Highlights on molecular identification of closely related species

Infection, Genetics and Evolution 13 (2013) 67–75

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Review

Highlights on molecular identification of closely related species Lígia A. Almeida a,b, Ricardo Araujo a,c,⇑ a

IPATIMUP, Institute of Molecular Pathology and Immunology, University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal ICBAS, Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira n.° 228, 4050-313 Porto, Portugal c Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal b

a r t i c l e

i n f o

Article history: Received 15 May 2012 Received in revised form 6 August 2012 Accepted 8 August 2012 Available online 12 September 2012 Keywords: Bacterial complex DNA–DNA hybridization Molecular identification Multilocus sequence typing Species definition Taxonomy

a b s t r a c t The term ‘‘complex’’ emerged in the literature at the beginning of the genomic era associated to taxonomy and grouping organisms that belong to different species but exhibited similar patterns according to their morphological, physiological and/or other phenotypic features. DNA–DNA hybridization values P70% and high identity on 16S rRNA gene sequences were recommended for species delineation. Electrophoretic methods showed in some cases to be useful for species identification and population structure but the reproducibility was questionable. Later, the implementation of polyphasic approaches involving phenotypic and molecular methods brought new insights into the analysis of population structure and phylogeny of several ‘‘species complexes’’, allowing the identification of new closely related species. Likewise, the introduction of multilocus sequence typing and sequencing analysis of several genes offered an evolutionary perspective to the term ‘‘species complex’’. Several centres worldwide have recently released increasing genetic information on distinct microbial species. A brief review will be presented to highlight the definition of ‘‘species complex’’ for selected microorganisms, mainly the prokaryotic Acinetobacter calcoaceticus – Acinetobacter baumannii, Borrelia burgdorferi sensu lato, Burkholderia cepacia, Mycobacterium tuberculosis and Nocardia asteroides complexes, and the eukaryotic Aspergillus fumigatus, Leishmania donovani and Saccharomyces sensu stricto complexes. The members of these complexes may show distinct epidemiology, pathogenicity and susceptibility, turning critical their correct identification. Dynamics of prokaryotic and eukaryotic genomes can be very distinct and the term ‘‘species complex’’ should be carefully extended. Ó 2012 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prokaryotic ‘‘species complex’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Burkholderia cepacia complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Acinetobacter calcoaceticus – Acinetobacter baumannii complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Borrelia burgdorferi sensu lato complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Mycobacterium tuberculosis complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Nocardia asteroides complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eukaryotic ‘‘species complex’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Saccharomyces sensu stricto complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Aspergillus fumigatus and section Fumigati. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Leishmania donovani complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding author at: IPATIMUP, Institute of Molecular Pathology and Immunology, University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. Tel.: +351 225570700; fax: +351 225570799. E-mail addresses: [email protected] (L.A. Almeida), ricjparaujo@ yahoo.com (R. Araujo). 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2012.08.011

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1. Introduction The first classification of microscopic organisms relied on morphological and physiological observations. The term ‘‘complex’’ emerged in the literature at the beginning of the genomic era associated to taxonomy in a wide variety of scenarios. The intended meaning was almost the same: grouping organisms belonging to different species but exhibited similar patterns according to their morphological, physiological or other phenotypic characteristics. The word ‘‘complex’’ was employed in different groups of microorganisms, such as bacteria, fungi, parasites, or higher organisms (Beaman and Beaman, 1994; Edwards-Ingram et al., 2004; Vandamme et al., 1997). With bacteria, constant changes in taxonomy were evident and, while some species were transferred from one genus to the other, new bacterial species and genera were also proposed. Wide consensus was sometimes difficult to achieve (Vandamme et al., 1997; Yabuuchi et al., 1992). Polyphasic taxonomy of prokaryotes was first proposed 40 years ago by Colwell (1970) and aimed to integrate different types of data. This approach proposed a taxonomic classification of any isolate according to a set of criteria (Colwell, 1970). The development of nucleic acid hybridization methodologies and their application to prokaryotes in the 1960’s allowed the first measurement of whole genome and gene sequence identities between strains. Complementary interactions between DNA–DNA and DNA–mRNA employing gel structures or membrane filters and radioactive measurements provided quantitative information on the genetic relatedness of several species (Brenner et al., 1967; McCarthy and Bolton, 1963). Since the 1970’s, DNA–DNA hybridization (DDH) has been used to compare nucleotide sequences and to delineate bacterial taxonomies (Colwell, 1970; Johnson et al., 1970). In 1987, DDH values P70% was finally recommended as criteria for standard species delineation by the ad hoc Committee of the International Committee for Systematic Bacteriology (Wayne et al., 1987). The 16S rRNA sequence-based methodologies, although only applied to prokaryote characterization by the late 1980s, were believed to be a valuable tool for establishing relationships between microorganisms through phylogenetic analysis (Collins et al., 1989). The 16S rRNA gene is present in the genome of all prokaryotes and its primary structure is highly conserved within organisms of the same genus. However, 16S rRNA gene analysis do not allow distinguishing many genetically related species, especially those presenting over 98% 16S rRNA sequence identity, such as the species complexes included in this review. Multilocus enzyme electrophoresis (MLEE), initially employed to study eukaryotes, was applied to prokaryotes by Milkman (1973). Milkman was a pioneer in the analysis of the electrophoretic motility of five loci of an extensive number of Escherichia coli strains. The MLEE methods enabled the detection of small alterations on nucleotide sequences of genes encoding enzymes and the electrophoretic patterns that were then correlated with the alleles of each locus. MLEE, by establishing a correlation between electrophoretic types (ETs) and alleles of housekeeping loci, allowed the evaluation of bacterial evolution and the inference of relationships between strains, proving in some cases to be more useful for species identification and analysis of population structure than DNA hybridization and 16S rRNA sequence analysis (Stackebrandt and Goebel, 1994). Methods based on enzymatic digestion of DNA molecules in combination with electrophoresis separation were developed in the 1980’s and turned out as relevant instruments for the analysis of larger DNA molecules (10 kb – 10 Mb). Pulsed field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) were the most widely used and often considered as ‘‘gold standard’’ methods for identifying varieties of bacteria, sometimes at strain level (Li et al., 2009; Olive and Bean, 1999).

The term ‘‘species complex’’ was proposed by Ursing et al. (1995) for grouping strains with distinct genomic characteristics, therefore, probably representing new distinct species. Each group would be named genomovar, followed by a roman number, being the first number attributed to the type strain of the species. The genomovars were, thus, transitory attributes of the putative species while they were waiting further confirmation of their phenotypic distinctiveness (Ursing et al., 1995). Subsequently, Vandamme et al. (1996) revised the concept proposing the normalization of taxonomic classification and ascertained coherency, reproducibility and uniformity of criteria. In addition, they applied the polyphasic approach, comprising a biochemical profile analysis, a whole cell protein profile, fatty acid analysis, sequencing of 16S rRNA and recA genes and DDH to the identification of Burkholderia cepacia complex (BCC) species (Vandamme et al., 1997). Multilocus typing methodologies, such as the multilocus sequence typing (MLST; http://www.mlst.net/), have significantly improved the accuracy of species characterization. MLST introduced by Maiden et al. (1998) to study Neisseria meningitis population, was based on the same principles of MLEE. But, instead of enzymes, MLST assigned the alleles of each locus of housekeeping genes by sequencing conserved fragments. The number of alleles identified was higher than in MLEE and this methodology was approved for strain genotyping, species identification and analysis of several bacterial populations (Maiden et al., 1998). With the introduction of the MLST and sequencing analysis of complete genomes, the term ‘‘species complex’’ gained a new perspective within the epidemiologic and evolutionary settings of bacterial biology. Closely related species may show distinct epidemiology, pathogenicity and susceptibility to the antibiotics and it is critical that correct identification of microbes be performed, particularly at clinical laboratories targeting pathogenic and relevant infectious agents. Now we have incoming genetic information available from several centres worldwide and it urges an overview of the findings that have been described by researchers in distinct microorganisms. Is taxonomic classification based on a set of criteria among bacteria or eukaryotic populations? How difficult is it to define a microbial species in prokaryotes and eukaryotes? A brief review of a few selected microorganisms is presented below to highlight the observations regarding the definition of ‘‘species complex’’. We selected organisms that currently show the most solid information regarding the definition of closely related species.

2. Prokaryotic ‘‘species complex’’ 2.1. Burkholderia cepacia complex B. cepacia complex (BCC) is a group of strictly aerobic, gramnegative, motile bacilli (Palleroni, 1984; Palleroni and Holmes, 1981). BCC also known as ‘‘B. cepacia like bacteria’’ have their taxonomic origin on Pseudomonas cepacia, first described in 1950 as phytopathogen by Burkholder (1950). Presently, the BCC includes 17 species: B. ambifaria, B. anthina, B. arboris, B. cepacia, B. cenocepacia, B. contaminans, B. diffusa, B. dolosa, B. lata, B. latens, B. metallica, B. multivorans, B. pyrrocinia, B. seminalis, B. stabilis, B. ubonensis, B. vietnamiensis (Vanlaere et al., 2009). The BCC was initially defined as a group of closely related species that were phenotypically similar and exhibited intermediate level of DDH values (30–60%), 98–100% identity on their 16S rRNA sequences and high recA sequence identity (94–95%). Lower values of DDH (below 30%) were observed between BCC strains and representatives of other species of the genus Burkholderia (Vanlaere et al., 2009).

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Vandamme et al. (1997) pioneered a polyphasic approach that enabled the distinction of five species or genomovars which they proposed to be collectively considered a species complex. Thus, the initial BCC accommodated genomovar I (B. cepacia), genomovar II (B. multivorans), genomovar III (B. cenocepacia), genomovar IV (B. stabilis) and genomovar V (B. vietnamiensis). Sequencing analysis of specific genes (e.g. 16S rRNA, recA, rpoB, gyrB) in combination with RFLP, AFLP and PFGE allowed the distinction of various BCC species (Coenye et al., 2001a, 2002; Vandamme et al., 2002; Vermis et al., 2002). Additionally, the recA gene sequencing discriminated some BCC species such as B. ambifaria, B. anthina and B. dolosa (Coenye et al., 2001b; Dalmastri et al., 2005; Vandamme et al., 2002). However, sequences from a single locus, such as 16S rRNA or recA were deemed insufficient to identify all 17 BCC species (Payne et al., 2005). On the other hand, the MLST scheme by using nucleotide sequences from the internal regions (400–500 bp) of seven housekeeping genes (atpD, gltB, gyrB, recA, lepA, phaC, trpB) allowed the identification of the two newest species B. contaminans and B. lata, therefore, revealing its ability to discriminate the 17 species of the BCC (Spilker et al., 2009; Vanlaere et al., 2009). Alternatively, methods based on identification of genetic polymorphisms such as the multiplex single nucleotide primer extension (mSNuPE), which detects DNA sequence variations in six gyrB polymorphic regions, was employed by Ferri et al. (2010) to identify different BCC bacteria to the species level.

2.2. Acinetobacter calcoaceticus – Acinetobacter baumannii complex Acinetobacter calcoaceticus – Acinetobacter baumannii (ACB) complex is a group of phenotypically similar gram-negative bacteria that are characterized as aerobic, non-motile, rod shaped with a polar fimbriae, positive catalase and negative oxidase reactions (Dijkshoorn et al., 1996; Juni, 1984). Micrococcus calcoaceticus was the first identified strain of the genus Acinetobacter, which was isolated from the soil and named by Martinus Willenm Beijerink in 1911 (Baumann, 1968). Baumann’s results were later confirmed by DDH studies in collaboration with other laboratories (Baumann et al., 1968; Johnson et al., 1970). The ACB complex, first proposed by Gerner-Smidt and colleagues (1991), comprises four phenotypically similar and genotypically closely related species: A. baumannii, A. calcoaceticus, Acinetobacter pittii (formerly Acinetobacter genomic species 3), and Acinetobacter nosocomialis (formerly Acinetobacter genomic species 13TU) (Bouvet and Grimont, 1986; Nemec et al., 2011). The respective sequences exhibited interspecies DDH values between 65% and 75% and 16S rRNA sequence identity values ranging between 97% and 99.9% (Nemec et al., 2011; Tjernberg and Ursing, 1989). Different genomic methods, such as AFLP, ribotyping and PFGE, have been referred to successfully identify the ACB complex strains with high discriminatory power. However, these results are difficult to interpret and difficult to compare between laboratories (Dijkshoorn et al., 1996; van Dessel et al., 2004). In contrast, sequence-based methods have revealed higher discrimination within the ACB complex, especially the MLST scheme for A. baumannii developed by Bartual et al. (2005). This method has been considered a promising standard method for ACB complex identification at the species and strain level. A. baumannii MLST scheme is based on nucleotide sequences of seven housekeeping loci (gltA, gyrB, gdhB, recA, cpn60, gpi, rpoD) (Bartual et al., 2005). The subsequent exclusion of two genes (gyrB, gpi) from the initial MLST scheme improved the accuracy of the method and showed a good correlation with PFGE (Hamouda et al., 2010). Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) performs base composition analysis of six housekeeping genes (trpE, adk, efp,

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mutY, fumC, and ppa) and it discriminates the four species within ACB complex (Ecker et al., 2006). Nevertheless, PCR/ESI-MS is not as informative as MLST scheme and yields a slightly lower resolution power than MLST for ACB complex strain typing.

2.3. Borrelia burgdorferi sensu lato complex Borrelia burgdorferi sensu stricto (B. burgdorferi s. s.) is a gramnegative, spiral-shaped or helical bacteria, belonging to the family of spirochaetaceae (Kelly, 1984). B. burgdorferi s. s. belongs to a group of spirochaetes also known as Lyme disease spirochaetes, which are kept in nature by numerous vertebrate hosts and are transmitted by different ticks from the Ixodes persulcatus species complex (Margos et al., 2010). B. burgdorferi s. s., was first retrieved from Ixodes dammini ticks by Burgdorfer et al. (1982) and later recovered from skin, blood and cerebrospinal fluid of patients suffering from Lyme disease (Steere et al., 1983) and named B. burgdorferi sp. nov. by Johnson et al. (1984). Currently, the B. burgdorferi s. l. complex comprises 18 species (B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii, B. burgdorferi s. s., B. californiensis, B. carolinensis, B. garinii, B. japonica, B. kurtenbachii, B. lusitaniae, B. sinica, B. spielmanii, B. tanukii, B. turdae, B. valaisiana, B. yangtze) and several of them, namely B. afzelii, B. burgdorferi s. s., B. garinii, and B. spielmanii, have been associated with human Lyme disease (Kurtenbach et al., 2006; Margos et al., 2010). The B. burgdorferi s.l. spirochaetes have been shown to evolve clonally (Dykhuizen et al., 1993). Yet, they have also been described as a heterogeneous group of bacteria with frequent occurrence of lateral transfer events (Fraser et al., 2007; Marconi et al., 1996). This heterogeneity has been demonstrated by different approaches including serotyping, MLEE, electrophoretic and sequencing methods (Boerlin et al., 1992; Wilske et al., 1995, 1993b). These methodologies targeted, respectively, the expression of proteins encoded by plasmid genes (i.e. the outer surface proteins: ospA, ospB, ospC), genomic markers (clpA, clpX, nifS, pepX, pyrG, recA, rplB, uvrA, groEL, hbb or flaB) or the ribosomal regions of the bacterial genome for instance,16S rRNA, 23S rRNA and 16S-to-23S rRNA interspersed region (ITS) (Brisson and Dykhuizen, 2004; Liveris et al., 1995; Richter et al., 2006; Rudenko et al., 2009). Serotyping was the first widely used method to characterize B. burgdorferi s. l. complex strains based on the heterogeneity of the outer surface proteins (OspA and OspC) (Wilske et al., 1993a,b). However, false positive results due to lack or altered proteins following culture were observed (Wang et al., 1999). DDH and sequence analysis of the 16S rRNA locus showed limited ability to discriminate within the B. burgdorferi s. l. complex (Gevers et al., 2005; Stackebrandt and Jonas, 2006). Thus, multilocus approaches, namely MLST scheme (Margos et al., 2011), have been shown to improve the accuracy towards identification of B. burgdorferi s. l. complex species. MLSA was proposed by Richter et al. (2006) to replace DDH as an excellent alternative for B. burgdorferi s. l. species delineation. This proposal was based on the analysis of seven genes in a large number of B. burgdorferi s. l. strains. MLSA successfully enabled the identification of six new species within B. burgdorferi s. l. complex: B. spielmanii sp. nov., B. californiensis sp. nov., B. americana sp. nov., B. carolinensis sp., nov., B. bavariensis sp. nov., Borrelia kurtenbachii sp. nov. (Margos et al., 2010). Remarkably, Crowder et al. (2010) have reported the successful identification of various species within the B. burgdorferi s. l. by PCR/ESI-MS. In this assay the authors targeted seven Borrelia genes, six genes from the chromosomal region (gyrB, rpoC, rplB, leuS, flaB, hbb) and one gene from the hypervariable region (ospC) to identify simultaneous infection by different species of B. burgdorferi s. l. in the same host (Crowder et al., 2010). This methodology still needs to be tested in multiple laboratories.

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2.4. Mycobacterium tuberculosis complex Mycobacterium tuberculosis or M. tuberculosis sensu stricto (s. s.) is a non-motile, slow growing, aerobic and typically acid fast staining bacillus of the phylum Actinobacteria (Wayne and Kubica, 1984). M. tuberculosis s. s., the predominant cause of human tuberculosis worldwide, was first identified by Robert Koch in 1882 and named by Lehmann and Neumann in 1896 (Collins et al., 1982). M. tuberculosis s. s. is also the first member of the M. tuberculosis complex (MTBC), along with other seven genetically closely related species: M. bovis, M. africanum, M. microti, M. caprae, M. pinnipedii, M. canettii, M. mungi (Alexander et al., 2010). Although genetically related, MTBC species differ in host preference, geographic range and pathogenicity. M. tuberculosis s. s. is exclusively found in humans, while M. mungi seems to be confined to animals (Alexander et al., 2010; Bouakaze et al., 2011; Reddington et al., 2011). The MTBC concept had its origin in the identification of two genetically close but distinct species (M. tuberculosis and M. bovis) of slow growing mycobacteria, which were found to be distant from the other species (Tsukamura, 1976). These achievements relied on experimental findings that were solely based on a broad battery of 88 characters (biochemical tests) and numerical analysis and in conformity with the degree of DDH (86%) observed between the strain M. tuberculosis H37Ra and M. bovis BCG obtained by Bradley (1973). A more comprehensive investigation, regarding the elucidation of the genetic relationship among the slow growing mycobacteria, enabled the identification of two new MTBC species, M. africanum and M. microti, with 85–100% sequence identity between M. tuberculosis and these species (David et al., 1978; van Soolingen et al., 1998). For almost three decades, four members of the MTBC (M. tuberculosis s. s., M. bovis, M. africanum and M. microti) were considered to be phylogenetically related on the basis of DDH, MLEE, sequencing of the 16S rRNA and ITS (Collins et al., 1982; Feizabadi et al., 1996; Frothingham et al., 1994). In fact, the MTBC genome has revealed a remarkable low number of silent substitutions (1 nucleotide difference every 2–28 K base pairs) when compared with other pathogenic bacteria (Sreevatsan et al., 1997). Such lack of neutral diversity (considered to be a characteristic of genetically monomorphic organisms) hampered the use of molecular typing methods based on sequence variation of structural genes, such as MLST (Comas et al., 2009). Therefore, molecular methodologies to study MTBC have been focusing on repetitive elements which are characteristically observed in the MTBC genome, like the transposable sequences (IS6110, IS1081) and direct repeat (DR) regions. The transposable element IS6110 is found in MTBC organisms with a variable number of copies between 0 and 25; IS6110 – RFLP was considered the gold standard for MTBC characterization since the early 1990s (McEvoy et al., 2007). M. canettii was distinguished by a specific IS1081 RFLP band and unique synonymous mutations on recA gene sequence; in contrast, M. tuberculosis, M. bovis, and M. microti showed recA gene nule sequence variation (Van Soolingen et al., 1997). DR regions have also been recognised as an important feature of the MTBC bacteria. DRs are constituted by multiples of 36 bp, interspersed by unique spacers (34–41 bp in length). The different sizes of the 43 MTBC spacers are strain specific and may be associated to different hybridization patterns, the spoligotype signatures, which are traceable by spoligotyping (Groenen et al., 1993; Kamerbeek et al., 1997). Single or multiple genes (such as pncA, gyrB, gyrA, oxyR, katG, rpoB, hsp65 and 16S rRNA) may allow the distinction of some MTBC species (Aranaz et al., 2003; Arnold et al., 2005; Goh et al., 2006; Huard et al., 2006; Niemann et al., 2000). For instance, Niemann et al. (2000) used gyrB RFLP to discriminate M. tuberculosis/M. africanum type II, M. africanum type I, M. microti, M. bovis, and M. bovis BCG. M. caprae revealed a specific arrangement within a five gene

set (pncA, katG, oxyR, gyrA, gyrB) and a characteristic spoligotype pattern (represented by the lack of the spacers 1, 3–16, 30–33 and 39–43) (Aranaz et al., 2003). For M. pinnipedii, a particular fluorescent amplified fragment length polymorphism (FAFLP) was identified, suggesting it should be considered an independent species within MTBC (Cousins et al., 2003). A panel of 27 silent nucleotide substitutions (sSNP) enabled Gutacker et al. (2002) to identify genetic clusters among strains collected from a wide geographical area and to distinguish five known MTBC species (M. tuberculosis s. s., M. africanum, M. microti, M. bovis, M. canettii). Djelouadji et al. (2008) presented a singlestep sequencing method to identify seven MTBC species by sequencing the exact tandem repeat D (ETR-D) spacer in combination with six specific single nucleotide polymorphisms (SNPs) within four genes (oxyR, pncA, hsp65 and gyrB) and two deletions/insertions. Bouakaze et al. (2010) simplified the procedure and proposed a SNaPshot minisequencing-based assay to distinguish the seven MTBC species. Nonetheless, the eighth species belonging to MTBC (M. mungi) was identified by a polyphasic approach that confirmed the relatedness of a specific group of strains that clustered together apart from other strains (Kamerbeek et al., 1997; Supply et al., 2006). This cluster was confirmed to belong to the MTBC by 16S rRNA sequence identity, amplification of the MPB70 gene and yield of IS6110 and showed distinctiveness from the other MTBC members by a unique spoligotype (absence of RD9) (Alexander et al., 2010). At present, MALDI TOF mass spectrometry-based SNP assay in combination with iPLEX gold technology is capable to identify all the eight MTBC members to the species level and their lineages (Bouakaze et al., 2011). 2.5. Nocardia asteroides complex Nocardia species are recognised as slow growing, non-motile and aerobic actinomycetes which are gram-positive and acid-fast staining (Lechevalier, 1984). Nocardia species are ubiquitous in environment, although species distribution varies with different geographical locations (Tan et al., 2010). Several species have been found to cause disease in immunocompromised people, namely pulmonary, central nervous and cutaneous infections. Clinical manifestation of nocardiosis and susceptibility to antibiotics are reported to be species specific (Brown-Elliott et al., 2006; Wallace et al., 1988). The first microorganism of the Nocardia asteroides complex was identified in 1888 by Edmund Nocard (Beaman and Beaman, 1994). Almost hundred years later, Franklin and McClung (1976) were able to identify two distinct groups among ten isolates of putative N. asteroides species by employing DDH and a set of physiological tests. They also found a correlation between their groups and Tsukamura’s N. farsinica and N. asteroides groups (Franklin and McClung, 1976). N. asteroides complex was first proposed by Wallace and colleagues (1988) to accommodate N. asteroides strains grouped according to six antimicrobial susceptibility patterns. Subsequently, molecular methods identified different species and species complexes emerged from each of the six drug patterns. The species were validated by DDH, 16S rRNA sequence analysis and other molecular methods such as RFLP, PFGE, spoligotyping, restriction endonuclease analysis (REA) (Brown-Elliott et al., 2006). The observation of high heterogeneity among 16S rRNA sequences and DDH results within some Nocardia species provided evidence that additional species remained to be characterised (Yassin et al., 2000a,b). Sequencing of conserved gene regions, REA and RFLP led to the recognition of three new clusters of Nocardia species and to the proposal of N. nova complex, N. transvalensis complex, and N. otitidiscavarium complex (Patel et al., 2004). A study led by Liu et al. (2011) which applied 16S

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rRNA sequence analysis on samples stored in Taiwan hospitals from 1998, revealed that 35 out of 36 isolates had been erroneously classified as N. asteroides. Thus, N. cyriacigiorgica an underreported species was found to be the second most relevant species (18%) responsible for nocardiosis in Taiwan (Liu et al., 2011). Although 16S rRNA sequence identity remained the gold standard for Nocardia species identification, it failed to identify many species (Brown-Elliott et al., 2006; McTaggart et al., 2010). The accurate identification of Nocardia species has been achieved by MLSA developed to characterize Nocardia microorganisms recovered from clinical isolates, at the strain and species level. The five loci (gyrB, secA1, hsp65, rpoB and 16S rRNA) scheme was revealed to be more discriminatory than the other approaches for species identification. Although the studies performed did not contemplate comparison with DDH analysis, MLSA has corroborated previously identified groups (McTaggart et al., 2010). 3. Eukaryotic ‘‘species complex’’ 3.1. Saccharomyces sensu stricto complex Saccharomyces cerevisiae is a unicellular eukaryote that belongs to the Fungi Kingdom. It is also known as budding yeast (reproduces by a division process producing buds) (Louis, 2011; Rainieri et al., 2003). This species, along with five other phenotypically similar fermenting yeasts – S. cariocanus, S. bayanus, S. kudriavzevii, S. mikatae, S. paradoxus, have been confirmed as distinct biological species, constituting the Saccharomyces sensu stricto complex (SSSC) (Naumov et al., 2000). SSSC yeasts show great ability to transform carbohydrates into ethanol and CO2 via the fermentation process. This feature has been applied by humans in bread, wine and beer processing (Sicard and Legras, 2011). The six species constituting the SSSC displayed intraspecific high DNA sequence identity and low to moderate interspecies DNA identity. High values of 18S rRNA sequence identity were also reported (Naumov et al., 2000). SSSC is in accordance with the biological concept of species for higher eukaryotes, which advocates that crossings between members of different species would produce sterile hybrids with non-viable and infertile offspring while intraspecies crossings would originate viable and fertile offspring (Naumov et al., 2000; Naumova et al., 2003; Rainieri et al., 2003). Several molecular approaches have been used to identify the species composing the SSSC. DDH, electrophoretic karyotyping, 18S rRNA sequencing and rRNA restriction analysis, have been successfully used to distinguish between S. paradoxus, S. cerevisiae, and S. bayanus (Naumova et al., 2003). Other molecular methods like PFGE, AFLP, RFLP, gene sequencing (such as b-tubulin), MLST, microsatellite markers, SNPs, universally primed polymerase chain reaction (UP-PCR) analysis and MLEE were proven to successfully distinguishing the six ‘‘sibling species’’ composing the SSSC (Huang et al., 2009; Naumova et al., 2003). 26S rRNA sequencing, a simple, fast and reliable method for distinguishing yeast species, was considered unsuitable to distinguish the closely related species that constitute the SSSC (Chang et al., 2007). MLST that includes the genes CDC19, PHD1, FZF1 and SSU1, is believed to be the ideal methodology to study the population structure and phylogeny of the SSSC (Aa et al., 2006).

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Fumigati includes 35 species containing anamorphous Aspergillus species and teleomorphic species of the genus Neosartorya (Samson et al., 2007; Varga and Tóth, 2003). The characteristics of those colonies on standard culture media are very similar to A. fumigatus, with a few differences in the shape of the released conidia. Section Fumigati comprises filamentous ascomycetes characterized by uniseriate aspergilli and flask shaped vesicles that produce small conidia (3–5 lm of diameter). In fact, a single colony may produce and release a large number of small conidia that under good conditions can rapidly germinate. Species within section Fumigati are saprophytic moulds that easily colonize indoor environments and common substrates such as furniture, paints, plants or water conducts. A. fumigatus is capable of germinating and growing at high temperatures (up to 48 °C) which confers an adaptive advantage for the invasion of the human body in comparison to other Aspergillus species (Araujo and Rodrigues, 2004). For years this temperature was used as a restricted condition for the identification of A. fumigatus in clinical laboratories. Misidentification of fungal species has been sporadically reported in clinical laboratories over the last 10 years, particularly of isolates of Aspergillus lentulus, Aspergillus viridinutans, Neosartorya pseudofischeri and Neosartorya udagawae that are identified as A. fumigatus (Balajee et al., 2005a,b; Hong et al., 2008). Therefore, molecular identification is at present recommended for the correct identification of species within section Fumigati. Sequencing of genes, namely actin, calmodulin, ITS, rodlet A and/or b-tubulin, has been proposed for a correct identification of species belonging to ‘‘A. fumigatus complex’’ (Samson et al., 2007; Yaguchi et al., 2007). Random amplified polymorphic DNA (Brandt et al., 1998), restriction fragment length polymorphism (Staab et al., 2009) microsphere-based Luminex assay (Etienne et al., 2009) and a recent electrophoretic strategy employing specific primers for A. fumigatus (Serrano et al., 2011) may allow molecular identification of A. fumigatus and the subdivision of other isolates within the section Fumigati. MLST developed for A. fumigatus was confirmed as highly specific for this mould species. Some difficulties associated to the amplification of MLST genes proved to be useful in the past for the identification of new species within section Fumigati, namely N. pseudofischeri (Balajee et al., 2006). Attending to other markers, few microsatellites have been described as specific for fungal species, as it was the case of markers CNG1, CNG2 and CNG3 in Cryptococcus neoformans (Hanafy et al., 2008). Additionally, few microsatellites have been described as always found within groups of closely related species (Cristancho and Escobar, 2008; Kuhls et al., 2007). A set of microsatellites combined in a multiplex PCR were previously proposed for specific genotyping of A. fumigatus (Araujo et al., 2009). The complete genome sequence of Neosartorya fischeri, a closely related species to A. fumigatus, revealed a group of three markers previously described for A. fumigatus (Araujo et al., 2012). By employing A. fumigatus microsatellite-based multiplex PCR in less restrictive conditions, few microsatellites could be amplified and identified as transversal to species within ‘‘A. fumigatus complex’’. It is likely that some microsatellites had evolved and became specific for single species, e.g. markers MC1, MC2 and MC7 in A. fumigatus, while other microsatellites are more extensively recognized in a group of closely related species, e.g. marker MC6b in section Fumigati (Araujo et al., 2012).

3.2. Aspergillus fumigatus and section Fumigati

3.3. Leishmania donovani complex

Aspergillus fumigatus is an important pathogen responsible for more than 90% of mould infections in immunocompromised patients, even after the administration of appropriate antifungal treatment (Araujo et al., 2010). A. fumigatus is by far the most relevant species belonging to section Fumigati. At present, section

Leishmania donovani is an obligate intracellular, protozoan flagellate or kinetoplastid belonging to the Trypanosomatid family and to the Eukaryote domain of life (Ahmad et al., 2010). The L. donovani complex (LDC) is constituted by four phenotypically indistinguishable Leishmania species (Leishmania archibald, Leish-

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mania chagasi, L. donovani, and Leishmania infantum). These protozoa are responsible for visceral leishmaniasis, cutaneous and mucosal leishmaniasis. The disease is transmitted by the bite of infected females of the subfamily of Phlebotominae known as sand flies (Bastien, 2011; Lukeš et al., 2007). Complementary molecular methods have been reported for L. donovani complex strain typing such as PCR-RFLP of kDNA minicircles, random amplified polymorphic DNA (RAPD), multilocus microsatellite typing (MLMT), RFLP of antigen coding genes (gp63 and cpb) or sequence analysis of the amplified ITS1 (Botilde et al., 2006; Schönian et al., 2011). These methods have shown inability to distinguish different species from the L. donovani complex. In contrast, MLEE, MLST and MLMT have been referred to be able to distinguish the Leishmania species and detect all three diploid allele combinations (Schönian et al., 2011). In endemic regions molecular methods provide good markers for detection of asymptomatic and symptomatic infections. However, these results should be interpreted with caution because only symptomatic patients should pursue treatment (the available drugs are still very toxic) (Deborggraeve et al., 2008). Although MLEE is still considered the reference method for Leishmania strain typing and species identification (Schönian et al., 2011), it was found to lack discriminatory power when dealing with closely related species such as those constituting the LDC (Botilde et al., 2006). For instance, different LDC genotypes were identified within the most common MLEE zymodeme phenotype (MON-1) (Lukeš et al., 2007). MLST is currently considered the most powerful phylogenetic approach, it has been shown to have high discriminatory power, reproducibility and transportability of results between laboratories (Schönian et al., 2008). MLMT, allowed the identification of six genetically distinct populations, which corroborated with MLST results. In addition, Leishmania microsatellites yield high rates of mutation, empowering the discrimination ability of MLMT to the strain and species level (Kuhls et al., 2007; Schönian et al., 2011).

4. Conclusion Recently, MLST and MLSA have been systematically used to separate and identify species within prokaryotic and eukaryotic species complexes. Genomic data are largely supporting a redefinition of previous concepts and can bring more consensual classifications among taxonomists. In fact, the availability of more complete genomes and the possibility to compare closely related species will certainly reveal increasing data and, therefore, encourage additional discussion on the definition of ‘‘species’’ among researchers. Interesting and distinct genomic situations observed until now, such as M. tuberculosis complex, support the expectation that more studies and data are needed to completely evaluate biodiversity in microbiology. Next generation sequencing enables fast and high throughput sequencing of large numbers of gene sequences and may allow the extension of MLST up to hundreds of target genes. Furthermore, NGS will enable the simultaneous sequencing of genes potentially informative in crucial biological functions like virulence and drug resistance (Schönian et al., 2011). The pan-genome has been more intensively assessed in the latest years but information still emerges regarding closely related species; the pan-genomes regularly integrating new genes may indicate species exploring several environments and exposed to strong evolution driving forces. Bacterial core genome analyses may reorganize phylogenies and mark the definition of closely related species. The term ‘‘species complex’’ should be carefully extended to eukaryotes as the dynamics of the prokaryotic and the eukaryotic genomes are very distinct. Horizontal gene transfer is very com-

mon among bacteria, while in eukaryotes the rate of these events is relatively low. The presence of SNPs is extensively used for assessing diversity within bacterial species, while in eukaryotes such markers do not reach the diversity found in bacteria due to the huge differences on the mechanisms responsible for maintaining DNA integrity. Transposable and repeated elements, mainly microsatellites (short tandem repeats), can be present in prokaryotic and eukaryotic genomes, but certainly the acquisition of genetic diversity involving these elements has great differences on both genomes (Richard et al., 2008). Some microsatellites have been widely described as species-specific, while others can be found across closely related species. The dynamic of these repeated elements in the genome can be very useful for the definition of species in eukaryotes. In addition, eukaryotic complete genomes have revealed several cases of chromosomal rearrangement that suggest ‘rare-mating’ event among closely related species (Borneman et al., 2012). In prokaryotic and eukaryotic genomes huge challenges are still being faced in order to understand the complete value of these phenomena on closely related species. We are sure that challenging and surprising observations will emerge in upcoming years. Acknowledgements The authors thank to Fernando Tavares (Faculty of Science, University of Porto) and both reviewers by the valuable suggestions that certainly improved the manuscript. This work was supported by grants from Fundação Calouste Gulbenkian (No. 35-9924-S/ 2009). R.A. is supported by Fundação para a Ciência e a Tecnologia (FCT) Ciência 2007 and by the European Social Fund. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Education and Science, Technology and Higher Education and is partially supported by FCT. References Aa, E., Townsend, J.P., Adams, R.I., Nielsen, K.M., Taylor, J.W., 2006. Population structure and gene evolution in Saccharomyces cerevisiae. FEMS Yeast Res. 6, 702–715. Ahmad, N., Plorde, J.J., Drew, L.W., 2010. Flagellates. In: Ryan, K.J., Ray, C.G. (Eds.), Sherris Medical Microbiology, fifth ed. McGraw Hill Companies, New York, pp. 813–834. Alexander, K.A., Laver, P.N., Michel, A.L., Williams, M., van Helden, P.D., Warren, R.M., Gey van Pittius, N.C., 2010. Novel Mycobacterium tuberculosis complex pathogen, M. mungi. Emerg. Infect. Dis. 16, 1296–1299. Aranaz, A., Cousins, D., Mateos, A., Domínguez, L., 2003. Elevation of Mycobacterium tuberculosis subsp. caprae Aranaz et al. 1999 to species rank as Mycobacterium caprae comb. nov., sp. nov. Int. J. Syst. Evol. Microbiol. 53, 1785–1789. Araujo, R., Amorim, A., Gusmão, L., 2012. Diversity and specificity of microsatellites within Aspergillus section Fumigati. BMC Microbiol. 12, 154. Araujo, R., Pina-Vaz, C., Rodrigues, A.G., 2010. Mould infections: a global threat to immunocompromised patients. In: Ahmad, I., Owais, M., Shahid, M., Aqil, F. (Eds.), Combating Fungal Infections: Problems and Remedy. Springer-Verlag, Berlin, Heidelberg, pp. 1–19. Araujo, R., Pina-Vaz, C., Rodrigues, A.G., Amorim, A., Gusmão, L., 2009. Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing. Clin. Microbiol. Infect. 15, 260–266. Araujo, R., Rodrigues, A.G., 2004. Variability of germinative potential among pathogenic species of Aspergillus. J. Clin. Microbiol. 42, 4335–4337. Arnold, C., Westland, L., Mowat, G., Underwood, A., Magee, J., Gharbia, S., 2005. Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum. Clin. Microbiol. Infect. 11, 122–130. Balajee, S.A., Gribskov, J., Brandt, M., Ito, J., Fothergill, A., Marr, K.A., 2005a. Mistaken identity: Neosartorya pseudofischeri and its anamorph masquerading as Aspergillus fumigatus. J. Clin. Microbiol. 43, 5996–5999. Balajee, S.A., Gribskov, J.L., Hanley, E., Nickle, D., Marr, K.A., 2005b. Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot. Cell 4, 625–632. Balajee, S.A., Nickle, D., Varga, J., Marr, K.A., 2006. Molecular studies reveal frequent misidentification of Aspergillus fumigatus by morphotyping. Eukaryot. Cell 5, 1705–1712. Bartual, S.G., Seifert, H., Hippler, C., Luzon, M.A.D., Wisplinghoff, H., RodriguezValera, F., 2005. Development of a multilocus sequence typing scheme for characterization of clinical of Acinetobacter baumannii. J. Clin. Microbiol. 43, 4382–4390.

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