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Evaluation of pyrrolidonyl arylamidase for the identification of nonfermenting Gram-negative rods
Diagnostic Microbiology and Infectious Disease 57 (2007) 101 – 103 www.elsevier.com/locate/diagmicrobio
Evaluation of pyrrolidonyl arylamidase for the identification of nonfermenting Gram-negative rods Karina A. Bombicino4, Marisa N. Almuzara, Angela M.R. Famiglietti, Carlos Vay Laboratorio de Bacteriologı´a Clı´nica, Departamento de Bioquı´mica Clı´nica, Facultad de Farmacia y Bioquı´mica, Hospital de Clı´nicas Jose´ de San Martı´n, Universidad de Buenos Aires (UBA), Co´rdoba 2351, Co´digo Postal 1120, Capital Federal, Buenos Aires, Argentina Received 15 September 2005; accepted 28 February 2006
Abstract To evaluate the activity of pyrrolidonyl arylamidase (PYR) for the differentiation and identification of nonfermenting gram negative rods (NFGNR), 293 isolates were tested. A 24 h culture of each test organism was prepared. From this a 108–109 cfu/mL suspension was added to 0.25 mL of sterile physiologic solution. A PYR disk was then added and the test was incubated for 30 minutes at 35–378C, at environmental atmosphere. Reading was done by adding 1 drop of cinnamaldehyde reagent. Strains of Acinetobacter baumannii, Acinetobacter haemolyticus, Alcaligenes faecalis, Bergeyella zoohelcum, Bordetella bronchiseptica, Bordetella hinzii, Brevundimonas diminuta, Brevundimonas vesicularis, Brucella ovis, Brucella spp., Brucella suis, Burkholderia cepacia complex, Moraxella catarrhalis, Moraxella lacunata, Moraxella nonliquefaciens, Moraxella osloensis, Oligella ureolytica, Pseudomonas alcaligenes, Pseudomonas mendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas Vb3, Psychrobacter phenylpyruvicus, and Stenotrophomonas maltophilia were PYR negative. On the other hand Achromobacter piechaudii, Achromobacter denitrificans, Achromobacter xylosoxidans, Burkholderia gladioli, Chryseobacterium gleum-indologenes, Comamonas testosroni, Cupriavidus pauculus, Delftia acidovorans, Elizabethkingia meningoseptica, Myroides spp., Ochrobactrum anthropi, Pseudomonas oryzihabitans, Ralstonia pickettii, Rhizobium radiobacter, Shewanella spp., Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Weeksella virosa were PYR positive. Finally, Acinetobacter lwoffii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Roseomonas spp., and Sphingomonas paucimobilisparapaucimobilis were PYR variable. PYR testing should be considered as a useful tool to facilitate the identification of NFGNR. D 2007 Elsevier Inc. All rights reserved. Keywords: Nonfermenting gram-negative rods (NFGNR); Pyrrolidonyl arylamidase (PYR)
Szewczuk and Mulczyk (1969, 1970) described the synthesis of l-pyrrolidonyl-a- and h-naphthylamides as chromogenic substrates for the detection of pyrrolidonyl peptidase activity. The utility of the pyrrolidonyl arylamidase (PYR) test to identify nonfermenting Gram-negative rods (NFGNRs) has previously had limited evaluation (Laffineur et al., 2002; Nicola et al., 1995). Two hundred ninety-three strains of NFGNR were tested for PYR test. All isolates, except Brucella ovis, were obtained from clinical specimens and isolated at our laboratory between 1994 and 2003. Stocked strains were frozen at 70 8C in skim milk and were first subcultured on trypticase
4 Corresponding author. Tel.: +54-11-5950-8663; fax: +54-11-59508694. E-mail address: [email protected] (K.A. Bombicino). 0732-8893/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.02.012
soy agar and Columbia blood agar for Brucella spp. before being tested. The isolates were identified by conventional methods according to the scheme of Schreckenberger (1997) and the working scheme for NFGNR developed in our laboratory. A PYR disk (Lab. Britania, Buenos Aires, Argentina) was placed in 0.25 mL of a 3- to 4-McFarland bacterial suspension and incubated at 37 8C for 30 min. One drop of cinnamaldehyde reagent was then added, and the solution was allowed to sit at room temperature for 10 min. Disks that turned pink were considered positive, and those that turned yellow were considered negative. The results of PYR test of 293 cultures are presented in Table 1. Identification of NFGNR by conventional testing methods is difficult and requires trained operators, specific tests, and many days of incubation. The identification process continues to increase in complexity as taxonomic studies contribute to our knowledge of new genera and species.
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Table 1 Number of isolates for each microorganism and their results for the PYR test Microorganism
No. of strains
PYR+
PYR
A. denitrificans A. piechaudii A. xylosoxidans Acinetobacter baumannii Acinetobacter haemolyticus Acinetobacter lwoffii A. faecalis Bergeyella zoohelcum B. bronchiseptica Bordetella hinzii Brevundimonas diminuta Brevundimonas vesicularis B. ovis Brucella spp. Brucella suis B. gladioli B. cepacia complex Chryseobacterium gleum-indologenes C. testosteroni C. pauculus Delftia acidovorans E. meningoseptica Moraxella catarrhalis Moraxella lacunata Moraxella nonliquefaciens M. osloensis Myroides spp. Ochrobactrum anthropi O. ureolytica Pseudomonas aeruginosa P. alcaligenes P. fluorescens Pseudomonas mendocina Pseudomonas oryzihabitans Pseudomonas pseudoalcaligenes P. putida Pseudomonas stutzeri Pseudomonas Vb3 Psychrobacter phenylpyruvicus R. pickettii R. radiobacter Roseomonas spp. Shewanella spp. Sphingobacterium multivorum Sphingobacterium spiritivorum Sphingomonas paucimobilisparapaucimobilis S. maltophilia Weeksella virosa
4 5 25 9 2 8 17 3 5 1 1 3 1 2 1 3 25 15 1 2 5 9 9 2 1 6 4 7 2 13 2 7 2 4 2 14 15 7 1 3 2 3 13 3 1 5
4 5 25 0 0 2 0 0 0 0 0 0 0 0 0 3 0 15 1 2 5 9 0 0 0 0 4 7 0 5 0 5 0 4 0 0 0 0 0 3 2 1 13 3 1 3
0 0 0 9 2 6 17 3 5 1 1 3 1 2 1 0 25 0 0 0 0 0 9 2 1 6 0 0 2 8 2 2 2 0 2 14 15 7 1 0 0 2 0 0 0 2
16 2
0 2
16 0
Identification of Burkholderia cepacia complex is difficult, particularly for those genomospecies that do not decarboxylate lysine (LDC negative) (Henry et al., 2001; Mukwaya and Welch, 1989; Stead, 1992). Members of this complex cause nosocomial infections and have emerged as serious pathogens in patients with the cystic fibrosis (CF) and chronic granulomatous disease (CGD) (Gilligan, 1991; LiPuma, 1998; Pegues et al., 1993; Winkelstein et al., 2000) Burkholderia gladioli, Ralstonia pickettii, and Ralstonia mannitolilytica have similar
phenotypic features to that of the isolates of B. cepacia LDC negative, and they have also been isolated from the respiratory tracts of patients with CGD and CF and cause nosocomial acquired infections (Barker et al., 1997; Ferna´ndez et al., 1996; Kahyaoglu et al., 1995; Raveh et al., 1993; Ross et al., 1995; Trotter et al., 1990). Hydrolysis of PYR represents a new tool with which to differentiate B. cepacia complex (PYR ) from B. gladioli, R. mannitolilytica, and R. pickettii (all PYR+) (this study and Laffineur et al., 2002). Rhizobium radiobacter and Agrobacterium spp. yellow group also have biochemical features similar to B. cepacia LDC negative. The 3-ketolactonate test must be performed to correctly identify these strains. This test requires at least 5 days of incubation to get to a negative result (Schreckenberger, 1997; Schreckenberger et al., 2003). B. cepacia complex is naturally resistant to colistin, and R. radiobacter is presumed to be susceptible. However, R. radiobacter was shown to be resistant (Laffineur et al., 2002). In our study, both strains of R. radiobacter were PYR+, which is supported by a previous work performed by Laffineur et al., where 19 strains were also all PYR+. This suggests that the PYR test can be a new method to distinguish between R. radiobacter and B. cepacia LDC negative. Results of the PYR test on Pseudomonas were variable. But PYR test could be used as a complementary test to distinguish between Pseudomonas fluorescens and Pseudomonas putida. In the event of a positive PYR reaction, P. putida could be excluded more rapidly. A similar result was previously described (Laffineur et al., 2002). Elizabethkingia meningoseptica has been described as an etiologic agent of neonatal meningitis and pneumonia and catheter-related blood stream infections in adults. (Bloch et al., 1997; Chiu et al., 2000). E. meningoseptica can be misidentified as Stenotrophomonas maltophilia because they have similar phenotypic attributes, including appearance of culture, hydrolysis of esculin and DNA, and multiple antibiotic resistance. Oxidase, motility, and indole tests are also very useful to differentiate between these species and are less labor intensive. The PYR test could also be useful in this setting because E. meningoseptica is PYR+ and S. maltophilia is PYR . Alcaligenes faecalis and Myroides spp. can be difficult to distinguish because both species produce a fruity odor on young cultures. The PYR test, however, can rapidly identify A. faecalis (PYR ) from Myroides spp. (PYR+). Within the asaccharolytic, mobile, rapidly urease-positive NFGNR, the PYR test can be useful to separate Cupriavidus pauculus (PYR+) from Bordetella bronchiseptica and Oligella ureolytica (both PYR ). Pyrrolidonyl arylamidase test could therefore replace the reduction of nitrate, which is time consuming (Vandamme et al., 1999). Recent studies have shown that Achromobacter xylosoxidans, Achromobacter denitrificans, and Achromobacter piechaudii all belong to the same genus, Achromobacter, rather than the genus Alcaligenes to which they were
K.A. Bombicino et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 101 – 103
formerly assigned (Yabuuchi et al., 1998). A. faecalis remains in the genus Alcaligenes. It is noteworthy that all Achromobacter spp. hydrolyze PYR, whereas A. faecalis does not. Comamonas testosteroni, Pseudomonas alcaligenes, and A. piechaudii cannot be properly identified by using biochemical tests because they are inactive to most substrates (Kiska and Gilligan, 2003). Identifying the number and location of their flagella remains the only reliable method by which to distinguish these species (Kiska and Gilligan, 2003). C. testosteroni is characterized as having 2 or more polar flagella. P. alcaligenes has only 1 and A. piechaudii possesses peritrich flagellum (Kiska and Gilligan, 2003). If a determination of the characterization of flagellum cannot be made, special tests such as the assimilation of testosterone or the utilization of dlnorleucine must be performed (Kiska and Gilligan, 2003). Our research demonstrates that PYR is hydrolyzed by A. piechaudii but not C. testosteroni. Laffineur et al. (2002) have similarly described 14 strains of A. piechaudii that hydrolyze PYR and 15 strains of C. testosteroni that do not. As mentioned earlier, all our strains of P. alcaligenes were PYR in accordance with the results of Laffineur et al. (2002). Currently, Ochrobactrum and Brucella belong to the same family, Brucellaceae. Our research shows that the PYR test can aid in distinguishing between these genera (Table 1). Our results show that 18 isolates of Moraxella were all PYR (including Moraxella osloensis). However, De Baere et al. (2002) have shown that Moraxella atlantae, a new specie with similar biochemical features to M. osloensis, is PYR+. According to our results and those from De Baere et al., the PYR test could be a new method by which to differentiate between M. osloensis and M. atlantae. Considering the short time needed to perform the PYR test, its ease of handling, and its low cost, the test proposed in this study could be included as a complementary test in conventional schemes of identification of NFGNR. Acknowledgments This work was supported by the Proyect UBACYT B 080. References Barker PM, Wood RE, Gilligan PH (1997) Lung infection with Burkholderia gladioli in a child with cystic fibrosis: acute clinical and spirometric deterioration. Pediatr Pulmonol 24:123 – 125. Bloch KC, Nadarajah R, Jacobs R (1997) Chryseobacterium meningosepticum: an emerging pathogen among immunocompromised adults. Medicine 76:30 – 40. Chiu YS, Hsueh PR, Wu JJ, Ho SW, Hsieh WC, Luh KT (2000) Atypical Chryseobacterium meningosepticum and meningitis and sepsis in newborns and the immunocompromised. Taiwan Emerg Infect Dis 6:481 – 486. De Baere T, Muylaert A, Everaert E, Wauters G, Claeys G, Verschraegen G, Vaneechoutte M (2002) Bacteremia due to Moraxella atlantae in cancer patient. J Clin Microbiol 40:2693 – 2695. Ferna´ndez C, Wilhelm I, Andradas E, Gaspar C, Gomez J, Romero J, Mariano JA, Corral O, Rubio M, Elviro J, Fereres J (1996) Nosocomial outbreak
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of Burkholderia pickettii infection due to a manufactured intravenous product used in three hospitals. Clin Infect Dis 22:1092 – 1095. Gilligan PH (1991) Microbiology of airway disease in patients with cystic fibrosis. Clin Microbiol Rev 4:35 – 51. Henry DA, Mahenthiralingam E, Vandamme P, Coenye T, Speert DP (2001) Phenotypic methods for determining genomovar status of the Burkholderia cepacia complex. J Clin Microbiol 39:1073 – 1078. Kahyaoglu O, Nolan B, Kumar A (1995) Empyema and bloodstream infection caused by Burkholderia gladioli in a patient with cystic fibrosis after lung transplantation. Pediatr Infect Dis J 15:637 – 638. Kiska DL, Gilligan PH (2003) Pseudomonas. In: Manual of Clinical Microbiology. (8th ed.). Eds, PR Murray, EJ Barron, JH Jorgensen, MA Pfaller, and RH Yolken. Washington (DC): ASM Press, pp 719 –728. Laffineur K, Janssens M, Charlier J, Avesani V, Wauters G, Delme´e M (2002) Biochemical and susceptibility tests useful for identification of nonfermenting gram-negative rods. J Clin Microbiol 40:1085 – 1087. LiPuma JJ (1998) Burkholderia cepacia: management issues and new insights. Clin Chest Med 19:473 – 486. Mukwaya GM, Welch DF (1989) Subgrouping of Pseudomonas cepacia by cellular fatty acid composition. J Clin Microbiol 27:2640 – 2646. Mulczyk M, Szewczuk A (1970) Pyrrolidonyl peptidase in bacteria: a new colorimetric test for differentiation of Enterobacteriaceae. J Gen Microbiol 61:9 – 13. Nicola F, Centorbi H, Bantar C, Smayevsky J, Bianchini H (1995) Utilidad de la deteccio´n de pirrolidonil-aril-amidasa en la diferenciacio´n de enterobacterias y bacilos gram-negativos no fermentadores. Rev Argent Microbiol 27:204 – 209. Pegues DA, Carson LC, Anderson RL, Norgard MJ, Argent TA, Jarvis WR, Woernle CH (1993) Outbreak of Pseudomonas cepacia bacteremia in oncology patients. Clin Infect Dis 16:407 – 411. Raveh D, Simhon A, Gimmon Z, Sacks T, Sapiro M (1993) Infectious caused by Pseudomonas pickettii in association with permanent indwelling intravenous devices: four cases and a review. Clin Infect Dis 17:877 – 880. Ross JP, Holland SM, Gill VJ, De Carlo ES, Gallin JI (1995) Severe Burkholderia gladioli infection in chronic granulomatous disease: report of two successfully treated cases. Clin Infect Dis 21:1291 – 1293. Schreckenberger PC (1997) Glucose nonfermenting gram-negative rods. Symposium 252-C Update on Gram-Negative Rods: Taxonomy, Identification, and Clinical Significance of the 97th General Meeting. Washington (DC)7 American Society for Microbiology. Schreckenberger PC, Daneshvar MI, Weyant MI, Hollis DG (2003) Acinetobacter, Achromobacter, Chryseobacterium, Moraxella, and other nonfermentative gram negative rods. In: Manual of Clinical Microbiology. Eds, PR Murray, EJ Baron, JH Jorgensen MA Pfaller, and RH Yolken.Washington (DC)7 ASM Press, pp 749 – 779. Stead DE (1992) Grouping of plant-pathogen and some other Pseudomonas spp. by using cellular fatty acid profiles. Int J Syst Bacteriol 42:281 – 285. Szewczuk A, Mulczyk M (1969) Pyrrolidonyl-peptidase in bacteria. The enzyme from Bacillus subtilis. Eur J Biochem 8:63 – 67. Trotter JA, Kuhis TL, Pickett DA, Reyes de la Rocha S, Welch DF (1990) Pneumonia caused by a newly recognized pseudomonad in a child with chronic granulomatous disease. J Clin Microbiol 28:1120 – 1124. Vandamme PJ, Goris J, Coenye T, Hoste B, Janssens D, Kersters K, De Vos P, Falsen E (1999) Assignment of Centers for Disease Control group IV c-2 to the genus Ralstonia as Ralstonia paucula sp. nov. Int J Syst Bacteriol 49:663 – 669. Winkelstein JA, Marino MC, Johnston Jr. RB, Boyle J, Curnutte J, Gallin JI, Malech HL, Holland SM, Ochs H, Quie P, Buckley RH, Foster CB, Chanock SJ, Dickler H (2000) Chronic granulomatous disease. Medicine 79:155 – 159. Yabuuchi E, Kawamura Y, Kosako Y, Ezaki T (1998) Emendation of genus Achromobacter and Achromobacter xylosoxidans (Yabuuchi and Yano) and proposal of Achromobacter ruhlandii (Packer and Vishniac) comb. nov., Achromobacter piechaudii (Kiredjian et al) comb. nov, and Achromobacter xylosoxidans subsp. denitrificans (Ruger and Tan) comb. nov. Microbiol Immunol 42:429 – 438.
Evaluation of pyrrolidonyl arylamidase for the identification of nonfermenting Gram-negative rods Karina A. Bombicino4, Marisa N. Almuzara, Angela M.R. Famiglietti, Carlos Vay Laboratorio de Bacteriologı´a Clı´nica, Departamento de Bioquı´mica Clı´nica, Facultad de Farmacia y Bioquı´mica, Hospital de Clı´nicas Jose´ de San Martı´n, Universidad de Buenos Aires (UBA), Co´rdoba 2351, Co´digo Postal 1120, Capital Federal, Buenos Aires, Argentina Received 15 September 2005; accepted 28 February 2006
Abstract To evaluate the activity of pyrrolidonyl arylamidase (PYR) for the differentiation and identification of nonfermenting gram negative rods (NFGNR), 293 isolates were tested. A 24 h culture of each test organism was prepared. From this a 108–109 cfu/mL suspension was added to 0.25 mL of sterile physiologic solution. A PYR disk was then added and the test was incubated for 30 minutes at 35–378C, at environmental atmosphere. Reading was done by adding 1 drop of cinnamaldehyde reagent. Strains of Acinetobacter baumannii, Acinetobacter haemolyticus, Alcaligenes faecalis, Bergeyella zoohelcum, Bordetella bronchiseptica, Bordetella hinzii, Brevundimonas diminuta, Brevundimonas vesicularis, Brucella ovis, Brucella spp., Brucella suis, Burkholderia cepacia complex, Moraxella catarrhalis, Moraxella lacunata, Moraxella nonliquefaciens, Moraxella osloensis, Oligella ureolytica, Pseudomonas alcaligenes, Pseudomonas mendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas Vb3, Psychrobacter phenylpyruvicus, and Stenotrophomonas maltophilia were PYR negative. On the other hand Achromobacter piechaudii, Achromobacter denitrificans, Achromobacter xylosoxidans, Burkholderia gladioli, Chryseobacterium gleum-indologenes, Comamonas testosroni, Cupriavidus pauculus, Delftia acidovorans, Elizabethkingia meningoseptica, Myroides spp., Ochrobactrum anthropi, Pseudomonas oryzihabitans, Ralstonia pickettii, Rhizobium radiobacter, Shewanella spp., Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Weeksella virosa were PYR positive. Finally, Acinetobacter lwoffii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Roseomonas spp., and Sphingomonas paucimobilisparapaucimobilis were PYR variable. PYR testing should be considered as a useful tool to facilitate the identification of NFGNR. D 2007 Elsevier Inc. All rights reserved. Keywords: Nonfermenting gram-negative rods (NFGNR); Pyrrolidonyl arylamidase (PYR)
Szewczuk and Mulczyk (1969, 1970) described the synthesis of l-pyrrolidonyl-a- and h-naphthylamides as chromogenic substrates for the detection of pyrrolidonyl peptidase activity. The utility of the pyrrolidonyl arylamidase (PYR) test to identify nonfermenting Gram-negative rods (NFGNRs) has previously had limited evaluation (Laffineur et al., 2002; Nicola et al., 1995). Two hundred ninety-three strains of NFGNR were tested for PYR test. All isolates, except Brucella ovis, were obtained from clinical specimens and isolated at our laboratory between 1994 and 2003. Stocked strains were frozen at 70 8C in skim milk and were first subcultured on trypticase
4 Corresponding author. Tel.: +54-11-5950-8663; fax: +54-11-59508694. E-mail address: [email protected] (K.A. Bombicino). 0732-8893/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.02.012
soy agar and Columbia blood agar for Brucella spp. before being tested. The isolates were identified by conventional methods according to the scheme of Schreckenberger (1997) and the working scheme for NFGNR developed in our laboratory. A PYR disk (Lab. Britania, Buenos Aires, Argentina) was placed in 0.25 mL of a 3- to 4-McFarland bacterial suspension and incubated at 37 8C for 30 min. One drop of cinnamaldehyde reagent was then added, and the solution was allowed to sit at room temperature for 10 min. Disks that turned pink were considered positive, and those that turned yellow were considered negative. The results of PYR test of 293 cultures are presented in Table 1. Identification of NFGNR by conventional testing methods is difficult and requires trained operators, specific tests, and many days of incubation. The identification process continues to increase in complexity as taxonomic studies contribute to our knowledge of new genera and species.
102
K.A. Bombicino et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 101 – 103
Table 1 Number of isolates for each microorganism and their results for the PYR test Microorganism
No. of strains
PYR+
PYR
A. denitrificans A. piechaudii A. xylosoxidans Acinetobacter baumannii Acinetobacter haemolyticus Acinetobacter lwoffii A. faecalis Bergeyella zoohelcum B. bronchiseptica Bordetella hinzii Brevundimonas diminuta Brevundimonas vesicularis B. ovis Brucella spp. Brucella suis B. gladioli B. cepacia complex Chryseobacterium gleum-indologenes C. testosteroni C. pauculus Delftia acidovorans E. meningoseptica Moraxella catarrhalis Moraxella lacunata Moraxella nonliquefaciens M. osloensis Myroides spp. Ochrobactrum anthropi O. ureolytica Pseudomonas aeruginosa P. alcaligenes P. fluorescens Pseudomonas mendocina Pseudomonas oryzihabitans Pseudomonas pseudoalcaligenes P. putida Pseudomonas stutzeri Pseudomonas Vb3 Psychrobacter phenylpyruvicus R. pickettii R. radiobacter Roseomonas spp. Shewanella spp. Sphingobacterium multivorum Sphingobacterium spiritivorum Sphingomonas paucimobilisparapaucimobilis S. maltophilia Weeksella virosa
4 5 25 9 2 8 17 3 5 1 1 3 1 2 1 3 25 15 1 2 5 9 9 2 1 6 4 7 2 13 2 7 2 4 2 14 15 7 1 3 2 3 13 3 1 5
4 5 25 0 0 2 0 0 0 0 0 0 0 0 0 3 0 15 1 2 5 9 0 0 0 0 4 7 0 5 0 5 0 4 0 0 0 0 0 3 2 1 13 3 1 3
0 0 0 9 2 6 17 3 5 1 1 3 1 2 1 0 25 0 0 0 0 0 9 2 1 6 0 0 2 8 2 2 2 0 2 14 15 7 1 0 0 2 0 0 0 2
16 2
0 2
16 0
Identification of Burkholderia cepacia complex is difficult, particularly for those genomospecies that do not decarboxylate lysine (LDC negative) (Henry et al., 2001; Mukwaya and Welch, 1989; Stead, 1992). Members of this complex cause nosocomial infections and have emerged as serious pathogens in patients with the cystic fibrosis (CF) and chronic granulomatous disease (CGD) (Gilligan, 1991; LiPuma, 1998; Pegues et al., 1993; Winkelstein et al., 2000) Burkholderia gladioli, Ralstonia pickettii, and Ralstonia mannitolilytica have similar
phenotypic features to that of the isolates of B. cepacia LDC negative, and they have also been isolated from the respiratory tracts of patients with CGD and CF and cause nosocomial acquired infections (Barker et al., 1997; Ferna´ndez et al., 1996; Kahyaoglu et al., 1995; Raveh et al., 1993; Ross et al., 1995; Trotter et al., 1990). Hydrolysis of PYR represents a new tool with which to differentiate B. cepacia complex (PYR ) from B. gladioli, R. mannitolilytica, and R. pickettii (all PYR+) (this study and Laffineur et al., 2002). Rhizobium radiobacter and Agrobacterium spp. yellow group also have biochemical features similar to B. cepacia LDC negative. The 3-ketolactonate test must be performed to correctly identify these strains. This test requires at least 5 days of incubation to get to a negative result (Schreckenberger, 1997; Schreckenberger et al., 2003). B. cepacia complex is naturally resistant to colistin, and R. radiobacter is presumed to be susceptible. However, R. radiobacter was shown to be resistant (Laffineur et al., 2002). In our study, both strains of R. radiobacter were PYR+, which is supported by a previous work performed by Laffineur et al., where 19 strains were also all PYR+. This suggests that the PYR test can be a new method to distinguish between R. radiobacter and B. cepacia LDC negative. Results of the PYR test on Pseudomonas were variable. But PYR test could be used as a complementary test to distinguish between Pseudomonas fluorescens and Pseudomonas putida. In the event of a positive PYR reaction, P. putida could be excluded more rapidly. A similar result was previously described (Laffineur et al., 2002). Elizabethkingia meningoseptica has been described as an etiologic agent of neonatal meningitis and pneumonia and catheter-related blood stream infections in adults. (Bloch et al., 1997; Chiu et al., 2000). E. meningoseptica can be misidentified as Stenotrophomonas maltophilia because they have similar phenotypic attributes, including appearance of culture, hydrolysis of esculin and DNA, and multiple antibiotic resistance. Oxidase, motility, and indole tests are also very useful to differentiate between these species and are less labor intensive. The PYR test could also be useful in this setting because E. meningoseptica is PYR+ and S. maltophilia is PYR . Alcaligenes faecalis and Myroides spp. can be difficult to distinguish because both species produce a fruity odor on young cultures. The PYR test, however, can rapidly identify A. faecalis (PYR ) from Myroides spp. (PYR+). Within the asaccharolytic, mobile, rapidly urease-positive NFGNR, the PYR test can be useful to separate Cupriavidus pauculus (PYR+) from Bordetella bronchiseptica and Oligella ureolytica (both PYR ). Pyrrolidonyl arylamidase test could therefore replace the reduction of nitrate, which is time consuming (Vandamme et al., 1999). Recent studies have shown that Achromobacter xylosoxidans, Achromobacter denitrificans, and Achromobacter piechaudii all belong to the same genus, Achromobacter, rather than the genus Alcaligenes to which they were
K.A. Bombicino et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 101 – 103
formerly assigned (Yabuuchi et al., 1998). A. faecalis remains in the genus Alcaligenes. It is noteworthy that all Achromobacter spp. hydrolyze PYR, whereas A. faecalis does not. Comamonas testosteroni, Pseudomonas alcaligenes, and A. piechaudii cannot be properly identified by using biochemical tests because they are inactive to most substrates (Kiska and Gilligan, 2003). Identifying the number and location of their flagella remains the only reliable method by which to distinguish these species (Kiska and Gilligan, 2003). C. testosteroni is characterized as having 2 or more polar flagella. P. alcaligenes has only 1 and A. piechaudii possesses peritrich flagellum (Kiska and Gilligan, 2003). If a determination of the characterization of flagellum cannot be made, special tests such as the assimilation of testosterone or the utilization of dlnorleucine must be performed (Kiska and Gilligan, 2003). Our research demonstrates that PYR is hydrolyzed by A. piechaudii but not C. testosteroni. Laffineur et al. (2002) have similarly described 14 strains of A. piechaudii that hydrolyze PYR and 15 strains of C. testosteroni that do not. As mentioned earlier, all our strains of P. alcaligenes were PYR in accordance with the results of Laffineur et al. (2002). Currently, Ochrobactrum and Brucella belong to the same family, Brucellaceae. Our research shows that the PYR test can aid in distinguishing between these genera (Table 1). Our results show that 18 isolates of Moraxella were all PYR (including Moraxella osloensis). However, De Baere et al. (2002) have shown that Moraxella atlantae, a new specie with similar biochemical features to M. osloensis, is PYR+. According to our results and those from De Baere et al., the PYR test could be a new method by which to differentiate between M. osloensis and M. atlantae. Considering the short time needed to perform the PYR test, its ease of handling, and its low cost, the test proposed in this study could be included as a complementary test in conventional schemes of identification of NFGNR. Acknowledgments This work was supported by the Proyect UBACYT B 080. References Barker PM, Wood RE, Gilligan PH (1997) Lung infection with Burkholderia gladioli in a child with cystic fibrosis: acute clinical and spirometric deterioration. Pediatr Pulmonol 24:123 – 125. Bloch KC, Nadarajah R, Jacobs R (1997) Chryseobacterium meningosepticum: an emerging pathogen among immunocompromised adults. Medicine 76:30 – 40. Chiu YS, Hsueh PR, Wu JJ, Ho SW, Hsieh WC, Luh KT (2000) Atypical Chryseobacterium meningosepticum and meningitis and sepsis in newborns and the immunocompromised. Taiwan Emerg Infect Dis 6:481 – 486. De Baere T, Muylaert A, Everaert E, Wauters G, Claeys G, Verschraegen G, Vaneechoutte M (2002) Bacteremia due to Moraxella atlantae in cancer patient. J Clin Microbiol 40:2693 – 2695. Ferna´ndez C, Wilhelm I, Andradas E, Gaspar C, Gomez J, Romero J, Mariano JA, Corral O, Rubio M, Elviro J, Fereres J (1996) Nosocomial outbreak
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