Canadian Journal of Microbiology. Utilization of carbon sources by clinical isolates of Aeromonas. Journal: Canadian Journal of Microbiology

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1 Canadian Journal of Microbiology Utilization of carbon sources by clinical isolates of Aeromonas Journal: Canadian Journal of Microbiology Manuscript ID cjm r1 Manuscript Type: Note Date Submitted by the Author: 21-Nov-2016 Complete List of Authors: Prediger, Karoline; Universidade Federal do Paraná, Análises Clínicas Surek, Monica; Universidade Federal do Paraná, Análises Clínicas Dalagassa, Cibelle; Universidade Federal do Paraná, Análises Clínicas Assis, Flávia; Universidade Federal do Paraná, Análises Clínicas Piantavini, Mário; Universidade Federal do Paraná, Farmácia Souza, Emanuel; Universidade Federal do Paraná, Bioquímica e Biologia Molecular Pedrosa, Fabio O.; Universidade Federal do Paraná, Bioquímica e Biologia Molecular Farah, Sônia; Laboratório Central do Estado do Paraná Alberton, Dayane; Universidade Federal do Paraná, Análises Clínicas Fadel-Picheth, Cyntia ; Universidade Federal do Paraná, Análises Clínicas Keyword: Aeromonas, Biolog, Carbon source, Mucin

2 Page 1 of 15 Canadian Journal of Microbiology 1 Utilization of carbon sources by clinical isolates of Aeromonas Authors: Karoline C. Prediger, 1 Monica Surek, 1 Cibelle B. Dallagassa, 1 Flávia E. A. Assis, 1 Mario S. Piantavini, 2 Emanuel M. Souza, 3 Fábio O. Pedrosa, 3 Sônia M. S. S. Farah, 4 Dayane Alberton, 1 Cyntia M. T. Fadel-Picheth 1 1 Departamento de Análises Clínicas, Universidade Federal do Paraná 2 Departamento de Farmácia, Universidade Federal do Paraná 3 Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná 4 Laboratório Central do Estado do Paraná (LACEN-PR) Karoline C. Prediger: karol_obs@hotmail.com Monica Surek: monicasurek@ufpr.br Cibelle B. Dallagassa: cibelledallagassa@yahoo.com.br Flávia E. A. Assis: flaviaemanoelli@uol.com.br Mario S. Piantavini: mario.s.piantavini@gmail.com Emanuel M. Souza: souzaem@ufpr.br Fábio O. Pedrosa: fpedrosa@ufpr.br Sônia M. S. S. Farah: farahos@uol.com.br Dayane Alberton: dayanealberton@ufpr.br Cyntia M. T. Fadel-Picheth: fpicheth@ufpr.br Correspondence: Cyntia M. T. Fadel-Picheth, Departamento de Análises Clínicas, Universidade Federal do Paraná, Av. Lothário Meissner 632, Curitiba, Paraná, Brasil, CEP fpicheth@ufpr.br Telephone: ; Fax number:

3 Canadian Journal of Microbiology Page 2 of 15 2 Abstract Bacteria in the genus Aeromonas are primarily aquatic organisms; however, some species can cause diseases in humans, ranging from wound infections to septicemia, of which diarrhea is the most common condition. The ability to use a variety of carbon substrates is advantageous for pathogenic bacteria. Therefore, we used Biolog GN2 microplates to analyze the ability of 103 clinical, predominantly diarrheal, isolates of Aeromonas to use various carbon sources and verified whether among the substrates metabolized by these strains there were some endogenous to the human intestine. Results indicate that Aeromonas present great diversity in the utilization of carbon sources, and that they preferentially use carbohydrates and amino acids as carbon sources. Among the carbon sources metabolized by Aeromonas in vitro, some were found to be components of intestinal mucin including aspartic acid, glutamic acid, L-serine, galactose, N- acetyl-glucosamine, and glucose, which were used by all strains tested. Additionally, mannose, D- serine, proline, threonine, and N-acetyl-galactosamine were used by several strains. The potential to metabolize substrates endogenous to the intestine may contribute to Aeromonas capacity to grow in and colonize the intestine. We speculate that this may help explain the ability of Aeromonas to cause diarrhea. Keywords: Aeromonas; Carbon source; Biolog; Mucin.

4 Page 3 of 15 Canadian Journal of Microbiology 3 Bacteria in the genus Aeromonas are primarily aquatic organisms, most frequently found in fresh and estuarine waters and in association with aquatic animals. Moreover, these bacteria are found in a wide variety of fresh products such as meat and dairy, and can be isolated from virtually every environmental niche where bacterial ecosystems exist (Martin-Carnahan and Joseph 2005; Janda and Abbott 2010; Horneman and Ali 2011). In addition, some Aeromonas species are able to cause diseases in humans such as wound infection, septicemia, pneumonia, and ocular and urinary tract infections (Janda and Abbott 2010; Parker and Shaw 2011; Chao et al. 2013), of which diarrhea is the most common condition (Parker and Shaw 2011). Aeromonas are non-sporeforming gram-negative rods and facultative anaerobes that ferment glucose to acids or acids with gas and reduce nitrates to nitrites. They are catalase positive, usually oxidase positive, and generally resistant to 150 µg of the vibriostatic agent O/129 (Abbott et al. 2003; Carnahan and Joseph 2005; Horneman and Ali 2011). Ammonium salts are used by most isolates as the sole source of nitrogen (Carnahan and Joseph 2005). Biochemical studies on Aeromonas have shown that they can use a number of other carbohydrates in addition to glucose (Abbott et al. 1992; Abbott et al. 2003; Carnahan and Joseph 2005; Horneman and Ali 2011). However, knowledge on the use of other carbon sources by Aeromonas species is still limited (Carnahan et al. 1989). Furthermore, carbon metabolism is considered essential during the early stages of many bacterial infections, and the ability to use a wide variety of carbon substrates may be advantageous for pathogenic bacteria (Fabich et al. 2008; Fuchs et al. 2012). The objectives of this work were to evaluate the ability of clinical isolates of Aeromonas to metabolize various carbon sources and to verify whether some of the substrates metabolized were components of intestinal mucin. One hundred and three Aeromonas strains, previously identified by using biochemical methods (Abbott et al. 2003), 16S rrna restriction fragment length polymorphism (RFLP) assay, and/or gyrb sequencing (Borrell et al. 1997; Yáñez et al. 2003) were used in this study. These strains included A. caviae (63 strains; all diarrheal isolates), A. hydrophila (23 strains; 3 wound isolates and 20 diarrheal isolates), A. veronii bv. sobria (14 strains; 1 blood isolate and 13 diarrheal isolates), and A. trota (3 strains; 1 cerebrospinal fluid isolate and 2 diarrheal isolates) (Surek et al. 2010; Assis et al. 2014; our unpublished data Vizzotto, B.S.; Dallagassa, C. B.). Additionally, A.

5 Canadian Journal of Microbiology Page 4 of 15 4 hydrophila ATCC 7966 and A. caviae ATCC were also tested, bringing the total number of tested strains up to 105. Strains were tested using Biolog GN2 microplates (Biolog, Hayward, USA). The GN2 microplates contained 95 distinct carbon substrates including carbohydrates, alcohols, organic acids, amino acids, and polymeric compounds. Positive reactions are indicated by the development of a purple color resulting from the simultaneous oxidation of the carbon source and reduction of a tetrazolium dye (Jones et al. 1993). Strains were grown overnight at 36 C on tryptic soy agar (Difco-Becton, Dickinson and Co., Le Pont-de-Claix, France), and isolated colonies were used to prepare suspensions in 0.9% sterile saline with 58% transmittance at 590 nm (Tang et al. 1998). Aliquots (150 µl) of the bacterial suspensions were inoculated onto GN2 microplates and incubated at 36ºC in a moist chamber. The results of this assay were determined visually after 24 and 48 hours of incubation. Tests with any development of purple tone were considered positive. Two independent assays were performed for each strain. Results were analyzed with BioNumerics 7.5 (Applied Maths, Keistraat, Belgium) using Jaccard similarity coefficient and a representative dendrogram was constructed with the unweighted pair group method with arithmetic mean (UPGMA). All strains yielded negative results for 15 substrates: α-cyclodextrin, adonitol, D-arabitol, i-erythritol, xylitol, D-galactonic acid lactone, γ-hydroxybutyric acid, itaconic acid, α-ketovaleric acid, quinic acid, sebacic acid, hydroxy-l-proline, L-phenylalanine, L-pyroglutamic acid, and phenylethylamine. Moreover, all strains were able to oxidize 20 carbon sources, including Tween 40 and Tween 80; the carbohydrates dextrin, glycogen, N-acetyl-D-glucosamine, D-fructose, D- galactose, α-d-glucose, maltose, sucrose, D-trehalose, and D-mannitol; D-gluconic acid; the amino acids L-asparagine, L-aspartic acid, L-glutamic acid, and L-serine; the dipeptides glycyl-l-aspartic acid and glycyl-l-glutamic acid; and inosine. Utilization rates for 60 carbon sources varied and are indicated in Table 1. When these results were compared with the results of another study in which Biolog GN microplates were used (Carnahan et al. 1989), differences were found in the number of substrates for which all strains tested positive (20 in the present study and 10 in the prior study using GN microplates) and in the number of substrates yielding uniformly negative results (15 in

6 Page 5 of 15 Canadian Journal of Microbiology 5 this study and 29 in the prior study). These distinctions may be associated with differences in the media composition of GN and GN2 microplates, criteria used to interpret test results, the additional 24 hours of incubation used in our study that facilitated the interpretation of weak positive results, or variability among the strains analyzed. All strains were able to metabolize several classes of carbon sources, as expected. However, only a few acids were used by most of the strains, what was also observed for sugar alcohols. In contrast, most strains were able to metabolize several amino acids and carbohydrates, mainly hexoses (Table 1), suggesting that Aeromonas preferentially uses these compounds as carbon sources. These results are in agreement with data suggesting that Aeromonas strains can ferment most hexoses but rarely attack sugar alcohols (Janda et al. 1985). Several other carbohydrates were metabolized presenting variable results among the strains (Table 1). However, even cellobiose and gentiobiose which have been suggested as useful for Aeromonas species identification (Carnahan et al. 1989), with the inclusion of A. trota and the higher frequencies of positive results for A. hydrophila and A. veronii bv. sobria found in the present study had the potential for species discrimination reduced. Although D,L-lactic acid has potential to separate A. veronii bv sobria, no single compound alone was able to distinguish the species (Table 1). Overal, the results of Biolog GN2 testing indicate that the Aeromonas strains analyzed present a high diversity (Table 1, Figure 1). Extensive phenotypic diversity among Aeromonas that were tested with several distinct substrates was also observed by Abbott and coworkers (2003). The similarity coefficient among all strains was 40% and most A. caviae (53/64) was grouped together, including A. caviae ATCC 14486; part of A. hydrophila (13/ 24) was also clustered together. However, there was no absolute separation of species and A. hydrophila ATCC 7966 was grouped with bacteria of other species (Figure 1), confirming the biological heterogeneity of the genus Aeromonas. Finally, among the 105 strains tested, the same metabolic profile was shared by only 2 A. caviae strains (Figure 1). Regarding the biochemical behavior of strains based on isolation source, it was not possible to distinguish among the intestinal and extra-intestinal isolates of A. hydrophila and A. veronii bv. sobria.

7 Canadian Journal of Microbiology Page 6 of 15 6 The ability to use a broad variety of carbon substrates, especially glycoproteins that are widely available in hosts, is advantageous for pathogenic bacteria and fundamental to establishing infection (Fabich et al. 2008; Fuchs et al. 2012). Since most of the Aeromonas strains (98/103, 95%) analyzed were intestinal isolates, we verified whether any of the carbon sources metabolized by these strains in vitro were endogenous to the human intestine. The intestinal epithelium is covered by a mucus layer whose main components are mucins, heavily glycosylated proteins. Serine and threonine are the amino acids prevalent in the core protein of mucin that also contain a high percentage of proline, aspartic acid, and glutamic acid. The oligosaccharide chains of mucin are formed by fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, sialic acid (Podolsky et al. 1983; Kim and Khan 2013) mannose and glucose (Podolsky et al. 1983). Several primary components of intestinal mucin, including aspartic acid, glutamic acid, L-serine, galactose, N-acetyl-glucosamine, and glucose, were used by all strains tested. Additionally, mannose, D- serine, proline, threonine, and N-acetyl-galactosamine were used by several strains (Table 1). These mucin components, released by the action of mucin-degrading bacteria present in the human digestive tract, can play an important role in bacterial colonization by providing a carbon and energy source (Derrien et al. 2010). Recently, recognition of the importance of metabolic pathways that are required for bacteria to colonize hosts has led to the redefinition of virulence as the sum of classical virulence factors, requisite metabolic pathways, and key import and export pumps (Fabich et al. 2008; Fuchs et al. 2012; Mobley 2015). Our data suggest that these Aeromonas strains may potentially use some components of mucin, but this needs to be experimentally confirmed. The ability to metabolize substrates that are endogenous to the host may contribute to Aeromonas capacity to colonize the intestine and cause diarrhea. Acknowledgements This work was supported by the Brazilian Program of National Institutes of Science and Technology - INCT/Brazilian Research Council - CNPq/MCT and Fundação Araucária. We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarships.

8 Page 7 of 15 Canadian Journal of Microbiology 7 Conflict of interest: The authors declare no conflict of interest. References Abbott, S.L., Cheung, W.K.W., Kroske-Bystrom, S., Malekzadeh, T. e Janda, J.M Identification of Aeromonas strains to the genospecies level in the clinical laboratory. J Clin Microbiol 30(5): Abbott, S.L., Cheung, W.K.W e Janda, J.M The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes. J Clin Microbiol 41(6): Assis, F.E.A, Wolf, S., Surek, M., De Toni, F., Souza, E.M., Pedrosa, F.O., Farah, S.M.S.S., Picheth, G. e Fadel-Picheth, C.M.T Impact of Aeromonas and diarrheagenic Escherichia coli screening in patients with diarrhea in Paraná, southern Brazil. J Infect Dev Ctries 8(12): Borrell N, Acinas SG, Figueras MG e Martinez-Murcia AJ (1997) Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rrna genes. J Clin Microbiol 35(7): Carnahan, A.M., Joseph, S.W. e Janda, J.M Species identification of Aeromonas strains based on carbon substrate oxidation profiles. J Clin Microbiol 27(9): Chao, C.M., Lai, C.C., Tsai, H.Y., Wu, C.J., Tang, H.J., Ko, W.C. e Hsueh, P.R Pneumonia caused by Aeromonas species in Taiwan, Eur J Clin Microbiol Infect Dis 32(8): Derrien, M., Passel, M.W.J.V., Bovenkamp, J.H.B.V., Schipper, R.G., Schipper, R.G., Vos, W.M.D. e Dekker, J Mucin-bacterial interactions in the human oral cavity and digestive tract. Gut Microbes 1(4): Fabich, A.J., Jones, S.A., Chowdhury, F.Z., Cernosek, A., Anderson, A., Smalley, D., McHargue, J.W., Hightower, G.A., Smith, J.T., Autieri, S.M., Leatham, M.P., Lins, J.J., Allen, R.L., Laux, D.C., Cohen, P.S. e Conway, T Comparison of carbon nutrition for pathogenic

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11 Canadian Journal of Microbiology Page 10 of FIGURE 1. Dendrogram showing the similarity of Aeromonas spp. regarding the use of carbon source

12 Page 11 of 15 Canadian Journal of Microbiology Table 1. Utilization of carbon sources by Aeromonas strains Total A. caviae A. hydrophila A. veronii bv. sobria A. trota Carbon source (n = 105) (n = 64) (n = 24) (n = 14) (n = 3) N-Acetyl-D-galactosamine L-Arabinose D-Cellobiose L-Fucose Gentiobiose m-inositol α-d-lactose Lactulose D-Mannose D-Melibiose β-methyl-d-glucoside D-Psicose D-Raffinose

13 Canadian Journal of Microbiology Page 12 of 15 L-Rhamnose D-Sorbitol Turanose Methyl pyruvate Mono-methyl-succinate Acetic acid γ-amino butyric acid cis-aconitic acid Citric acid Formic acid D-Galacturonic acid D-Glucosaminic acid D-Glucuronic acid α-hydroxybutyric acid β-hydroxybutyric acid p-hydroxyphenylacetic acid α-ketobutyric acid

14 Page 13 of 15 Canadian Journal of Microbiology α-ketoglutaric acid D,L-Lactic acid Malonic acid Propionic acid D-Saccharic acid Succinic acid Bromosuccinic acid Succinamic acid Urocanic acid Glucuronamide L-Alaninamide D-Alanine L-Alanine L-Alanyl-glycine L-Histidine L-Leucine L-Ornithine

15 Canadian Journal of Microbiology Page 14 of 15 L-Proline D-Serine L-Threonine D,L-Carnitine Uridine Thymidine Putrescine Aminoethanol ,3-Butanediol Glycerol D,L-α-Glycerol phosphate Glucose-1-phosphate Glucose-6-phosphate Numbers indicate the percentage of strains that tested positive for the use of that source.

16 Page 15 of 15 Canadian Journal of Microbiology *Indicate reference strains. 401x1300mm (72 x 72 DPI)