Taxonomic characterisation of ceftazidime-resistant Brevundimonas isolates and description of Brevundimonas faecalis sp. nov.

Systematic and Applied Microbiology 34 (2011) 408–413

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Taxonomic characterisation of ceftazidime-resistant Brevundimonas isolates and description of Brevundimonas faecalis sp. nov. Claudia Scotta a , A. Bennasar a,b , E.R.B. Moore c,d , J. Lalucat a,e , M. Gomila f,∗ a

Microbiologia, Departament de Biologia, Universitat de les Illes Balears, 07122 Palma de Mallorca, Illes Balears, Spain Institut Universitari d’ Investigació en Ciències de la Salut (IUNICS-UIB), 07122 Palma de Mallorca, Illes Balears, Spain Culture Collection University of Gothenburg (CCUG), Department of Clinical Bacteriology, Sahlgrenska University Hospital, Gothenburg, Sweden d Sahlgrenska Academy of the University of Gothenburg, Gothenburg, Sweden e Institut Mediterrani d’Estudis Avanc¸ats, IMEDEA (CSIC-UIB), 07122 Palma de Mallorca, Illes Balears, Spain f Unitat d’ Investigació – Microbiologia, Fundació Hospital Son Llàtzer, 07198 Palma de Mallorca, Illes Balears, Spain b c

a r t i c l e

i n f o

Article history: Received 10 February 2011 Received in revised form 23 May 2011 Accepted 1 June 2011 Keywords: Brevundimonas faecalis sp. nov. Ceftazidime resistance Sewage 16S rRNA gene sequence Phylogeny ITS1 gyrB gene rpoB gene DNA–DNA hybridisation CFA Biolog MALDI-TOF MS

a b s t r a c t Three ceftazidime-resistant strains isolated from the sewage water of a municipal hospital in Palma de Mallorca, Spain, were analysed phenotypically and genotypically to clarify their taxonomic positions. Sequence determinations and phylogenetic analyses of the 16S rRNA genes indicated that strains CS20.3T , CS39 and CS41 were affiliated with the species of the alphaproteobacterial genus Brevundimonas, most closely related to B. bullata, B. diminuta, B. naejangsanensis and B. terrae. Additional sequences analyses of the ITS1 region of the rRNA operon and the genes for the housekeeping enzymes DNA gyrase ␤subunit and RNA polymerase ␤-subunit, genomic DNA–DNA hybridisation similarities, cell fatty acid profiles and physiological and biochemical characterizations supported the recognition of CS20.3T (CCUG 58127T = CECT 7729T ) as a distinct and novel species, for which the name Brevundimonas faecalis sp. nov. is proposed. Strains CS39 and CS41 were ascribed to the species B. diminuta. © 2011 Elsevier GmbH. All rights reserved.

Two species described originally as Pseudomonas diminuta and Pseudomonas vesicularis were reclassified by Segers et al. [34] into a new genus, Brevundimonas, with B. diminuta defined as the type species of the genus. In 1999, several species of Caulobacter were transferred to Brevundimonas and the description of the genus was emended markedly [1]. At the time of this study, Brevundimonas was comprised of 21 validly published species isolated from various sources: B. diminuta (fresh water) [23,34] and B. vesicularis (urinary bladder epithelium) [6,11,34]; B. alba (soil) [1,31]; B. aurantiaca (Chlorella culture) [1,31]; B. aveniformis (activated sludge) [33]; B. bacteroides (fresh water) [1,31]; B. basaltis (sand) [7]; B. bullata (soil) [15,20]; B. halotolerans (brackish water) [2]; B. intermedia (fresh water) [1,31]; B. kwangchunensis (alkaline soil) [40]; B. lenta (soil)

Abbreviations: ITS1, internally transcribed spacer 1; LB, Luria Bertani; CCUG, Culture Collection of the University of Göteborg; ATCC, American Type Culture Collection; KCTC, Korean Collection for Type Cultures. ∗ Corresponding author. Tel.: +34 971 172068; fax: +34 971 173184. E-mail address: [email protected] (M. Gomila). 0723-2020/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2011.06.001

[42]; B. mediterranea (marine water) [10]; B. naejangsanensis (soil) [20]; B. nasdae (condensation water) [24]; B. poindexterae (activated sludge) [2]; B. staleyi (activated sludge) [2]; B. subvibroides (fresh water) [1,31]; B. terrae (alkaline soil) [41]; B. vancanneytii (clinical blood sample) [8]; and B. variabilis [1,31]. Brevundimonas species have rarely been associated with human infections and their clinical significance, if any, remains undetermined [4]. Nevertheless, hospital-acquired Brevundimonas infections have been reported [12,17,27,30]. Moreover, intrinsic resistance to quinolones has been noted in B. diminuta ATCC 11568T , while susceptibilities to aminoglycosides and cephalosporins have been reported among strains of B. diminuta and B. vesicularis [12,17]. The role of non-clinical niches as reservoirs for bacteria carrying the genes responsible for antibiotic resistance and the dynamics of the spread of these genes is a focus that is being studied in detail by several research groups [32]. In this study, the taxonomic status of three Gram-negative strains, CS20.3T , CS39 and CS41, isolated during an investigation of ceftazidime-resistant micro-organisms in the sewage water of one of the largest hospitals in Palma de Mallorca (Spain) has been

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Fig. 1. Neighbor-joining tree based on 16S rRNA gene sequences showing phylogenetic relationships between Brevundimonas faecalis CS20.3T and closest related Brevundimonas species. Bootstrap values greater than 450, based on 1000 replications, are indicated at branching nodes as percentages. Bar, 0.01 nt substitutions per site.

assessed. A polyphasic taxonomic study showed that one of the strains, CS20.3T , represents a distinct and new species of the genus Brevundimonas within the Alphaproteobacteria. Water obtained from a constant-flow sewer that collects waste water from the Hospital Son Llàtzer in Mallorca was plated, in duplicate, on Cetrimide agar (Merck, Darmstadt, Germany), Gould S1 agar [14] and King B agar [21], which were all supplemented with 30 mg L−1 ceftazidime (Combinopharm, Madrid, Spain). Plates were incubated at 30 ◦ C for 48 h. Individual cultures were checked for purity by plating on the same medium used for isolation, and stored in glycerol at −80 ◦ C. Isolate redundancy was assessed using ERIC PCR [38], and isolates were identified through partial 16S rRNA gene amplification and sequencing. Isolates CS20.3T , CS39 and CS41 that appeared on King B agar medium supplemented with ceftazidime (30 mg L−1 ) were related to the genus Brevundimonas by their partial 16S rRNA gene sequences and they were selected for further analysis. The closest type strains of Brevundimonas species were also included in the study for comparison purposes: B. diminuta ATCC 11568T , B. bullata CCUG 57113T , B. naejangsanensis CCUG 57609T and B. terrae KCTC 12418T . All strains were maintained on Luria Bertani (LB) agar [26] at 30 ◦ C. Colonies from the three isolates grown on LB agar after 48 h were circular, convex, light yellow, with a smooth edge and 2 mm in diameter, except CS20.3T , which was 1 mm in diameter. After four days incubation, a light brown, raised centre appeared in each colony. None of the strains produced fluorescent diffusible pigments. Morphology and flagella insertion were determined, using transmission electron microscopy of cells at the exponential growth phase in LB broth (Sharlau). The samples were negatively stained with phosphotungstic acid (1% w/v, pH 7.0), as described by Lalucat [22]. A Hitachi model H600 electron microscope was used at 75 kV. Cells were observed to be rod-shaped (1.2–1.5 ␮m

long, 0.5 ␮m wide), and motile by means of a single polar flagellum (Supplementary Fig. S1). Total DNA was extracted using a method described previously [39]. In addition to 16S rRNA genes, the rRNA operon internally transcribed spacer 1 (ITS1) region, fragments of the genes for DNA gyrase subunit ␤ (gyrB) and RNA polymerase subunit ␤ (rpoB) were amplified and sequenced as previously described [13,16,35]. As necessary, gyrB PCR products were cloned into the pGEM-T easy vector system, following the manufacturer’s instructions (Promega, Spain). The gene sequences were aligned with closely related sequences retrieved from the GenBank Nucleotide Sequence Database (http://www.ncbi.nlm.nih.gov/genbank/). Alignments of the sequences were carried out using a hierarchical method for multiple alignments, implemented in the program CLUSTALX [36]. Alignments obtained automatically were checked manually. Evolutionary distances, derived from sequence-pair dissimilarities, were calculated using the Jukes and Cantor correction [18] included in the program DNADIST of the Phylogenetic Inference Package (PHYLIP version 3.5c, [9]). Phylogenetic trees were constructed using the neighbor-joining distance method. Topologies of the trees were analysed using TreeView [28], and bootstrap (1000 replications) estimates of the branching order were calculated [9]. The sequences for the nearly complete (1320 nucleotide positions) 16S rRNA genes of the isolates revealed that the strains CS20.3T , CS39 and CS41 belonged to the Brevundimonas genus (Fig. 1). In the phylogenetic tree, generated using the neighborjoining evolutionary distance algorithm, the three isolates were placed in the clade of species comprising B. diminuta, B. naejangsanensis, B. terrae and B. vancanneytii. The same topology was obtained using other algorithms: DNA maximum likelihood and DNA parsimony. Strains CS39 and CS41 clustered with B. diminuta

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ATCC 11568T , with 16S rRNA gene sequence similarities of 99.9% (one nucleotide difference). Strain CS20.3T appeared in a separate branch of the dendrogram (bootstrap value of 48%). Strain CS20.3T 16S rRNA gene sequence similarities were 99.3% with B. diminuta ATCC 11568T , 98.6% with B. bullata CCUG 57113T , 98.5% with B. naejangsanensis CCUG 57609T , 98.5% with B. terrae KCTC 12418T and 98.0% with B. vancanneytii CCUG 1797T . The sequence similarities of strain CS20.3T with all other species of Brevundimonas were less than 97.5%. ITS1 gene sequences (approximately 700 nucleotide positions) were analysed and the branch lengths between strains were deeper, reflecting the higher level resolution offered by ITS1 sequence comparisons. Strains CS39 and CS41 appeared in the same branch as B. diminuta ATCC 11568T , with sequence similarities of 97.3% and 99.4%, respectively. CS20.3T appeared in a distinct branch of the tree, with ITS1 sequence similarities of 87.2% to those of B. bullata CCUG 57113T , 82.2% to B. diminuta ATCC 11568T , 79.9% to B. naejangsanensis CCUG 57609T and 74.9% to B. terrae KCTC 12418T (Supplementary Fig. S2 and Table S1). The PCR-amplification primers targeting the gyrB gene generated PCR-products of approximately 400 nucleotide positions, although non-specific products were also produced when amplifying some Brevundimonas species (strains CS20.3T , CS39, CS41, B. diminuta ATCC 11568T and B. terrae KCTC 12418T ). The PCR products from the gyrB amplification of strain CS20.3T and B. diminuta ATCC 11568T were cloned into the pGEM-T easy vector system and sequenced. In the gyrB sequence phylogenetic tree, strain CS20.3T clustered with B. diminuta ATCC 11568T , B. naejangsanensis CCUG 57609T and B. bullata CCUG 57113T , with sequence similarity values lower than 85% (Supplementary Fig. S3 and Table S2). This degree of gyrB sequence similarity suggested that strain CS20.3T was genotypically sufficiently different to be considered distinct at the species level. A fragment of approximately 700 nucleotide positions of the rpoB gene sequence was also analysed for strains CS39 and CS41, which showed similarities of 98.4% to each other (20 nucleotide differences) and 98.9% and 99.0%, respectively, with the sequence of B. diminuta ATCC 11568T . The rpoB sequences of the two isolated strains exhibited more than 5% sequence difference with the sequences of all other Brevundimonas species analysed but it was not possible to amplify the rpoB gene for strain CS20.3T . All sequences determined in this study have been deposited in the public database EMBL under the following accession numbers: FR775448–FR775451 for the 16S rRNA gene, FR775452–FR775458 for ITS1, FR775459–FR775462 for the gyrB gene and FR775463–FR775467 for the rpoB gene. Based on the 16S rRNA gene sequence comparative analyses, genomic DNA–DNA hybridization studies were carried out on the isolated strains with the most closely related species of Brevundimonas (i.e., with 16S rRNA gene similarity values greater than 98.0%). Genomic DNAs were isolated by the method of Marmur [25]. DNA–DNA hybridizations were performed as described by Urdiain et al. [37], using the genomic DNAs of Brevundimonas faecalis CS20.3T and B. diminuta ATCC 11568T as probes against the DNAs from other strains of Brevundimonas spp. The genomic DNA–DNA similarities of strain CS20.3T with the type strains of the phylogenetically closest species were less than 50%, confirming that it was genotypically sufficiently divergent to be considered as a distinct and novel species. Isolates CS39 and CS41 showed DNA–DNA relatedness levels greater than 70% with B. diminuta ATCC 11568T , indicating that these two strains should be assigned to this species (Supplementary Table S3). A previously reported reversed-phase high-performance liquid chromatography method was followed, with the modifications reported by Urdiain et al. [37], to determine the base compositions of the DNAs. The G + C content of the genomic DNA of CS20.3T was

determined to be 65.1%, which was within the range of 62–68% [29] that has been recorded for the species of Brevundimonas. Biochemical characterisations of substrate utilization and metabolite production were performed for the three isolates and the type strains of the most closely related species of Brevundimonas, using the API 20NE identification system for Gram-negative non-enterobacterial rods, and the API ZYM system for the characterisation of 19 enzymatic reactions according to the instructions of the manufacturer (bioMérieux). The GN2 MicroPlateTM assay of substrate oxidation [5] was also carried out, according to the manufacturer’s instructions (Biolog Inc.), with homogeneous suspensions of cells grown on blood agar. Growth at various temperatures, from 4 ◦ C to 42 ◦ C, was tested on LB medium (Sharlau) and growth at 0.5–6% (w/v) NaCl was tested for the three isolates. Strain CS20.3T differed from all other species of Brevundimonas, with respect to several metabolic-phenotypic characteristics (Table 1). It could be distinguished from the phylogenetically most closely related species by its inability to catalyze the decomposition of hydrogen peroxide, to reduce nitrate, to produce acid from glucose and to grow at 3% NaCl. In addition, it did not grow at 4 ◦ C or 42 ◦ C, and was differentiated from the most closely related Brevundimonas species by its ability to grow with N-acetyl-glucosamine or with capric acid, and its inability to grow with malic acid as sole carbon sources. Typically, strains of B. diminuta should not reduce nitrate but, as shown in Table 1, strains CS39 and CS41 were able to do so. However, a case of a strain of B. diminuta with this ability was also reported by Ballard et al. [3]. Thus, it seems that nitrate reduction is a variable characteristic in B. diminuta strains. Although gelatinase activity has been reported as negative in B. diminuta [29], positive reactions for this enzyme were observed for B. diminuta ATCC 11568T and isolate CS39. This result was also reported by Li et al. [24]. The API ZYM did not contribute any differential characteristics (see Supplementary Table S4). Substrate oxidation profiles, obtained through Biolog GN2, are listed in Supplementary Table S5. Strain CS20.3T could be differentiated from the B. diminuta cluster by its ability to assimilate l-histidine, hydroxy-l-proline, l-rhamnose and propionic acid, as well as its inability to assimilate acetic acid, urocanic acid, dextrin, ␣-ketobutyric acid, ␣-ketoglutaric acid, ␣-ketovaleric acid, l-phenylalanine, ␣-d-glucose, turanose, ␣-d-glucose-1-phosphate, cellobiose, succinic acid mono-methyl-ester, succinic acid and dglucose-6-phosphate. Gas chromatography of cell fatty acid (CFA) methyl esters was performed for the three isolates using a standardized protocol, similar to that of the MIDI Sherlock MIS system (http://www.ccug.se/pages/CFA method 2008.pdf), and was compared with the results for the type strains of the most closely related species. Cell biomasses for all strains compared were harvested from blood agar medium, incubated at 30 ◦ C, except for B. diminuta, which was incubated at 37 ◦ C. CFAs were identified, quantified and the relative amount of each fatty acid was expressed as a percentage of the total fatty acids in the profile of that strain. The major cellular fatty acids (CFAs) detected in CS20.3T were C18:1 ␻7c and C16:0 (59.0 and 25.5%, respectively, of the total CFAs), as indicated in Supplementary Table S6. These CFA profiles were similar to those reported for Brevundimonas species [1]. Few chemotaxonomicphenotypic differences were detected among the type strains of the most closely related species. The CFA profiles of strains CS39 and CS41 were practically indistinguishable from that of the B. diminuta type strain. Matrix-assisted linear desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) assays for isolates CS20.3T , CS39 and CS41, as well as the type strains of the most closely related species, were performed at Anagnostec, GmbH, Germany [19]. The cells were analysed on a Flexi Mass stainless steel target, using a whole-cell protocol with a 1 ␮L matrix solution

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Table 1 Differential phenotypic characteristics between the isolates and closest related Brevundimonas species. Characteristic Oxidase Catalase Growth at 4 ◦C 20 ◦ C 30 ◦ C 37 ◦ C 42 ◦ C Growth at 3% NaCl Hydrolysis of Tween 80 (OF) Urea Gelatine Nitrate reduction Glucose fermentation Growth in Glucose N-acetylglucosamine Capric acid Adipic acid Malic acid

1

2

3

4 a

5

6

8g

7 b

+ −

+ +

+ +

+ +

+ +

+ +

+/w w/−b

+ −c

− + + + − −

− + + + + +

− + + + + +

− + + + + nde

+ + + + − −d

+ + + + + +d

w + + + − −d

nd nd nd nd nd +

− − − − −

− − + + −

− − − + +

− −f + − −

+ − − − −

+ − − − −

− − − − −

− − − + −

− + + − −

− − + − −

− − + − −

− − +f − −

− − − − −

− − + − +

w − − w −

− − + − +

Strains: 1. B. faecalis sp. nov. CS20.3T ; 2. B. diminuta CS39; 3. B. diminuta CS41; 4. B. diminuta ATCC 11568T ; 5. B. terrae KCTC 12418T ; 6. B. naejangsanensis CCUG 57609T ; 7. B. bullata CCUG 57113T ; 8. B. vancanneytii CCUG 1797T . Data has been obtained in this study unless indicated. +, positive, −, negative, w, weak, nd, not determined. a Data from Palleroni [29] and Segers et al.[34]; different results for the type strains were obtained by Li et al. [24]. b Data taken from Kang et al., 2009 [20]. c Different results at the CCUG webpage. d Data taken from the original description article in each case. e There is no reference, but it is a weak positive at the CCUG webpage for B. diminuta CCUG 1427T . f Different results reported by Li et al. [24]. g Data taken from Estrela and Abraham [8].

of saturated ␣-cyano-4-hydroxy-cinnamic acid in a mixture of acetonitrile:ethanol:water (1:1:1), acidified with 3% (v/v) trifluoroacetic acid. For each strain, mass spectra were prepared in duplicate and analysed on an AXIMA Confidence instrument (Shimadzu/Kratos, Manchester, UK), in the linear positive ion extraction mode. Mass spectra were accumulated from 100 profiles, each from five nitrogen laser pulse cycles, by scanning the entire sample spot. Ions were accelerated with pulsed extraction at a voltage of 20 kV. Raw mass spectra were processed automatically for baseline correction and peak recognition. Resulting mass fingerprints were exported to SARAMIS (Spectral Archiving and Microbial Identification System, Release 3.36, Anagnostec GmbH, Germany) and then analysed. The percentage similarities of identical mass peaks were calculated and used to generate dendrograms, applying single-linkage agglomerate calculations. In the MALDI-TOF MS cluster analysis derived from mass signals of whole-cell protein profiles, the duplicate spectra profiles for each strain analysed clustered at a similarity of 75% (B. naejangsanensis CCUG 57609T ) or greater. The different species differentiated from

one another with 50% (between B. diminuta ATCC 11568T and B. naejangsanenssis CCUG 57609T ), or less, peak profile similarity. Strains CS39 and CS41 clustered with B. diminuta ATCC 11568T (approximately 80% similarity), further supporting the observations that these isolated strains represented strains of B. diminuta. Strain CS20.3T constituted a distinct branch of the dendrogram, with mass peak profiles most similar (approximately 35% similarity) to those of B. terrae KCTC 12418T and with approximately 30% similarity to those of B. diminuta (Fig. 2). MALDI-TOF MS profiles for all the strains studied have been included as Supplementary Fig. S4. These data further supported the conclusion that strain CS20.3T comprised a distinct and separate species from all other Brevundimonas species, even at the level of expression of the most abundant cellular proteins. Considering all the genotypic and phenotypic data, including genomic DNA–DNA hybridization similarity results and assessments of phylogenetic relationships, strains CS39 and CS41 were classified as members of B. diminuta species. However, we suggest that strain CS20.3T represents a distinct and novel species

Fig. 2. Dendogram of relatedness between B. faecalis CS20.3T and phylogenetically closest members of Brevundimonas based on MALDI-TOF MS analysis.

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within the genus Brevundimonas, for which the name Brevundimonas faecalis sp. nov. is proposed. Brevundimonas faecalis CS20.3T was deposited in the CCUG, Culture Collection of the University of Göteborg (CCUG 58127T ) and in the Spanish Culture Collection (CECT 7729T ). Isolates CS39 and CS41 were deposited in the CCUG as CCUG 58128 and CCUG 58129, respectively.

[3]

Description of Brevundimonas faecalis sp. nov.

[4]

Brevundimonas faecalis (fa.e.ca’lis. L.n. faex faecis, dregs, faeces; L. fem. suff. -alis, suffix denoting pertaining to; N.L. fem. adj. faecalis, pertaining to faeces). Cells are Gram-negative, aerobic rods (1.2–1.5 ␮m long, 0.5 ␮m wide), motile by means of a short-wavelength single polar flagellum. Colonies on LB agar medium are circular with a smooth border, slightly convex, creamy yellow in colour and 1.0 mm in diameter after 48 h incubation at 30 ◦ C. The strain does not grow at 4 ◦ C or at 42 ◦ C, and the optimal growth temperature oscillates between 30 and 37 ◦ C. Growth occurs in the presence of 0–1.5% but not 3% NaCl. Nitrate and nitrite are not utilized as terminal electron acceptors. Oxidase activity is positive but catalase is negative. Indole formation, glucose fermentation, arginine dihydrolase, urease, ␤glucosidase, gelatinase and PNPG tests are negative. In the API ZYM assay, alkaline phosphatase, esterase (C4), ester lipase (C8), leucine arylamidase, trypsin, chymotrypsin, acid phosphatase and phosphoamidase activities were detected, but lipase (C14), valine arylamidase, cysteine arylamidase, ␣- and ␤-galactosidase, ␤glucuronidase, ␣- and ␤-glucosidase, N-acetyl-␤-glucosaminidase, ␣-mannosidase and ␣-fucosidase activities were absent. It grows with N-acetyl-glucosamine and capric acid but not with glucose, arabinose, mannose, mannitol, maltose, gluconate, adipic acid, malic acid, citrate and phenylacetic acid. It is able to assimilate lhistidine, hydroxyl-l-proline, l-leucine, l-alaninamide, Tween 40 and 80, l-rhamnose, d-alanine, l-alanine, l-proline, l-alanylglycine, propionic acid, l-asparagine, l-aspartic acid, l-serine, l-glutamic acid, l-threonine, pyruvic acid methyl ester, ␤-hydroxybutyric acid, glycyl-l-aspartic acid, and glycyl-l-glutamic acid as carbon sources. The main cellular fatty acids are C18:1 ␻7c and C16:0 . The G + C mol % of the DNA is 65.14. Strain CS20.3T (CCUG 58127T = CECT 7729T ) is the type strain, isolated from sewage water from a hospital in Palma de Mallorca (Spain).

[2]

[5] [6] [7]

[8]

[9] [10]

[11] [12]

[13]

[14]

[15]

[16]

[17] [18]

[19]

[20]

Acknowledgments This work was supported by projects CGL 2009-12180 from the CICYT (Spain) and Consolider (CSD 2009-00006), as well as by FEDER funding. Claudia Scotta was the recipient of a pre-doctoral fellowship from the Conselleria d’Interior, Direcció General de Recerca, Desenvolupament Tecnològic i Innovació del Govern de les Illes Balears (FPI07). M. Gomila was the recipient of a postdoctoral contract from the Juan de la Cierva program of the Ministerio de Ciencia e Innovación of Spain. M. Gomila and E. Moore acknowledge the support of the FEMS Research Fellowship 2009-1. The authors acknowledge the technical assistance of the CCUG staff.

[21]

Appendix A. Supplementary data

[27]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.syapm.2011.06.001.

[28]

[22]

[23] [24]

[25] [26]

[29]

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