in this niche environment is essential towards integrated quality assurance programmes. For selected species, the 5.8S-ITS rDNA sequence analysis is a rapid and accurate means.     

IDENTIFICATION OF WHALE SPECIES BY LIQUID CHROMATOGRAPHIC ANALYSIS OF SARCOPLASMIC PROTEINS

Liquid chromatography (LC) was applied to identify whale species by analyzing water-soluble sarcoplasmic proteins in skeletal muscles. Twenty-five samples from four baleen whale species (fin whale, sei whale, Bryde's whale, and minke whale) and eight toothed whale species (sperm whale, Baird's beaked whale, short-finned pilot whale, Dall's porpoise, northern right whale dolphin, Pacific white-sided dolphin, common dolphin, and striped dolphin) were analyzed. Water-soluble sarcoplasmic proteins were extracted from each sample and analyzed using a UV-VIS spectrophotometric detector at 280 nm and a pho-todiode array detector. The chromatographic profiles of each sample showed distinctive qualitative and quantitative characteristics for each whale species, making species identification possible. A photodiode array detector was useful for further accurate identification of whale species by obtaining the absorption spectra of separated protein peaks. These results suggest that the LC method is readily applicable to rapid, simple, and reliable identification of whale species.     

Refined identification of Vibrio bacterial flora from Acanthasther planci based on biochemical profiling and analysis of housekeeping genes

We used a polyphasic approach for precise identification of bacterial flora (Vibrionaceae) isolated from crown-of-thorns starfish (COTS) from Lizard Island (Great Barrier Reef, Australia) and Guam (U.S.A., Western Pacific Ocean). Previous 16S rRNA gene phylogenetic analysis was useful to allocate and identify isolates within the Photobacterium, Splendidus and Harveyi clades but failed in the identification of Vibrio harveyi-like isolates. Species of the V harveyi group have almost indistinguishable phenotypes and genotypes, and thus, identification by standard biochemical tests and 16S rRNA gene analysis is commonly inaccurate. Biochemical profiling and sequence analysis of additional topA and mreB housekeeping genes were carried out for definitive identification of 19 bacterial isolates recovered from sick and wild COTS. For 8 isolates, biochemical profiles and topA and mreB gene sequence alignments with the closest relatives (GenBank) confirmed previous 16S rRNA-based identification: V. fortis and Photobacterium eurosenbergii species (from wild COTS), and V natriegens (from diseased COTS). Further phylogenetic analysis based on topA and mreB concatenated sequences served to identify the remaining 11 V harveyi-like isolates: V. owensii and V. rotiferianus (from wild COTS), and V. owensii, V. rotiferianus, and V. harveyi (from diseased COTS). This study further confirms the reliability of topA-mreB gene sequence analysis for identification of these close species, and it reveals a wider distribution range of the potentially pathogenic V. harveyi group.     

Yeast species associated with orange juice: evaluation of different identification methods

Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.     

Molecular characterization of Trichinella genotypes by inter-simple sequence repeat polymerase chain reaction (ISSR-PCR)

A bulk analysis of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) provides a quick, reliable, and highly informative system for DNA banding patterns that permit species identification. The present study evaluates the applicability of this system to Trichinella species identification. After a single amplification carried out on a single larva with the primer 816([CA]nRY) under high stringency conditions, which provide high reproducibility, we were able to identify by consistent banding patterns 5 sibling species: Trichinella spiralis (ISS48), 2 Trichinella britovi isolates (ISS11 and ISS86), Trichinella murrelli (ISS35), Trichinella nativa (ISS71), Trichinella nelsoni (ISS29); 3 additional Trichinella genotypes: T8 (ISS149), T9 (ISS408 and ISS409), and T6 (ISS34); and the nonencapsulated species Trichinella pseudospiralis (ISS13). Moreover, 33 new Trichinella isolates from 2 zoogeographical regions were unequivocally identified. All Trichinella isolates have shown an identical pattern with those produced by the reference strain. According to these data, we have demonstrated that ISSR-PCR is a robust technique that emerges as a useful new application for the molecular identification of Trichinella isolates in epidemiological studies.     

RecA gene sequence and Multilocus Sequence Typing for species-level resolution of Burkholderia cepacia complex isolates

Aim:  To identify, by means of recA sequencing and multilocus sequence typing (MLST), Burkholderia cepacia complex (BCC) isolates of environmental and clinical origin, which failed to be identified by recA RFLP and species-specific PCR.
Methods and Results:  By using recA sequence-based identification, 17 out of 26 BCC isolates were resolved at the level of species and lineage (ten Burkholderia cenocepacia IIIB, two Burkholderia arboris and five Burkholderia lata ). By using MLST method, 24 BCC isolates were identified. MLST confirmed recA sequence results, and, furthermore, enabled to identify isolates of the BCC5 group, and showed relatedness with Burkholderia contaminans for one of the two isolates not identified.
Conclusions:  recA sequence-based identification allowed to resolve, at the level of species and lineage, 65·4%, of the BCC isolates examined, whilst MLST increased this percentage to 88·5%.
Significance and Impact of the Study:  BCC isolates previously not resolved by recA RFLP and species-specific PCR were successfully identified by means of recA sequencing and MLST, which represent the most appropriate methods to identify difficult strains for epidemiological purposes and cystic fibrosis patients management.