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The Phylum

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Chapter Title Spirochaetaceae Phylum Copyright Year 2014 Copyright Holder Springer-Verlag Berlin Heidelberg Corresponding Author Family Name Karami Particle Given Name Ali Suffix Division/Department Molecular Biology Research Center Organization/University Baqiyatallah University of Medical Sciences Street Molasadra Postcode 1993 City Tehran Country Iran Phone 00982188039883 Fax 00982188039883 Email [email protected] Email [email protected] Author Family Name Sarshar Particle Given Name Meysam Suffix Division/Department Molecular Biology Research Center Organization/University Baqiyatallah University of Medical Sciences Street Molasadra Postcode 1993 City Tehran Country Iran Email [email protected] Author Family Name Ranjbar Particle Given Name Reza Suffix Division/Department Molecular Biology Research Center Organization/University Baqiyatallah University of Medical Sciences Street Molasadra Postcode 1993 City Tehran Country Iran Phone +98-21-88039883 Fax +98-21-88039883 Email [email protected] Author Family Name Zanjani Particle Given Name Rahim Sorouri Suffix Division/Department Department of Microbiology, Faculty of Medicine Organization/University Baqiyatallah University of Medical Sciences City Tehran Country Iran Email [email protected] Abstract Spirochaetaceae is a family of spirochetes that cause syphilis, Lyme disease, epidemic and endemic relapsing fever, leptospirosis, swine dysentery, and periodontal disease. The spirochetes are presently classified as members of class in the order Spirochaetales and are divided into three major phylogenetic groupings or families. The first family, Spirochaetaceae, contains in the genera Borrelia, Brevinema, Cristispira, Spirochaeta, Spironema, and Treponema. The second family, Brachyspiraceae, contains the genus Brachyspira (Serpulina). The third family, Leptospiraceae, contains species of the genera Leptonema and Leptospira. One of the unique features of spirochetes is motility mediated by axial flagella with a rapid drifting rotation. The DNA of the Spirochaeta species contains guanine (G) + cytosine (C) ranging from 51 % to 65 mol %. The presence of several linear plasmids seems to cause the segmentation of Borrelia DNA into several linear pieces. This has led to the suggestion that the relatively small linear chromosome and the linear plasmids actually are minichromosomes. Various molecular and immunological detection methods have been developed for detection and identification of spirochetes. Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:12 Page Number: 1

1 156 Spirochaetaceae Phylum 2 Ali Karami1 . Meysam Sarshar1 . Reza Ranjbar1 . Rahim Sorouri Zanjani2 1 3 Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 2 4 Department of Microbiology, Faculty of Medicine, Baqiyatallah University of Medical Sciences, 5 Tehran, Iran

7 Basic Biology of the Spirochetal ...... 1 endemic relapsing fever, leptospirosis, swine dysentery, and 46 periodontal disease (> Table 156.1) (Cabello et al. 2001; Cullen 47 8 Molecular Analysis of Spirochetes ...... 3 et al. 2004; Li et al. 2000; Leschine et al. 2006; Paster and 48 9 DNA-DNA Hybridization Studies ...... 3 Dewhirst 2000; Pavia et al. 1994). 49 10 Linear and Circular Plasmids in Borrelia burgdorferi ..... 4 All members of the spirochetes phylum range from 0.1 50 to 0.5 mm in width and from 10 to 50 mm in length (Pavia 51 11 Molecular Diagnostics of Spirochetes ...... 7 et al. 1994). For example, Leptospira are so thin (0.12 mm in 52 12 Borrelia spp...... 7 diameter) that they cannot be seen with a light microscope. 53 13 Leptospira spp...... 8 Cristispira and Spirosymplokos are so large (>100 mm long) 54 14 Treponema Pallidum ...... 8 that they approach the size of multicellular organisms (Li et al. 55 15 Pulsed-Field Gel Electrophoresis Method ...... 9 2000). Use of dark-field or phase-contrast microscopy or 56 16 Multilocus Sequence Typing (MLST) Analysis ...... 11 staining with a fluorochrome dye such as acridine orange and 57 then viewing under a microscope equipped for fluorescence 58 microscopy is the best way to visualize spirochetes 59 17 Abstract (> Fig. 156.1) (Pavia et al. 1994). Spirochetes are difficult to 60 18 Spirochaetaceae is a family of spirochetes that cause syphilis, grow in the laboratory; therefore, specialized media and culture 61 19 Lyme disease, epidemic and endemic relapsing fever, leptospi- conditions (such as low oxygen tension) are essential to optimize 62 20 rosis, swine dysentery, and periodontal disease. The spirochetes their replicating capabilities (Hardham and Rosey 2000; Pavia 63 21 are presently classified as members of class Spirochaetes in the et al. 1994). 64 22 order Spirochaetales and are divided into three major phyloge- Pathogenic spirochetes exhibit remarkable structural and 65 23 netic groupings or families. The first family, Spirochaetaceae, physiological variability (> Table 156.2). One of the unique fea- 66 24 contains species in the genera Borrelia, Brevinema, Cristispira, tures of spirochetes is their motility, which is mediated by axial 67 25 Spirochaeta, Spironema, and Treponema. The second family, flagella (> Fig. 156.2) with a rapid drifting rotation that allows 68 26 Brachyspiraceae, contains the genus Brachyspira (Serpulina). strains of the Treponema, Borrelia, and Leptospira genera to 69 27 The third family, Leptospiraceae, contains species of the genera invade and colonize host tissues, resulting in diseases such as 70 28 Leptonema and Leptospira. One of the unique features of spiro- Lyme disease and syphilis. Moreover, some can swim in a highly 71 29 chetes is motility mediated by axial flagella with a rapid drifting viscous, gel-like medium, such as that found in connective tissue, 72 30 rotation. The DNA of the Spirochaeta species contains guanine and inhibit the motility of most other bacteria (Caro-Quintero 73 31 (G) + cytosine (C) ranging from 51 % to 65 mol %. The presence et al. 2012; Lux et al. 2001; Rosey et al. 1996; Sadziene et al. 1991). 74 32 of several linear plasmids seems to cause the segmentation of The periplasmic flagella (PFs) consist of a core of at least 75 33 Borrelia DNA into several linear pieces. This has led to the three related proteins (FlaB1, FlaB2, and FlaB3) and a sheath of 76 34 suggestion that the relatively small linear chromosome and the FlaA protein. In all species of spirochetes, the PFs are essential 77 35 linear plasmids actually are minichromosomes. Various for motility; they are subterminally attached near each end and 78 36 molecular and immunological detection methods have been extend toward the center of the cell (Charon et al. 2012; Li et al. 79 37 developed for detection and identification of spirochetes. 2000, 2008; Rosey et al. 1996; Sadziene et al. 1991). The latest 80 study indicates that flagellar stiffness directly affects the spiro- 81 chete’s swimming speed (Li et al. 2008). 82 Basic Biology of the Spirochetal Bacteria Spirochetal outer membranes (> Fig. 156.3) are composed 83 38 primarily of phospholipids, outer membrane proteins (OMPs), 84 39 Spirochaetaceae is a family of spirochetes that cause Lyme and, in some instances, lipopolysaccharides (LPS). However, 85 40 disease and relapsing fever. The spirochetes are free-living or many spirochetes, including Treponema and Borrelia, lack 86 41 host-associated, saccharolytic, nonpathogenic, obligate or outer membrane LPS. Some members of the Spirochetes phy- 87 42 facultative anaerobic spiral-shaped bacteria of high motility lum, including Leptospira and Brachyspira, do synthesize LPS, 88 43 and a variable number of periplasmic flagella. Some are the and LPS is the principal surface antigen displayed by these 89 44 etiological agents of several important animal and human organisms and of considerable importance to diagnostics and 90 45 diseases, including syphilis, Lyme disease, epidemic and immunity (Cullen et al. 2004). 91

E. Rosenberg et al. (eds.), The Prokaryotes – Actinobacteria, DOI 10.1007/978-3-642-38954-2_156, # Springer-Verlag Berlin Heidelberg 2014 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:12 Page Number: 2

2 156 Spirochaetaceae Phylum

. t1:1 Table 156.1 Characteristics of spirochetes (Cabello et al. 2001)

No. of flagella Human Family Genera Species Chromosome % G+C content Bacteriophages Plasmids (per extremity) disease t1:2 t1:3 Spirochaetaceae Borrelia B. burgdorferi One linear 27–32 + Linear 7–30 Lyme disease, t1:4 and relapsing fever circular

t1:5 Brevinema 34–36 1

t1:6 Cristispira 25–53 >100

t1:7 Spirochaeta 51–65 1

t1:8 Spironema Treponema T. pallidum One circular 52 Circular 4–8 Syphilis, pinta, t1:9 yaws T. denticola One circular 35–37 2 Periodontal

t1:10 disease

t1:11 Brachyspiraceae

t1:12 Brachyspira B. hyodysenteriae One circular 25–27 + 10–13

t1:13 Leptospiraceae

t1:14 Leptospira L. biflexa Two circular 53–57 + 1 Leptonema t1:15

. Fig. 156.1 Electron microscopy of unfixed, negative stained DK1 strain (skin isolate from Denmark). This strain consists of two morphologically distinct Borrelia:(A) small and (B) large. Scale bar ¼ 1 m; magnification ¼ 10,260 (Karami et al. 2007)

92 According to conventional phenotypic and genotypic char- ribosomal (r) RNA cataloging and sequencing, characterization 100 93 acteristics and analysis, including cellular lipids, carbohydrates, of intergenic spacer regions of rRNA genes, OspA, lipoprotein 101 94 enzymes, cell proteins, cytoplasmic fibrils, metabolites, genome and flagellin gene sequencing, and rRNA gene organization, the 102 95 size, structure and base composition, restriction endonuclease spirochetes are presently classified in the class Spirochaetes in 103 96 analysis, restriction fragment length polymorphisms, multilocus the order Spirochaetales and are divided into three major phy- 104 97 enzyme electrophoresis, pulse-field gel electrophoresis, DNA- logenetic groupings or families. The first family, Spirochaetaceae, 105 98 DNA hybridization, arbitrarily primed polymerase chain contains species of the genera Borrelia, Brevinema, Cristispira, 106 99 reaction/randomly amplified polymorphic DNA fingerprinting, Spirochaeta, Spironema, and Treponema. The second family, 107 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:13 Page Number: 3

Spirochaetaceae Phylum 156 3

108 Brachyspiraceae, contains the genus Brachyspira (Serpulina). The DNA of the Spirochaeta species has G+C contents ranging 116 109 third family, Leptospiraceae, contains species of the genera from 51 % to 65 mol% (Wang and Schwartz 2006). For example, 117 110 Leptonema and Leptospira (> Table 156.3) (Olsen et al. 2000; Treponema pallidum has a single circular chromosome encoding 118 111 Paster and Dewhirst 2000). 1,034 open reading frames (ORFs), with a G+C content of 119 112 The diversity of the spirochetes and the diseases they cause 52.8 %; Borrelia burgdorferi has a single linear chromosome 120 113 can be partly explained by the diversity of the structure and encoding 853 ORFs, with a G+C content of 28.6 %; and 121 114 composition of their genetic material. Using HPLC (high- Leptospira interrogans has two circular chromosomes of 122 115 performance liquid chromatography), it was found that the 4.6 Mb and 350 kb, with a G+C content of 51–54 %. The 123 presence of several ORFs lacking homologs in other bacteria and 124 . the presence of several families of paralogous genes are also 125 t2:1 Table 156.2 characteristic of spirochetal genomes (Cabello et al. 2001). 126 Unusual features of the pathogenic spirochetes (Pavia et al. 1994) Molecular genetic studies of other spirochetal species have 127 m Large bacteria: up to 50 m long but very small in diameter also been successful and include the use of electroporation, the 128 m compared with other bacteria (e.g., cocci and rods are 1-3 m long identification of several antibiotic resistance phenotypes as use- 129 and red blood cells are 6-8 mm in diameter); they also exhibit ful genetic markers, the development of extrachromosomal 130 a unique spiral, helical shape t2:2 cloning vectors, and the isolation of knockout mutants and 131 Most require special staining techniques such as silver stain or their complementation (> Table 156.4) (Cabello et al. 2001). 132 fluorescence, and microscopy or dark-field microscopy for

t2:3 visualization They exhibit a slow rate of growth, i.e., 24-33-h division time Molecular Analysis of Spirochetes in vivo, compared with Esherichia coli with 20-min division time. 133 They are extremely sensitive to elevated temperatures (38 C) t2:4 DNA-DNA Hybridization Studies Pathogenic treponemes cannot be cultivated on artificial media; 134 other spirochetes can be grown with some difficulty or with special Conventional phenotypic and genotypic characteristics have 135 t2:5 media been used successfully for the classification of the Spirochetes 136 They cause chronic, stage-related, and sometimes extremely phylum (Olsen et al. 2000; Paster et al. 2000). The most common 137 t2:6 debilitating or crippling disease in the untreated host analyses of the molecular and the chemotaxonomy of 138 They do not seem to produce toxins t2:7 spirochetes have been reported by Olsen et al. These analyses 139 The interplay or interrelationship between the invading include the investigation of cellular lipids, carbohydrates, 140 spirochetes and the subsequent host response as factors in the enzymes, cell proteins, cytoplasmic fibrils, metabolites, genome 141 disease process have yet to be clearly or fully defined t2:8 size, structure and base composition, restriction endonuclease 142 They have endoflagella, also called axial fibrils, intertwined analysis, restriction fragment length polymorphism, multilocus 143 between the cell wall and protoplasmic cylinder. Most bacterial enzyme electrophoresis, pulsed field gel electrophoresis, DNA- 144 flagella are extracellular t2:9 DNA hybridization, arbitrarily primed polymerase chain reac- 145 Most pathogenic spirochetes (Borrelia, Treponema) are tion/randomly amplified polymorphic DNA fingerprinting, and 146 microaerophilic (once thought to the anaerobes) t2:10 gene sequences, as well as 16S rRNA sequence comparisons 147 B. burgdorferi is the most unique organism in that it has linear (Olsen et al. 2000). The phylogenetic relationships of the spiro- 148 plasmids that code for outer-surface proteins > t2:11 chetal genera are shown in Fig. 156.4. 149

. Fig. 156.2 Spirochetes are distinguished from other bacterial phyla by the location of their flagella, sometimes called axial filaments, which run lengthwise between the bacterial inner membrane and outer membrane in periplasmic space Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:15 Page Number: 4

4 156 Spirochaetaceae Phylum

Axial filament

Endoflagella

Outer membrane

Cell membrane

. Fig. 156.3 Structure of the spirochete’s axial filament and endoflagella

150 Nevertheless, DNA-DNA hybridization of spirochetes seems to the genus Treponema. Species of a genus are not expected to 186 151 to be the gold-standard method for designation and evaluation differ from each other by more than 10 mol% (Stackebrandt and 187 152 of new species, particularly closely related ones (Fukunaga et al. Liesack 1993). A range of more than 15 mol% is usually taken as 188 153 1995; Hyde and Johnson 1984; Olsen et al. 2000; Paster and a sign of phylogenetic heterogeneity. 189 154 Dewhirst 2000). Three Lyme disease spirochetes isolated from The DNA relatedness between serogroups and serovars in 190 155 Ixodes ticks and from human spinal fluid, three species of North Leptospiraceae was examined by Yasuda et al. (1987), who con- 191 156 American Borrelia, four species of Treponema, and two species of firmed the validity of the species Leptospira parva and 192 157 Leptospira have been studied (Hyde et al. 1984). The mol% Leptonema illini. Leptospira interrogans and Leptospira biflexa 193 158 G+C values for Lyme disease spirochetes ranged from 27.3 % were extremely heterogeneous. Five new species were established 194 159 to 30.5 %, similar to the range of 28.0–30.5 % for the Borrelia from L. interrogans and two new species from L. biflexa. 195 160 species but different than the values for the Leptospira or Trep- DNA-DNA hybridization demonstrated a new Leptospira spe- 196 161 onema species which ranged from 35.3 % to 53 %. Lyme disease cies, Leptospira fainei sp. nov., isolated from pigs in Australia 197 162 spirochetes represent a new species of Borrelia, with DNA (Perolat et al. 1998). Brachyspira hyodysenteriae hyodysenteriae, 198 163 homology ranging from 31 % to 59 % in the three North with a G+C content of 26 mol%, obviously does not belong to 199 164 American strains of Borrelia studied. These studies also showed Treponema. Borrelia spp. hae a G+C content of 30 mol%, 200 165 that Lyme disease spirochetes from three sources constituted Leptospira spp. of 35 41 mol%, and L. illini of 53 mol% 201 166 a single species, with DNA homology ranging from 76 % to (Olsen et al. 2000). It is important to determine the phylogenetic 202 167 100 % (Paster et al. 1991; Richter et al. 2004). The levels of DNA diversity of spirochetes in order to begin assessing their potential 203 168 reassociation with the previously described Lyme disease role and significance in the ecosystem. Despite these develop- 204 169 Borrelia, B. burgdorferi,[B. garinii], and B. afzelii, were only ments, significant hitherto unrecognized spirochetal diversity 205 170 8–13 %. The 16S rRNA gene sequences were also determined probably exists. 206 171 and aligned with the 16S rRNA sequences of other Borrelia 172 species (Fukunaga et al. 1995). 173 A high degree of relatedness between the three North Amer- Linear and Circular Plasmids in Borrelia 207 174 ican Borrelia was also seen, with homology varying from 77 % to burgdorferi 208 175 95 %, indicating that these spirochetes represent a single species. 176 Lyme disease spirochetes and Borrelia species exhibited almost The presence of several linear plasmids that seem to segment 209 177 no homology with Leptospira and Treponema species (0-2 %). Borrelia DNA into several linear pieces has led to the suggestion 210 178 The type species of the genus Treponema, T. pallidum, has a that the relatively small linear chromosome and the linear plas- 211 179 G+C content of 52 mol% (Penn 1992). mids actually are minichromosomes. In B. hermsii it has been 212 180 There are significant differences between T. pallidum sub- shown that total cellular DNA organized into several complete 213 181 species and other spirochetal species. T. pallidum subsp. genomes, suggesting that linear plasmids are like small chromo- 214 182 pallidum has a 55 % DNA similarity with T. phagedenis, somes. The plasmid profile of B. burgdorferi from different 215 183 T. refringens, and B. hyodysenteriae (Miao and Fieldsteel 1980; geographic areas has revealed that there is significant heteroge- 216 184 Miao et al. 1978). In contrast, T. denticola has a G+C content of neity, a feature that can be used for classification of bacteria 217 185 38 mol%. Therefore, there is doubt whether T. denticola belongs within a given species. Another related spirochete, B. hermsii, 218 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:18 Page Number: 5

Spirochaetaceae Phylum 156 5

. t3:1 Table 156.3 Classification of the Spirochaetaceae phylum

219 like B. burgdorferi, has several linear and circular plasmids, and (Sigma-Aldrich, St. Louis, MO, USA) changes the plasmid pro- 224 220 the genes responsible for antigenic variation are located in the file, and loss of plasmids may change the infectivity of the 225 221 linear plasmids. In B. burgdorferi, a 49-kb linear plasmid carries organism. The linear plasmids of B. burgdorferi show 226 222 the genes for outer surface proteins A and B (OspA and OspB). a structure similar to that of eukaryotic viruses such as vaccinia 227 223 It has been shown that passage of B. burgdorferi in BSK medium and African swine fever virus in having covalently closed ends 228 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:24 Page Number: 6

6 156 Spirochaetaceae Phylum 7,16–19 7,10,11 7 14 7,24 a 12 9 22 15 8 20 13 21 Genetic complementation References cfpA , flaB1 nox , , flaB, gac, guaB, gyrB, oppAV, oppA1V, ospC, rpoS flaA1 nox flgE dmcA, msp, tap1 flaB flaA1 tlyA + + Heterologous gene expression Mutants isolated ExchangeExchange + + ExchangeAdditionExchange + + + AdditionAdditionAddition + + + Exchange + Addition + Addition + Exchange and addition AdditionExchange and addition + Process of gene recombination gfp, lacZ egfp Reporter gene Chloramphenicol, kanamycin kanamycin Erythromycin Erythromycin Chloramphenicol, kanamycin Coumermycin, kanamycin Antibiotic resistance markers ) 2001 flaB pER141, pER158, pER199 pER218 Chloramphenicol, Linear pHLfE DNApTS1 Erythromycin Various linear plasmid DNA pGKl2 Erythromycin pBSV2pED1 Erythromycin Kanamycin DNA circular plasmid DNA pPA2 Kanamycin Introduced DNA or cloning vector Transduction Linear chromosomal Electroporation pJBKs Kanamycin Electroporation pKTl20Electroporation Chloramphenicol Various linear and Electroporation pGKLep1 Kanamycin Methods of gene transfer Table 156.4 Brachyspira hyodysenteriae Treponema denticola Borrelia burgdorferi Leptospira biflexa Species M.L. Sartakova et al. unpublished . Genetic systems in spirochetes (Cabello et al. a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 : : : : : : : : : : : : : : : : : t4 t4 t4 t4 t4 t4 t4 t4 t4 t4 t4 t4 t4 t4 t4 t4 t4 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:24 Page Number: 7

Spirochaetaceae Phylum 156 7

5%

Treponema vincentii Uncultivable PROS treponeme Grp 1 Treponema medium Treponema denticola Treponema phagedenis Uncultivable oral treponeme Grp 3:B Treponema pallidum Uncultivable termite treponeme NL-1 Treponema succinifaciens (pig) Treponema maltophilum Treponema Uncultivable oral treponeme Grp 4:18 Treponema pectinovorum Treponema bryantii (bovine) Treponema Smibert-2 Uncultivable oral treponeme Grp 5:22 Treponema amylovorum Treponema Smibert-5 Treponema socranskii ss paredis Treponema socranskii ss buccale Treponema socranskii ss socranskii Spirochaeta bajacaliforniensis Spirochaeta Spirochaeta halophila Uncultivable Cristispira clone CPI Cristispira Borrelia hermsii Borrelia Borrelia burgdorferi Brevinema andersonii Brevinema Brachyspira (Serpulina) hyodysenteriae Brachyspira Brachyspira innocens Leptonema illini Leptonema Leptospira biflexa Leptospira Leptospira interrogans

. Fig. 156.4 16S rRNA dendrogram demonstrating the phylogenetic relationships of representatives of spirochetal genera. Note that not all cultivable spirochete species are included in this dendrogram. Representatives of ‘‘uncultivable’’ species are noted as such. There are presently about 50 species of uncultivable oral treponemes (unpublished data) and 68 species of uncultivable termite treponemes (J.A. Breznak, personal communication). TREECON, a software package for the Microsoft Windows environment, was used for the construction and drawing of this neighbor-joining tree. The scale bar represents a 5 % difference in nucleotide sequence determined by taking the sum of all of the horizontal lines connecting two species. Vertical distance has no meaning

229 like hairpin loops. Analysis of the profile of plasmids from e.g., 16S rRNA, 23S rRNA, and 16S-to-23S rRNA interspersed 241 230 different Danish isolates of B. burgdorferi isolated from skin region (ITS), are good targets for the detection and isolation of 242 231 and cerebrospinal fluid of Lyme disease patients reveals several Borrelia species (Brisson and Dykhuizen 2004; Liveris et al. 1995; 243 232 linear and circular plasmids with strong heterogeneity Richter et al. 2006; Rudenko et al. 2009a, b). In patients with skin 244 233 (> Figs. 156.5 and > 156.6) (Karami 2012; Karami et al. 2006, lesions, qPCR testing of a skin biopsy allows early detection of 245 234 2007). Borrelia DNA, whereas serological tests are usually negative at 246 this stage (Ivacic et al. 2007). The number of Borrelia sp. in other 247 infected tissues and body fluids is generally very low (Aguero- 248 Molecular Diagnostics of Spirochetes Rosenfeld et al. 2005). qPCR tests usually detect Borrelia DNA in 249 235 synovial fluid or synovial membrane biopsies of affected joints 250 Borrelia spp. (Aguero-Rosenfeld et al. 2005). Tests using the OspA-encoding 251 236 gene as the target were reported to be more sensitive than those 252 237 The expression of proteins encoded by plasmid genes (i.e., the using the 16S rDNA (Aguero-Rosenfeld et al. 2005). By contrast, 253 238 outer surface protein genes ospA, ospB, and ospC), genomic qPCR tests cannot detect Borrelia DNA in the cerebrospinal fluid 254 239 markers (clpA, clpX, nifS, pepX, pyrG, recA, rplB, uvrA, groEL, of most patients with neuroborreliosis (Babady et al. 2008). Tests 255 240 hbb,orflaB), or the ribosomal regions of the bacterial genome, for blood and urine samples also have low sensitivity (Aguero- 256 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:24 Page Number: 8

8 156 Spirochaetaceae Phylum

A BCDE F G

48 kb

23 kb

9.4 kb 16 kb 14 6.5 kb . Fig. 156.5 12 Electron micrograph of one supercoiled 25-kb plasmid extracted 10 from DK1 strain (magnification 52,000x) 3.3 kb 8.0 7.0 6.0 257 Rosenfeld et al. 2005). qPCR tests allow easier determination of 5.0 258 the Borrelia species involved (Ferdin et al. 2010). Remarkably, 259 Crowder et al. (2010) have reported the successful identification 2.3 kb 3.9 2.0 kb 260 of various species within B. burgdorferi sensu lato using PCR/ 2.9 261 ESI-MS. Seven Borrelia genes from the chromosomal and hyper- . Fig. 156.6 262 variable regions have been used to identify simultaneous infec- Plasmids isolated from different strains of Borrelia burgdorferi. 263 tion by different species of B. burgdorferi s. l. in the same host (A) Linear molecular markers (HindIII fragments of lDNA), (B) DK1 264 (Crowder et al. 2010). strain, (C) DK5 strain, (D) DK6 strain, (E) DK2 strain, (F) DK7 strain, and (G) a supercoiled circular molecular weight marker. Samples Leptospira spp. were separated in 0.3 % gel at 14 C for 20 h then stained with 265 ethidium bromide 266 Several molecular protocols for the detection of leptospiral DNA in 267 clinical material have been developedsincethe 1990s; most of them 268 have been reported to have high sensitivity. Restriction fragment 269 length polymorphism (RFLP) analysis (Herrmann et al. 1992), analyzing melting curves (Merien et al. 2005). Analysis of the 289 270 16S rRNA sequencing (Morey et al. 2006), variable number of secY DNA sequence amplified with the G1 and G2 primers can 290 271 tandem repeats (VNTR) (Majed et al. 2005), and multiple locus be used to detect pathogenic species (Gravekamp et al. 1993). 291 272 sequence typing (MLST) are the most reliable and robust Victoria et al. (2008) recently demonstrated that one gene from 292 273 current molecular methods for detection and identification of the S10-spc-a locus, which encodes the SecY preprotein 293 274 Leptospira sp. (Ahmed et al. 2006; Thaipadungpanit et al. 2007). translocase, gave a satisfactory identification to the species level. 294 275 Quantitative PCR tests allow detection of Leptospira DNA in 276 clinical samples (especially blood and serum samples) in the 277 early phase of the disease, before antibody titers are at detectable Treponema Pallidum 295 278 levels. These tests amplify target genes present only in patho- 279 genic strains of Leptospira sp. (Ahmed et al. 2009; Bourhy et al. The quantitative PCR test allows sensitive and rapid detection of 296 280 2011; Lin et al. 2009; Stoddard et al. 2009; Thaipadungpanit et al. T. pallidum DNA in syphilitic ulcers. The qPCR test is much less 297 281 2011). sensitive for the diagnosis of late manifestations of syphilis and 298 282 Amplification and sequencing of the 16S rRNA gene (Morey latent syphilis and such use of the qPCR test has not been fully 299 283 et al. 2006; Postic et al. 2000) with two-primer sets of IS elements validated (Gayet-Ageron et al. 2009; Heymans et al. 2010; Tipple 300 284 (and thus the large number of suitable priming sites) can be used et al. 2011). Occasionally, however, the qPCR test allows detec- 301 285 to discriminate between L. interrogans and L. kirschneri tion of T. pallidum in blood samples and cerebrospinal fluid of 302 286 (Cameron et al. 2008). infected patients during the secondary and latent phases of the 303 287 Quantitative real-time PCR with primers specific for Lfb1 disease and in infected pregnant women and neonates (Gayet- 304 288 can be used to distinguish between pathogenic species by Ageron et al. 2009; Tipple et al. 2011). 305 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:25 Page Number: 9

Spirochaetaceae Phylum 156 9

a b c

cN40 N40D10/E9B31 N40B N40C M kb cN40 N40D10/E9B31 N40B N40C kb λ cN40 N40D10/E9B31 N40B N40C OspD (Ip38) BBK32 (Ip36) VIsE1 (Ip28-1)/other BBG02 (Ip28–2) 23.7 9.4 145.5 BBE22 (Ip25) 6.6 97.0 BBU06 (Ip21) 4.4 BBD14 (IP17) 2.3 48.5 2.0

33.5 Ip28-2 Ip28-4 Ip28-5 15.0 PCR Probes 0.6

. Fig. 156.7 PFGE electrophoresis and Southern hybridization to determine plasmid profiles of B. burgdorferi strains and PCR for detection of the bbk32 gene

. t5:1 Table 156.5 Typing schemes for LB spirochetes using multilocus sequence analysis

Type of loci Loci Purpose Data References t5:2 Chromosomal housekeeping genes clpA, clpX, nifS, Taxonomy, borrelia.mlst.net, Margos et al. 2008, 2009, 2010; Hoen pepX, pyrG, recG, population >1,200 strains, et al. 2009, Ogden et al. 2010, Vollmer rplB, uvrA studies, 327 STs et al. 2011, Ogden et al. 2011, Takano t5:3 evolutionary et al. 2011 studies

t5:4 Plasmid-encoded Osp, chromomosal: ospA, 16S, p66, Taxonomy GenBank 110 Rudenko et al. 2009, 2010 rRNA, intergenic spacer, housekeeping 23S-5S IGS, flaB strains gene Plasmid-encoded Osp, chromosomal: ospA, 16S, 23S-5S Taxonomy 130 strains Richter et al. 2006, Postic et al. 2007,

t5:5 rRNA, intergenic spacer, housekeeping IGS, groEL, hbb, Chu et al. 2008 genes fla, recA

t5:6 17 plasmid-encoded loci, lp54, cp26, cp9, Population studies GenBank, 60 Qiu et al. 2004 chromosomal: housekeeping gene lp17, lp25, lp28-2, strains lp28-4, lp38, BB0082 Plasmid-encoded Osps, chromosomal: ospA, ospC, p66, Population studies GenBank, 115 Bunikis et al. 2004, Humphry et al. membrane protein, intergenic spacer 16S-23S IGS strains 2010 (except p66) t5:7

306 Leslie et al. (2007) developed a TaqMan real-time PCR assay Pulsed-Field Gel Electrophoresis Method 315 307 targeting the polA gene to directly detect the presence of 308 pathogenic T. pallidum in swabs and biopsy specimens Pulsed-field gel electrophoresis (PFGE) has been particularly 316 309 from genital and mucosal ulcers, placental specimens, valuable in epidemiological studies of spirochetal isolates and 317 310 and cerebrospinal fluid. They concluded that the T. pallidum for their molecular characterization. Macrorestriction 318 311 PCR will be a valuable addition to serology for the diagnosis fragment profile analysis by PFGE was used by Rayment et al. 319 312 of early syphilis and useful for the confirmation of other diag- (1997) to distinguish intestinal spirochetes. The restriction 320 313 nostic methods such as histopathology in late and congenital profile of SmaI and SmaII divided the isolates into two major 321 314 syphilis. clusters: Brachyspira pilosicoli and B. hyodysenteriae. Both species 322 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:25 Page Number: 10

10 156 Spirochaetaceae Phylum

BL206 1.00 B31 B515 1.00 16812UT ca6 1.00 1. ca5 ca4 MR623 1.00 B373 20604LT 22521LT 1.00 IPT191 IPT190 0.91 IPT137 = IGS1 IPT135 = IGS2 IPT69 = IGS3 0.78 IPT23 1.00 IPT2 = IGS4 21509LT = IGS5 1. IPT19 = IGS6 51405UT 0.99 114311UT = IGS7 1.00 JD1 = IGS8 BL515 = IGS9 BL538 1.00 Ca92–1096 0.75 B500 1.00 B331 1.00 B361 1.00 B509 0.92 519014UT 498801UT 297 0.72 1.00 1.00 B504 MR616 1.00 15912UT 0.86 Ca WTB27 0.98 Ca92–0953 47703UT 0.90 1.00 B485 0.93 MR607 MR662 1.00 1.00 20111LT IPT39 0.71 IPT58 1.00 B156 1.00 1.00 ca WTB32 15506UT MR654 1.00 MR661 1.00 B356 1.00 BL522 1.00 ca92–1337 1.00 48102UT 1.00 15903UT MR640 B348 1.00 N40 B418 1.00 Z41293 0.99 Z41493 1.00 NE49 IPT193 1.00 IPT198 B. garinii PBi

0.01 . Fig. 156.8 Bayesian phylogenetic inference of concatenated sequences of the housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA)of B. burgdorferi. Posterior probability values of clades are provided. Symbols refer to the major IGS genotypes as defined by Bunikis et al. (2004). Non-color-coded strains are from the Northeast and Midwest United States; yellow indicates strains from California, and blue indicates strains from Europe. The branch length of the outgroup B. garinii is not to scale (indicated by slashes). Scale bar ¼ 1 % divergence Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:26 Page Number: 11

Spirochaetaceae Phylum 156 11

100 typing method for classifying Leptospira strains (Ciceroni et al. 347 100 B. bissettii 2002; Naigowit et al. 2007; Trott et al. 2003). For example, PFGE 348 100 B. carolinensis 100 has been found to be unable to discriminate between 349 100 B. kurtenbachii 350 100 B. Californiensis L. interrogans serovars Icterohaemorrhagiae and Copenhageni. B. andersonii Nonetheless, almost 90 % can be identified on the basis of their 351 352 96 B. americana unique PFGE patterns (Galloway and Levett 2008). Trott et al. 52 genomospec2 (2003) reported that six Treponema phagedenis-like isolates from 353 73 B. burgdorferi cattle, of which four were from the same herd, were shown to 354 B. turdi have unique PFGE patterns after cleavage with XbaI, NotI, and 355 100 B. yangtze 356 100 B. valaisiana Sse8387I. In addition, PFGE can be used to construct physical 26 B. tanukii maps of the genomes and to define different groups of B. 357 B. lusitaniae burgdorferi sensu lato by combining the chromosomal LRFLPs 358 59 96 B. spielmanii resulting from digestion of a set of restriction enzymes, e.g., 359 B. afzelii MluI, SacI, BssHII, EagI, SmaI, ApaI, CspI, and 360 26 B. japonica SgraAI (Casjens and Huang 1993; Casjens et al. 1995; Davidson 361 37 B. garinii et al. 1992; Ojaimi et al. 1994). 362 100 B. bavariensis Mathiesen et al. (1997) reported that B. burgdorferi sensu 363 0.01 lato isolates from patients in the United States, irrespective of 364 . Fig. 156.9 their geographic region, belonged to a single rDNA cluster. 365 Neighbor-joining tree generated using concatenated sequences Karami et al. (2007) combined PFGE and Southern hybridiza- 366 of MLSA housekeeping genes showing LB group species. Black tion for the detection of genes encoding known virulence fac- 367 dots indicate species that occur in North America, circles indicate tors, ribosomal RNA gene spacer restriction fragment length 368 species that occur in Eurasia, and gray dots indicate species that polymorphism types (RSTs), OspC group determination, and 369 occur in the Old and New Worlds. Scale bar ¼ 1 % divergence. sequencing of the variable dbpA and ospC genes of B burgdorferi 370 Branch confidence values were calculated using a bootstrap strains. They concluded that use of PFGE and PCR of virulence 371 procedure with 100 repetitions (Original figure from Margos et al. factor genes followed by Southern hybridization is a useful strat- 372 (2010) Ticks and Tick-borne Diseases, doi:10.1016/j. egy to discriminate different strains of B. burgdorferi sensu 373 > ttbdis.2010.09.002, modified and reproduced with permission stricto ( Fig. 156.7) (Chan et al. 2012). 374 from Elsevier)

Multilocus Sequence Typing (MLST) Analysis 375

323 contained subspecific groups but they rarely correlated with MLST was originally meant to be used to enhance clinical diag- 376 324 the source of the isolates. PFGE has also been used for nosis and perform epidemiological monitoring, and population 377 325 subspecific differentiation of B. pilosicoli (Atyeo et al. 1996). studies (Urwin and Maiden 2003). However, the MLST concept 378 326 PFGE has been used to discriminate between strains within has since been broadened to include the analysis of closely 379 327 each Borrelia species. For example, 20 B. burgdorferi sensu stricto related species and this approach has been named multilocus 380 328 isolates were separated into 10 MluI LRFLPs, while 24 B. sequence analysis (MLSA) (Gevers et al. 2005; Hanage et al. 381 329 garinii and 6 B. japonica isolates exhibited four and two 2005, 2006). MLSA was developed with the goal of rapid and 382 330 LRFLPs, respectively (Belfaiza et al. 1993; Postic and Baranton robust hierarchical classification of all prokaryotic species 383 331 1994; Postic et al. 1993). PFGE was used to differentiate 25 (Gevers et al. 2005) and has been suggested as a solution to the 384 332 skin biopsy isolates of B. burgdorferi sensu lato, mainly from time-consuming and complicated method of defining 385 333 German patients with erythema migrans, borrelial prokaryote species using DNA–DNA hybridization (Bishop 386 334 lymphocytoma, and acrodermatitis chronica atrophicans et al. 2009; Gevers et al. 2005). 387 335 (Busch et al. 1996a, b). The study detected 15 B. afzelii,8[B. Since 2004 several multilocus schemes have been developed 388 336 garinii], and 2 B. burgdorferi sensu stricto species after MiuI to investigate the phylogenetic relationship of the Lyme 389 337 digestion. Twelve B. afzelii strains were further investigated borreliosis (LB) spirochetes. The greater amount of genetic 390 338 by digestion with five restriction enzymes. Most of these strains information obtained from several loci permits determination 391 339 revealed an individual PFGE pattern. PFGE was particularly of more subtle differences in and between species (Margos et al. 392 340 effective for detecting differences among B. afzelii 2011). For the LB group of spirochetes, five typing schemes 393 341 isolates. Busch et al. found large restriction fragment pattern using multiple loci have been developed (> Table 156.5) 394 342 (LRFP) analysis by PGFE to be a suitable tool for the molecular (Bunikis et al. 2004; Margos et al. 2008; Qiu et al. 2004; Richter 395 343 characterization of B. burgdorferi sensu lato strains (Busch et al. 2006; Rudenko et al. 2009a). The characteristics of the 396 344 et al. 1996a, b). eight chromosomal housekeeping genes of B. burgdorferi were 397 345 Genomic macrorestriction using rare cutting endonucle- located at the MLST website hosted by Imperial College London 398 346 ases such as NotI followed by PFGE is considered a powerful (www.mlst.net). 399 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:26 Page Number: 12

12 156 Spirochaetaceae Phylum

. Fig. 156.10 Phylogenetic trees based on concatenated sequences of seven MLST loci. Phylogenetic trees were constructed based on concatenated sequences of seven housekeeping loci for 189 unique STs using neighbor-joining (a) and maximum-likelihood (c) methods. Both trees show six distinct clusters for seven pathogenic Leptospira species. Relatedness between L. weilii isolates is different in the two trees. In the neighbor-joining tree, a cluster of L. weilii isolates with a highly diverse tpiA allele (tpiA 51) is distantly related to other L. weilii isolates, as shown in the enlarged view (b). The maximum likelihood tree shows that L. weilii isolates falls into three subgroups that cluster together, as shown in the enlarged view (d). Khaki, L. interrogans; dark blue, L. kirschneri; pink, L. nogouchii; dark green, L. santarosai; light green, L. borgpetersenii; brown, L. alexanderi; gray, L. weilii. Only bootstrap support values over 90 % are shown (doi:10.1371/journal.pntd.0001954.g001)

400 DNA-DNA hybridization and sequence analysis of the 16S vertebrate hosts (Vitorino et al. 2008; Vollmer et al. 2011). For 416 401 rRNA locus was shown to have limited ability to discriminate example, MLST data for B. burgdorferi showed that the B. 417 402 within the B. burgdorferi s. l. complex (Gevers et al. 2005; burgdorferi populations from Europe and North America are 418 403 Stackebrandt and Ebers 2006). However, MLSTanalysis (Margos genetically related but are currently separated with no or limited 419 404 et al. 2008, 2011) was shown to improve the accuracy in identi- gene flow between them (> Fig. 156.8) (Hoen et al. 2009; 420 405 fying B. burgdorferi s. l. complex species. MLSA was proposed by Margos et al. 2008; Ogden et al. 2011). 421 406 Richter et al. (2006) to replace DNA-DNA hybridization as an For phylogenetic analyses of the whole group of LB spiro- 422 407 excellent alternative for B. burgdorferi s. l. species delineation. chetes, housekeeping genes provide the benefit of defining 423 408 MLSA successfully enabled the identification of six new species outgroup species, as they are also present in the relapsing fever 424 409 within the B. burgdorferi s. l. complex: B. spielmanii sp. nov., spirochetes (B. hermsii, B. duttoni, and B. turicatae), allowing 425 410 B. californiensis sp. nov., B. americana sp. nov., B. carolinensis rooting of phylogenies. Several unrooted or midpoint-rooted 426 411 sp. nov., B. bavariensis sp. nov., and B. kurtenbachii sp. nov. phylogenies have been published for the LB group species 427 412 (Margos et al. 2010). (Margos et al. 2010; Richter et al. 2006; Rudenko et al. 2009b) 428 413 MLSA of housekeeping genes has revealed differences in the (> Fig. 156.9). These trees produced different topologies 429 414 level of geographic structuring of populations of LB species that compared to each other and to the concatenated housekeeping 430 415 are consistent with distribution patterns of their different gene trees. 431 Comp. by: DMuthuKumar Stage: Revises1 Chapter No.: 156 Title Name: HbPK-Vol11 Date:11/1/14 Time:02:28:29 Page Number: 13

Spirochaetaceae Phylum 156 13

432 MLST has also been used to type Leptospira spp. (Ahmed Bourhy P, Bremont S, Zinini F, Giry C, Picardeau M (2011) Comparison of real- 489 490 433 et al. 2006; Boonsilp et al. 2013; Margos et al. 2008; time PCR assays for detection of pathogenic Leptospira spp. in blood and identification of variations in target sequences. J Clin Microbiol 491 434 Thaipadungpanit et al. 2007). Seven loci (pntA, sucA, fadD, 49(6):2154–2160 492 435 tpiA, pfkB, mreA, and glmU) of the chromosome DNA of Brisson D, Dykhuizen DE (2004) ospC diversity in Borrelia burgdorferi. Genetics 493 436 Leptospira spp. based on the performance of primers as previ- 168:713–722 494 437 ously described (also can be obtained from the sharing website Bunikis J, Garpmo U, Tsao J, Berglund J, Fish D, Barbour AG (2004) Sequence 495 496 438 http://leptospira.mlst.net) (Zhang et al. 2009). The latest survey, typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe. Micro- 497 439 a Single Multilocus Sequence Typing (MLST) Scheme for Seven biology 150:1741–1755 498 440 Pathogenic Leptospira Species, was performed by Boonsilp et al. Busch U, Hizo-Teufel C, Boehmer R, Fingerle V, Nitschko H, Wilske B, Preac- 499 441 (2013). Phylogenetic trees reconstructed from concatenated Mursic V (1996a) Three species of Borrelia burgdorferi sensu lato 500 442 sequences of the seven loci of the modified scheme demon- (B. burgdorferi sensu stricto, B. afzelii, and B. garinii) identified from 501 502 443 strated the perfect classification of the isolates into seven path- cerebrospinal fluid isolates by pulsed-field gel electrophoresis and PCR. J Clin Microbiol 34:1072–1078 503 444 ogenic species, which resided in clearly distinct phylogenetic Busch U, Hizo-Teufel C, Bohmer R, Fingerle V, Rossler D, Wilske B, 504 445 clusters. The MLST scheme was used to gain new insight into Preac-Mursic V (1996b) Borrelia burgdorferi sensu lato strains isolated 505 446 the population genetic structure of the Leptospira species asso- from cutaneous Lyme borreliosis biopsies differentiated by pulsed-field gel 506 447 ciated with clinical disease and maintenance hosts in Asia electrophoresis. Scand J Infect Dis 28:583–589 507 508 448 (> Fig. 156.10). Cabello FC, Sartakova ML, Dobrikova EY (2001) Genetic manipulation of spiro- chetes–light at the end of the tunnel. Trends Microbiol 9(6):245–248 509 449 ˚ Rasba¨ck et al. (2007) compared the MLST results of 66 Cameron CE, Zuerner RL, Raverty S, Colegrove KM, Norman SA, Lambourn 510 450 carefully selected Brachyspira isolates and strains, with current DM, Jeffrie SJ, Gulland FM (2008) Detection of pathogenic Leptospira 511 451 classifications based on culture and biochemical tests, 16S rDNA bacteria in pinniped populations via PCR and identification of a source of 512 452 sequences, and strain-typing results from random amplified transmission for zoonotic leptospirosis in the marine environment. J Clin 513 514 453 polymorphic DNA (RAPD) analysis. The working group con- Microbiol 46:1728–1733 Caro-Quintero A, Ritalahti KM, Cusick KD, Lo¨ffler FE, Konstantinidis KT 515 454 cluded that MLST data are useful for molecular epidemiological (2012) The chimeric genome of Sphaerochaeta: nonspiral spirochetes that 516 455 studies, and, in particular, the eBURSTanalysis was shown to be break with the prevalent dogma in spirochete biology. 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