Moraxella porci sp. nov., isolated from pigs

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in International Journal of Systematic and Evolutionary Microbiology on 01 October 2010, available at: https://doi.org/10.1099/ijs.0.016626-0 .” 1 1 Moraxella porci sp. nov., a new Moraxella species isolated from pigs 2 3 4 5 1 Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Madrid, 6 Spain. 7 2 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, 8 Madrid, Spain. 9 3 Centre de Recerca en Sanitat Animal (CReSA), Campus de Bellaterra-Universitat Autónoma 10 de Barcelona, 08193-Bellaterra, Barcelona, Spain. 11 4 Departamento de Microbiología y Parasitología. Facultad de Farmacia. Universidad de 12 Sevilla. 41012 Sevilla, Spain. 13 14 15 Subject category: New Taxa - Proteobacteria 16 Running title: Moraxella porci sp. nov. 17 18 Author for correspondence: J.F. Fernández-Garayzábal 19 Tel: + 34 91 3943716 20 Fax: + 34 91 3943795 21 e-mail: [email protected] 22 23 24 25 26 27 28 29 The GenBank accession number for the 16S rRNA gene sequence of strain SN9-4MT is 30 FM872292 31 Nine isolates of Gram-negative, catalase-positive, oxidase-positive coccus-shaped 32 bacteria were cultured from pigs affected of different pathological processes. Phenotypic 33 2 and genetic methods were performed to determine the relationships of these isolates to 34 species belonging to the genus Moraxella. Analysis of the 16S rRNA gene sequence 35 demonstrated that the clinical isolates represent a new sub-line within the genus 36 Moraxella. The isolates were closely related to Moraxella cuniculi and Moraxella 37 pluranimalium with a 16S rRNA gene sequence similarity of 98.1% and 99.1%, 38 respectively. The isolates displayed high DNA-DNA relative binding ratios to each other 39 (74%) but distinctly lower levels of DNA homology with the closely related moraxellae 40 (lower to 32%). The new group of isolates were readily distinguished from all other 41 Moraxella species by physiological and biochemical tests. On the basis of the phenotypic 42 and molecular data, the nine Moraxella isolates from pigs represent a novel species 43 within the genus Moraxella, for which the name Moraxella porci sp. nov. is proposed. 44 The type strain is SN9-4MT (=CECT 7294T = CCUG 54912T). 45 46 47 The genus Moraxella currently comprises 18 species (Euzéby, 1997; Juni & Bøvre, 48 2005), some of them being described during the last decade (Vela et al. 2009; Angelos et al. 49 2007). The recognition of these novel species has been possible by the use of improved 50 taxonomic methods, such as chemotaxonomic and molecular-based approaches, especially 51 16S RNA gene sequencing. Although some of the Moraxella species have been isolated from 52 animal sources, most of them are usually found as part of the indigenous flora of animals 53 (Kodjo et al. 1995, 1997; Juni & Bøvre, 2005) and they are not considered of clinical 54 relevance in veterinary medicine. In fact, only Moraxella bovis is a well-established animal 55 pathogen, being responsible of ocular and respiratory infections (Brown et al., 1998, Quinn et 56 al., 1999, Lavin et al., 2000). In this study, we have characterized nine Moraxella-like 57 organisms isolated from different clinical specimens of pigs during the routine 58 microbiological diagnosis. On the basis of the phenotypic and phylogenetic results, a new 59 species of the genus Moraxella, Moraxella porci, is proposed. 60 The unidentified Gram-negative cocci organisms were recovered from the pleura (466-61 01 and 1335-02), lungs (1407-02 and 91-02), pericardium (43-02 and 464-01), brain (SN9-62 4MT), limphnodes (1008-04) and peritoneal cavity (N148-05CP-52G) of nine different pigs 63 with lesions of pleuritis, pneumonia, pericarditis, meningitis, arthritis and polyserositis, 64 respectively. Samples were collected and frozen at -40ºC until being processed in the 65 laboratory. Strains were isolated on Columbia blood agar plates (bioMérieux) after incubation 66 at 37ºC for 24-48 hours under aerobic conditions. The new isolates were Gram-stained and 67 3 assessed for the presence of oxidase and catalase tests as described by Barrow & Feltham 68 (1993). DNA hydrolysis was tested by using DNase test agar. Growth in brain heart infusion 69 broth was assessed at 4 and 15 ºC up to 14 d, at 22 ºC up to 7 d and at 30, 37 and 42 ºC for 48 70 h. Growth in the presence of 0.5, 1.5, 3, 4 and 6.5% NaCl and under anaerobic (with 4-10% 71 CO2) and microaerobic (with 5-15% O2 and 5-12% CO2) conditions, using GasPak Plus and 72 CampyPak Plus sytems (BBL), respectively was assessed at 37 ºC for 48 h. The growth on 73 MacConkey agar (bioMérieux) and chocolate agar (bioMérieux) were tested at 37 °C for 24 74 and 48 h. Additional biochemical tests were performed by using the API 20NE, API 20E and 75 API ZYM systems (bioMérieux) according to the manufacturer’s instructions, except that 76 incubation conditions for API 20NE was 37ºC for 72 h. The nine isolates exhibited similar 77 phenotypic characteristics, except for the assimilation of gluconate (1335-02 was positive) 78 and malate (the isolates 1335-02, 1407-02 y 43-02 were negative), growth in presence of 3% 79 NaCl (isolates 91-02, 466-01, 1008-04 and 1335-02 did not growth) AND reduction of nitrate 80 (43-02 and 1407-02 were negative). A detailed description of the morphological, 81 physiological and biochemical characteristics of the isolates is given in the species description 82 and in Table 1. 83 To establish the phylogenetic affinities of the unknown isolates, their 16S rRNA gene 84 sequences were determined as described previously (Vela et al., 2005) and subjected to a 85 comparative analysis. The almost complete sequences (1365 nucleotides) of the nine strains 86 were determined and pair-wise analysis revealed 99.4-100% 16S rRNA gene sequence 87 similarity among the ISOLTES. Sequence searches of GeneBank using the program FASTA 88 (Pearson, 1994) showed that the isolates were most closely related to members of the genus 89 Moraxella. The strain SN9-4MT displayed the closest 16S rRNA gene sequence similarity 90 with Moraxella pluranimalium CECT 7395T (99.1% sequence similarity) and Moraxella 91 cuniculi CCUG 2154T (98.1% sequence similarity). These sequences and those of other 92 known related strains were retrieved from GenBank and aligned with the newly determined 93 sequences using the program DNATools (Rasmussen, 1995). Phylogenetic trees were 94 constructed according to three different algorithms: neighbour-joining (Saitou & Nei, 1987) 95 using the programs DNATools and TREEVIEW (Page, 1996), maximum-likelihood using the 96 PHYML software (Guindon & Gascuel, 2003) and maximum-parsimony using the software 97 package MEGA (Molecular Evolutionary Genetics Analysis) version 3.1 (Kumar et al., 2004). 98 Genetic distances for the neighbour-joining and the maximum-likelihood algorithms were 99 calculated by the Kimura two-parameter (Kimura, 1980) and close-neighbour-interchange 100 (search level=2, random additions=100) was applied in the maximum-parsimony analysis. 101 4 The stability of the groupings was estimated by bootstrap analysis (1000 replications). 102 Phylogenetic trees obtained by using the neighbour-joining (Fig. 1) and the other two methods 103 (data not shown), revealed a clear affiliation of the swine isolates (as exemplified by strain 104 SN9-4MT) to the genus Moraxella, and placed them as a separate branch within an 105 intrageneric cluster containing the type strain of M. cuniculi CCUG 2154T and M. 106 pluranimalium CECT 7395T, which was supported by bootstrap re-sampling value of 100%. 107 The topology of the tree obtained by using the neighbour-joining method (Fig. 1) indicates 108 that the strain SN9-4MT formed a separate branch together with M. cuniculi, but it was 109 recovered with M. pluranimalium in the maximum-likelihood and maximum-parsimony trees. 110 DNA-DNA hybridization experiments were carried out between two isolates (SN9-4MT 111 and 466-01) and between strain SN9-4MT and its nearest phylogenetic neighbours, M. 112 cuniculi CCUG 2154T and M. pluranimalium CECT 7395T. DNA was extracted and purified 113 by the method of Marmur (1961). DNA-DNA hybridization studies were carried out by the 114 competition procedure of the membrane method of Johnson (1994), described in detail by 115 Arahal et al. (2001). The hybridization experiments were carried out under optimal 116 conditions, at a temperature of 49.5ºC, which is within the limits of validity for the filter 117 method (De Ley & Tijtgat, 1970). The percentage of hybridization was calculated as 118 described by Johnson (1994). Three independent determinations were carried out for each 119 experiment and the results reported are mean values. The DNA-DNA hybridization study 120 between the two new isolates showed 74% DNA relatedness to each other, demonstrating that 121 they are members of the same species (Wayne et al., 1987). The DNA-DNA reassociation 122 values between strain SN9-4MT and the type strains of M. cuniculi (CCUG 2154T) and M. 123 pluranimalium (CECT 7395T) were 17% and 31%, respectively, clearly confirming that the 124 new isolate constitute a separate species (Wayne et al., 1987). 125 The G+C content of the DNA of strain SN9-4MT was determined from the mid-point 126 value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962) obtained with a Perkin-127 Elmer UV-Vis Lambda 20 spectrophotometer at 260 nm. The G+C content of the type strain 128 was 46.6 mol%. This DNA G+C content is within the range reported for members of the 129 genus Moraxella (Juni & Bøvre, 2005). 130 Overall, the results of the present study show that the new isolates from pigs constitute a 131 distinct branch within the genus Moraxella (Fig. 1). Moreover, the new isolates could be 132 distinguished from their close phylogenetic relatives on the basis of phenotypic characteristics 133 (Table 1). Therefore, based on phenotypic, phylogenetic, and genotypic criteria it is clear that 134 the new isolates merit classification as a new species of the genus Moraxella, for which the 135 5 name Moraxella porci sp. nov. is proposed. Tests which are useful in differentiating M. porci 136 from other Moraxella species isolated from animals are shown in Table 1. 137 138 Description of Moraxella porci sp. nov. 139 Moraxella porci (por'ci. L. gen. n. porci, of a pig). 140 Cells are Gram-negative cocci (1 to 1.5 µm in diameter), nonspore-forming. Catalase-141 positive, oxidase-positive and aerobic. Colonies are circular, no-pigmented, smooth, entire 142 and do not produce a diagnostic odour on Columbia blood agar after 48 hours of incubation at 143 37ºC. Grows at 22ºC to 42ºC while no growth occurs at 15 ºC and 4 ºC. Growth is enhanced 144 in 5-10% CO2 (capnophilically) on 5% sheep blood agar and chocolate agar. Growth does not 145 occur on MacConkey agar. It grows on brain heart infusion broth containing 0.5-1.5 % NaCl, 146 but not with 4 and 6.5 % NaCl. Growth with 3 % NaCl was variable (SN9-4MT grew). 147 Colonies are non-haemolytic. With the API 20E and API 20NE kits, aesculin, gelatin and urea 148 are not hydrolyzed. Acetoin and indole are not produced (API 20 E). Acid is not produced 149 from D-glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, D-sucrose, D-melibiose, 150 amygdalin or L-arabinose (API 20E). Does not assimilate glucose, citrate, phenyl-acetate, L-151 arabinose, D-mannose, D-mannitol, N-acetyl-D-glucos amine, D-maltose, gluconate, caprate 152 or adipate (API 20NE). Assimilation of malate, and reduction of nitrate are variable (SN9-153 4MT was positive). Activity for ester lipase C8, esterase C-4, leucine arylamidase and 154 naphthol-AS-BI-phophohydrolase is detected (API ZYM). Valine arylamidase, lipase C14, 155 alkaline phosphatase cystine arylamidase, acid phosphatase, arginine dihydrolase, lysine 156 decarboxylase, ornithine decarboxylase, tryptophane deaminase, α-glucosidase, N-acetyl-β-157 glucosaminidase, β-glucosidase, β-glucuronidase, α-galactosidase, β-galactosidase, α-158 mannosidase, α-fucosidase, α-chymotrypsin and trypsin are not detected (API ZYM). The 159 clinical significance is uncertain. Isolated from pigs. 160 The type strain, SN9-4MT (=CECT 70294T = CCUG 54912T), was isolated from the 161 brain of a pig with meningitis. The DNA G+C content of this strain is 46.6 mol% (Tm). 162 163 ACKNOWLEDGEMENTS 164 The authors thank E. Arroyo and A. Casamayor for technical assistance. 165 166 REFERENCES 167 6 Angelos, J. A., Spinks, P. Q., Ball, L. M. & George1, L. W. (2007). Moraxella bovoculi sp. 168 nov., isolated from calves with infectious bovine keratoconjunctivitis. Int J Syst Evol 169 Microbiol 57, 789–795. 170 Arahal, D. R., García, M. T., Vargas, C., Canovas, D., Nieto, J. J. & Ventosa, A. (2001). 171 Chromohalobacter salexigens sp. nov., a moderately halophilic species that includes 172 Halomonas elongata DSM 3043 and ATCC 33174. Int J Syst Evol Microbiol 51, 1457-1462. 173 Barrow, G. I. & Feltham, R. K. A. (1993). Cowan and Steel's Manual for the Identification 174 of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. 175 Berrocal, A. Scott, M. I. U., Miller, D. & Flynn, H. W. (2001). Endophthalmitis caused by 176 Moraxella species. Am J Ophthalmol 132, 788-790. 177 Bøvre, K. (1984). Genus II Moraxella Lwoff 1939, 173 emend. Henriksen and Bøvre 1968, 178 391AL. In Bergey’s Manual of Systematic Bacteriology, vol 1, pp.297-302. Edited by N.R. 179 Krieg & J.G. Holt. New York: Springer. 180 Brown M. H., Brightman, A. H., Fenwick, B. W. & Rider, M. A. (1998). Infectious bovine 181 keratoconjunctivitis: a review. J Vet Inter Med 12, 259-266. 182 De Ley, J. & Tijtgat, R. (1970). Evaluation of membrane filter methods for DNA-DNA 183 hybridization. Antonie van Leeuwenhoek J Microbiol 36, 461-474. 184 Euzéby, J.P. (1997). List of Bacterial Names with Standing in Nomenclature: a folder 185 available on the Internet. Int J Syst Bacteriol 47,590-592. (List of Prokaryotic Names with 186 Standing in Nomenclature. Last full update May 03, 2007. [Online.] http://www.bacterio.net. 187 Guindon, S. & Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large 188 phylogenies by maximum likelihood. Syst Biol 52, 696–704. 189 Johnson, J. L. (1994). Similarity analysis of DNAs. In Methods for General and Molecular 190 Bacteriology, pp. 655-681. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. 191 Krieg. Washington, DC: American Society for Microbiology. 192 Juni, E. & Bøvre, K. (2005). Genus I. Moraxella Lwoff 1939, 173, emend. Henriksen and 193 Bøvre 1968, 391VP. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol 2 (The 194 Proteobacteria), part B (The Gammaproteobacteria), pp. 417-425. Edited by D. J. Brenner, 195 N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer 196 Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions 197 through comparative studies of nucleotide sequences. J Mol Evol 16, 111-120. 198 Kodjo, A., Tønjum, T., Richard, Y. & Bøvre, K. (1995). Moraxella caprae sp. nov., a new 199 member of the classical moraxellae. Int J Syst Bacteriol 45, 467–471. 200 7 Kodjo, A., Richard, Y. & Tonjum, T. (1997). Moraxella boevrei sp. nov., a new Moraxella 201 species found in goats. Int J Syst Bacteriol 47, 115–121. 202 Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular 203 evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163. 204 Lavin, S., Lastras, M. E., Marco, I. & Cabanes, F. X. (2000). Report of a case of 205 bronchopneumonia associated with Moraxella bovis isolation in a chamonis (Rupicrapa 206 pyrenaica). J Vet Med 47, 225-227. 207 Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from 208 microoganisms. J Mol Biol 3, 208-219. 209 Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic 210 acid from its thermal denaturation temperature. J Mol Biol 5, 109-118. 211 Olvera, A., Cerdà-Cuéllar, M., Nofrarías, M., Revilla, E., Segalés, J. & Aragon, V. 212 (2007). Dynamics of Haemophilus parasuis genotypes in a farm recovered from an outbreak 213 of Glässer's Disease. Vet Microbiol 123, 230-237. 214 Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal 215 computers. Comput Appl Biosci 12, 357–358. 216 Pearson, W. R. (1994). Using the FASTA program to search protein and DNA sequence 217 databases. Methods Mol Biol 24, 307–331. 218 Quinn, P. J., Carter, M. E., Markey, B. & Carter, G. R. (1999). Moraxella species. In 219 Clinical Veterinary Microbiology, pp. 284-286. Edited by P. J. Quinn, M. E. Carter, B. 220 Markey & G. R. Carter. Mosby, Edinburgh, Scotland. 221 Rasmussen, S. W. (1995). DNA Tools, a software package for DNA sequence analysis. 222 Carlsberg Laboratory, Copenhagen. 223 Rossau, R., Van Landschoot, A., Gillis, M. & De Ley, J. (1991). Taxonomy of 224 Moraxellaceae fam. nov., a new bacterial family to accommodate the genera Moraxella, 225 Acinetobacter, and Psychrobacter and related organisms. Int J Syst Bacteriol 41, 310-319. 226 Saitou, N. & Nei, M. (1987). The neighbour-joining method: a new method for 227 reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. 228 Vela, A. I., Collins, M. D., Lawson, P. A., García, N., Domínguez, L. & Fernández-229 Garayzábal, J. F. (2005). Uruburuella suis gen. nov., sp. nov., isolated from clinical 230 specimens of pigs. Int J Syst Evol Microbiol 55, 643-647. 231 Vela, A. I., Arroyo, E., Aragón, V., Sánchez-Porro, C., Latre, M. V., Cerdà-Cuéllar, M., 232 Ventosa, A., Domínguez, L. & Fernández-Garayzábal, J. F. (2009). Moraxella 233 8 pluranimalium sp. nov., isolated from animal specimens. Int J Syst Evol Microbiol 59, 671-234 674. 235 Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors. (1987). International 236 Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of 237 approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464. 238 239