Evaluation Ofapi Coryne System for Identifying Coryneform Bacteria

Home , Arcanobacterium, Corynebacterium jeikeium, Corynebacterium striatum, Nocardia asteroides, Nocardia brasiliensis, Rhodococcus equi

756 Y Clin Pathol 1994;47:756-759 Evaluation of API Coryne system for identifying

coryneform bacteria J Clin Pathol: first published as 10.1136/jcp.47.8.756 on 1 August 1994. Downloaded from

A Soto, J Zapardiel, F Soriano

Abstract that are aerobe or facultatively aerobe, non- Aim-To identify rapidly and accurately spore forming organisms of the following gen- coryneform bacteria, using a commercial era: Corynebacterium, Listeria, Actinomyces, strip system. Arcanobacterium, Erysipelothrix, Oerskovia, Methods-Ninety eight strains of Cory- Brevibacterium and Rhodococcus. It also per- nebacterium species and 62 additional mits the identification of Gardnerella vaginalis strains belonging to genera Erysipelorix, which often has a diphtheroid appearance and Oerskovia, Rhodococcus, Actinomyces, a variable Gram stain. Archanobacterium, Gardnerella and We studied 160 organisms in total from dif- Listeria were studied. Bacteria were ferent species of the Corynebacterium genus, as identified using conventional biochemi- well as from other morphological related gen- cal tests and a commercial system (API- era or groups, some of them not included in Coryne, BioMerieux, France). Fresh rab- the API Coryne database. bit serum was added to fermentation tubes for Gardnerella vaginalis isolates. Results-One hundred and five out ofthe Methods 160 (65.7%) organisms studied were cor- The study was carried out on Gram positive rectly and completely identified by the bacilli belonging to the genera Coryne- API Coryne system. Thirty five (21.8%) bacterium, Erysipelothrix, Oerskovia, Rhodococcus, more were correctly identified with addi- Actinomyces, Arcanobacterium, Gardnerella and tional tests. Seventeen (10-6%) organisms Listeria included in the API Coryne database were not identified by the system and (table 1). We also studied some organisms three (1.9%) were misidentified. belonging to genera that occasionally present Conclusions-The system was a good a diphtheroid appearance and are not alternative for identification of coryne- included in the system database (table 2). A form organisms. When occasionally per- total of 160 organisms were evaluated, includ- http://jcp.bmj.com/ formed with some additional tests, this ing 42 reference strains. Clinical isolates were method permits reliable and rapid identi- obtained from blood (seven isolates), skin (17 fication of coryneform organisms com- isolates), urine (13 isolates), calcule (one iso- pared with conventional methods. late), drainage (one isolate), exudate (one isolate) and abscess (one isolate). The rest of the (7 Clin Pathol 1994;47:756-759) organisms came from stock collections. All the strains were kept at - 70°C before use and on September 27, 2021 by guest. Protected copyright. cultivated either aerobically or, if necessary, in Human infections by Coryneform sp are not a CO, atmosphere for 24 to 48 hours at 35°C only increasing but microbiologists are more on heart-infusion agar supplemented with 5% aware of their possible importance, mainly in sheep blood. high risk and immunosuppressed patients. The strains were identified using tech- Over the past two decades other Coryne- niques cited by Hollis and Weaver,"3 Bayston bacterium species, different from Coryne- and Higgins,'4 Coyle' and others.'5-'7 The fol- bacterium diphtheriae, have been found in lowing tests were used: Gram staining; colony severe infections in people.'-3 Bacteraemia, pigmentation; haemolysis; catalase produc- endocarditis, peritonitis, osteomyelitis and tion; urease utilisation, gelatin, hippurate and infection of the urinary and respiratory tracts aesculin hydrolysis; the Voges-Proskauer reac- are the most common infections associated tion; nitrate reduction; acid production from Department of with Corynebacterium sp.4 l glucose, maltose, mannitol, xylose, sucrose, Clinical Microbiology, These bacteria, which are common in clini- lactose and glycogen in fermentation broth, Fundacion Jimenez Diaz, Madrid, Spain cal samples, may be disregarded by microbiol- with the addition of 10% rabbit serum for A Soto ogists partly because they are considered Gardnerella vaginalis. Oxidation and fermenta- J Zapardiel non-pathogenic or "contaminant," but also tion tests were performed for C aquaticum F Soriano because there are no simple methods to iden- strains. Casein, xanthine, and tyrosine hydrol- Correspondence to: Dr A Soto, Department of tify them correctly in a routine laboratory.2 ysis were used to identify Nocardia spp. Most Infectious Diseases and Interest is increasing in the isolation and iden- of the strains were identified to species level Bacteriology, Royal Postgraduate Medical tification of these organisms, and this led us to using these tests but Oerskovia spp were only School, Hammersmith evaluate the API Coryne system by comparing identified to genus level. Hospital, Du Cane Road, London W12 ONN it with conventional identification methods. The API Coryne system consists of 20 Accepted for publication This system is a micromethod for the identifi- microtubes containing dehydrated substrates 9 February 1994 cation of Gram positive Coryneform organisms for the demonstration of 11 enzymatic Evaluation ofAPI Cotyne system 757

Table 1 Corynebacterium species and related genera studied included in the API Coryne The readings, except for the aesculin, ure- database ase, and gelatin tests, were done after adding Strains Total Clinical Stock Reference the appropriate reagents. The fermentation J Clin Pathol: first published as 10.1136/jcp.47.8.756 on 1 August 1994. Downloaded from Corynebacterium urealyticum 27 22 2 ATCC 43042, reactions were considered positive when they ATCC 43043, ATCC 43044 turned yellow. Identification was made using Corynebacteriumjeikeium 22 15 5 ATCC 43734, CCUG 24871 Corynebacterium striatum 5 4 ATCC 6940 the table provided by BioMerieux and, when Corynebacterium xerosis 5 4 ATCC 373 there were difficulties, by contacting the API Corynebacterium diphtheriae 2 2 Corynebacterium pseudotuberculosis 4 3 ATCC 19410 computer service. The interpretation was car- Corynebactenium bovis 2 ATCC 7715, CCUG 2705 ried out adding data on macroscopic and Corynebacterium pseudodiphtheriticum 5 4 ATCC 10700 Corynebacterum kutscheri 2 1 ATCC 15677 microscopic morphology, catalase, and Corynebacterium renale 2 1 ATCC 19412 haemolysis, as well as the numerical profile of Corynebacterium cystitidis 1 ATCC 29593 Corynebacterium pilosum 1 ATCC 29592 the API Coryne system. Corynebacterium minutissimum 2 ATCC 23348, CCUG 541 All the strains with a profile of "acceptable" Corynebacterium ulcerans 6 4 CCUG 16556, NCTC 7907 Corynebacterium aquaticum 3 2 ATCC 14665 identification or better were considered cor- Coryneform CDC group F, 3 1 2 rectly identified. Some additional tests were Coryneforn CDC group A4 2 2 Corynefonn CDC group G, 1 1 performed on those strains with a profile of Oerskovia sp 4 3 ATCC 25835 "good identification to genus" within the Erysipelothrix rhusiopathiae 4 4 Rhodococcus equi 8 1 7 group C renaleiC cystitidis and C aquaticuml Listeria monocytogenes 11 9 ATCC 19111,NCTC 11994 Coryneform CDC group A, according to the Listeria innocua 2 2 Listeria murrayi 1 ATCC 25401 manufacturer's protocol. These tests included Listeria grayi 1 ATCC 25400 growth in 6% sodium chloride, production of Listeria ivannovii 1 1 Listeria seeligeri 2 2 acid from trehalose or fructose, the Voges- Arcanobacterium haemolyticum 5 4 ATCC 9345 Proskauer reaction, the CAMP test, growth at Actinomyces pyogenes 4 3 ATCC 19411 GardnereUla vaginalis 4 1 2 ATCC 14018 a pH of 5-4 and Tween-80 hydrolysis. The strains with a profile of "low", "doubtful" or "insufficient discrimination" were considered unidentified. When the profile was good at Table 2 Other species studied not included in the API Coryne database species level but did not match the conven- Strains Total Clinical Stock Reference tional identification, it was considered incorrectly identified. Corynebacterium ammoniogenes 1 ATCC 6871 Corynebacterium callunae 1 ATCC 15991 Corynebacteriumflavescens 1 ATCC 10340 Corynebacterium vitarumen 1 ATCC 10234 Clavibacter michiganense 1 CCUG 580 Results Curtobacteriumflaccumfaciens 1 CCUG 23824 Of the 160 organisms studied using the API Rhodococcus rhodochrous 1 ATCC 11048 Propionibacterium avidum 2 1 ATCC 25577 system, 105 (65-7%) of them were correctly Propionibacterium granulosum 1 ATCC 25564 and completely identified in 24 hours to Rothia dentocariosa 2 1 ATCC 17931 Nocardia asteroides 2 species level, 35 more (21-8%) were incom- 1 ATCC 19247 http://jcp.bmj.com/ Nocardia brasiliensis 1 ATCC 19296 pletely identified but finally correctly identi- Nocardiafarcinica 1 ATCC 3318 Lactobacillus acidophilus 2 1 ATCC 832 fied with additional tests, 17 (10.6%) were not identified as the profile number did not correspond to any organism, and in three (1 9%) cases the strains were misidentified. activities (nitrate reduction, pyrazinamidase, Most strains (87 5%) were correctly identified alkaline in 24 hours or after additional pyrrolidonyl arylamidase, phos- tests (table 3). on September 27, 2021 by guest. Protected copyright. phatase, glucuronidase, ,B galactosidase, Six strains of Cjeikeium had a good profile at a glucosidase, N-Acetyl-f,glucosaminidase, genus level (profile number 2100324) and aesculin, urease and hydrolysis of gelatin) or may represent a less common biotype. the fermentation of eight sugars (glucose, Additional tests were required for correct ribose, xylose, mannitol, maltose, lactose, identification (growth in 6% sodium chloride sucrose and glycogen). The catalase test was and the production of acid from fructose). performed by adding 1 drop of hydrogen per- One strain of C striatum required the Voges- oxide (3%) to the aesculin or gelatin test. Proskauer test as an additional test to differ- After one minute the appearance of bubbles entiate it from Coryneform CDC group G2. corresponded to a positive reaction. None of the C aquaticum strains was correctly The inoculum was prepared in distilled and completely identified by the API system. water with a turbidity greater than 6 on the One of them required growth at 42°C and McFarland scale measured by comparing it oxidation or fermentation tests as additional with the turbidity control included in the kit. tests. The other two were misidentified as R This inoculum was used for enzymatic tests. equi and Listeria spp. The strains belonging to To carry out the fermentation tests, about 0 5 the group C renaleiC cystitidis are indistin- ml of bacterial suspension was transferred to guishable using the API system, as they have an ampoule containing 2 ml of GP medium, the same profile. Additional tests such as with the addition of 10% rabbit serum for growth in pH 4-5 and Tween-80 hydrolysis Gardnerella vaginalis. After homogenisation, were required to differentiate both of them. this new suspension was distributed into the All the Coryneform CDC groups were cor- fermentation tubes and overlayed the cupules rectly identified in 24 hours. with mineral oil. The same was done for the The species of "related genera" included in urea hydrolysis tube. The strip was then incu- this study were correctly identified. L monocy- bated at 37°C for 24 hours. Blood agar was togenes and L innocua required the CAMP test also incubated as a control. and haemolysis to distinguish between them, 758 Soto, Zapardiel, Soriano

Table 3 Distibution ofAPI Coryne identification Previous investigations have done similar Correcdy Incompletely studies testing commercial systems for the and but study and identification of coryneforms.1820 completely correctdy Not Incorrecdy J Clin Pathol: first published as 10.1136/jcp.47.8.756 on 1 August 1994. Downloaded from Organisms studied Tested identified identified identified identified The API Coryne is a commercial system for the identification of aerobe or facultative, Corynebactenium urealyticum 27 27 Corynebacteriumjeikeium 22 16 6 non-branched and non-spore forming Gram Corynebacterium striatum 5 4 1 positive diphtheroid rods. In the clinical labo- Corynebacterium xerosis 5 5 Corynebacterium diphtheriae 2 2 ratory the isolation of organisms that have Corynebacterium these characteristics but are not truly coryne- pseudotuberculosis 4 4 Corynebacterium bovis 2 2 forms is common and could be tested with the Corynebacterium API Coryne system by mistake. This is why pseudodiphtheriticum 5 5 Corynebacterium kutscheri 2 2 we included in this study 146 strains of Coynebacterium renale 2 2 Corynebacterium species and related genera as Corynebacterium cystitidis 1 1 Corynebacterium pilosum 1 1 well as 14 strains of seven genera that occa- Corynebacterium minutissimum 2 2 sionally could be mistaken for a diphtheroid. Corynebacterium ulkerans 6 6 Corynebacterium aquaticum 3 1 2 The API Coryne system was able to iden- Coryneform CDC group F, 3 1 2 tify correctly and completely 105 (65 7%) out Coryneform CDC group A4 2 2 Coryneforn CDC group G, 1 1 of the total of the organisms studied. Thirty Oerskovia sp 4 2 2 five (21'8%) more strains were correctly iden- Erysipelothrix rhusiopathiae 4 4 Rhodococcus equi 8 8 tified with the aid of the additional tests or the Listeria monocytogenes 11 11 API computer database. Accordingly, the API Listeria innocua 2 2 Listeria murrayi 1 1 Coryne system identified 140 out of the 160 Listeria grayi 1 1 (87-5%) strains studied. Listeria ivannovii 1 1 Listeria seeligeri 2 2 The number of unidentified micro-organ- Arcanobacterium haemolyticum 5 4 1 isms was 17 out of 160 (10'6%). Unidentified Actinomyces pyogenes 4 4 Gardnerella vaginalis 4 4 organisms belonged to species not contained Corynebacterium ammoniogenes 1 1 in the database, such as non-pathogenic or Corynebacterium callunae 1 1 Corynebacteriumflavescens 1 1 plant pathogenic species of Corynebacterium or Corynebacterium vitarumen 1 1 related genera and unrelated organisms that Clavibacter michiganense 1 1 Curtobacteriumflaccumfaciens 1 1 occasionally present a diphtheroid morphol- Rhodococcus rhodochrous 1 ogy. An R rhodochrous strain was misidentified Propionibacterium avidum 2 2 Propionibacterium granulosum 1 1 as R equi. Only three (1 '9%) strains out of the Rothia dentocariosa 2 2 160 studied were misidentified with the API Nocardia asteroides 2 2 Nocardia brasiliensis 1 1 Coryne system. Nocardiafarcinica 1 1 Our study shows that the API Coryne sys- Lactobacillus acidophilus 2 2 160 105(65-7%) 35(21-8%) 17(10-6%) 3(1-9%) tem produced a similar or slightly lower per- 140(87-5%) centage of correctly identified organisms 157(98-1%) compared with other investigations,2' 24 but it http://jcp.bmj.com/ has to be remembered that many species we studied are not included in the API Coryne database. In general, most Corynebacterium as the manufacturer recommends. All the G species and related genera were correctly vaginalis strains required 10% rabbit serum to identified to species level with or without

be added to the inoculum, to be properly additional tests, as reported before.2' 22 Several on September 27, 2021 by guest. Protected copyright. identified. Oerskovia sp required the API data- species required additional tests especially in base. those genera other than Corynebacterium, such We also included four non-pathogenic as all the Listeria species.2' species of corynebacteria and 14 strains The main difference of our work is the belonging to seven genera of aerobic, aerotol- higher number of species that are not erant, or branched bacteria that can present a included in the API Coryne database and diphtheroid appearance and are not included were tested to challenge the system. Only one in the API Coryne system database. out of the 18 strains of species not included in Seventeen of these strains corresponded to the API Coryne database was incorrectly profiles of "low", "doubtful" or "insufficient identified. The rest were not identified with discrimination" or non-existent profiles and the system, but if we had checked whether were regarded as not having been identified they were anaerobic or aerobic, the Gram by the system. Rhodococcus rhodochrous was morphology, and for acid fast bodies, they misidentified as R equi (profile number probably would have been. 2151004). It is very important to follow the manufacturer's recommendations in respect of the preparation and amount of inoculum needed. Discussion A low inoculum gives no definitive results in Over the past decades an increase in oppor- bacteria with a slow or difficult growth. This tunistic infections by Gram positive diph- happened to us with G vaginalis for which we theroids has aroused interest in their had to add 10% rabbit serum to the GP identification in clinical laboratories. Con- medium. ventional methods are slow and complex We recommend that respiration, micro- because of the number of tests that have to be scopic morphology (coryneform), spores; performed which in the end can only identify macroscopic appearances-colony size, pig- between 40%-60% of the isolates.' mentation, and haemolysis should all be Evaluation ofAPI Coryne system 759

checked for optimal use of the API Coryne 9 Maskell R, Pead L. Corynebacteria as urinary pathogens. _lInfect Dis, 1990;162:781-2. system. 10 Nadal D, Schwobel A, von Graevenitz A. Coryne- The fact that it was possible to identify 140 bacterium group D2 and urolithiasis in a boy with megacalycosis. J Infect 1988;16:245-7. (87 5%) or 157 (98-1%) out of the 160 studied 11 Soriano F, Aguado JM, Ponte C, Fernandez-Roblas R, J Clin Pathol: first published as 10.1136/jcp.47.8.756 on 1 August 1994. Downloaded from micro-organisms with the API Coryne system, Rodriguez-Tudela JL. Urinary tract infection caused by Corynebacterium group D2. Report of 82 cases and depending on the inclusion of the unidentified review of the literature. Rev Infect Dis 1990;12:1019-34. organisms, shows that the API Coryne system 12 Clarridge J. When, why, and how far should coryneforms be identified? Clin Microbiol Newslet 1986;8:32-4. is very reliable and accurate. Most of the 13 Hollis DG, Weaver RE. Gram-positive organisms: a guide to Gram positive bacilli isolated from clinical identification. Centers for Disease Control: Atlanta, Georgia, 1985. samples were identified in under 48 hours, 14 Bayston R, Higgins J. Biochemical and cultural characteris- compared with at least one week by standard tics of "JK" coryneforms. J Clin Pathol 1986;39:654-60. 15 Finegold SM, Baron EJ. Diagnostic microbiology. Princeton: methods, an important factor to bear in mind. The CV Mosby Company 1986. 16 Keddie RM, Bousfield IJ. Cell wall composition for the classification and identification of coryneform bacteria. This work was supported by a Fundaci6n Conchita Rabago In: Goodfellow M, ed. Microbiological classification and grant from Fundaci6n Jimenez Diaz and Fondo de identification. London: Academic Press, 1980:167-87. Investigaci6n Sanitaria (FISS) from the Spanish Ministry of 17 Krech T, Hollis DG. Coryne bacterium and related organ- Health. isms. In: Manual of clinical microbiology. Washington, DC: American Society for Microbiology, 1991:277-86. 18 Grasmick AE, Bruckner DA. Comparison of rapid identification method and conventional substrates for identifi- 1 Coyle M, Lipsky BA. Coryneform bacteria in infectious cation of Corynebacterium group JK isolates. Jf Clin diseases: clinical and laboratory aspects. Clin Microbiol Microbiol 1987;25:1111-2. Rev 1990;3:227-46. 19 Kellie MC, Smith ID, Anstey RJ, Thornley JH, Rennie 2 Marshall RJ, Johnson E. Corynebacteria: incidence among RP. Rapid identification of antibiotic-resistant coryne- samples submitted to a clinical laboratory for culture. bacteria with the API 20S system. Jf Clin Microbiol Med Lab Sci 1990;47:36-41. 1984;19:245-7. 3 Young V, Meyers W, Moody M, Schimpf S. The emer- 20 Tillotson G, Arora M, Robbins M, Holton J. Identification gence of coryneform bacteria as a cause of nosocomial of Corynebacterium jeikeium and Corynebacterium infections in compromised hosts. Am Jf Med 1981;70: CDC group D2 with the API 20 Strep system. EurJ Clin 646-50. Microbiol Infect Dis 1988;7:675-8. 4 Ena J, Berenguer J, Pelaez T, Bouza E. Endocarditis 21 Freney J, Duperron MT, Courtier C, Hansen W, Allard F, caused by Corynebacterium group D2. Jf Infect 1991;22: Boeufgras JM, et al. Evaluation of API Coryne in com- 95-6. parison with conventional methods for identifying 5 Jackman P, Pitcher DG, Pelczynska S, Bormnan P. coryneform bacteria. Jf Clin Microbiol 1991;29:38-4 1. Classification of corynebacteria associated with endo- 22 Gavin SE, Leonard RB, Briselden AM, Coyle MB. carditis (Group JK) as a Corynebacterium jeikeium Evaluation of the Rapid CORYNE identification system sp.nov. System AppI Microbiol 1987;9:83-90. for Corynebacterium species and other coryneforms. 6 Jacobs NF, Perlino CA. "Diphtheroid" pneumonia. South J J Clin Microbiol 1992;30:1692-5. Med 1979;72:475-6. 23 Peloux P, Caniaux I. Identification des corynebacteries et 7 Langs JC, de Briel D, Sauvage C, Blickle JF, Akel H. germes apparentes. Apport d'une nouvelle galerie Endocardite a Corynebacterium du groupe D2, a point d'identification: API CORYNE. Feuillets de Biologie de depart urinaire. Med Mal Infect 1977;5:293-5. 1990;23:27-34. 8 Marshall RJ, Routh K, MacGowan AP. Corynebacterium 24 Morrison JRA, Tillotson GS. Identification of CDC group D2 bacteraemia. J Clin Pathol 1987;40: Actinomyces (Corynebacterium) pyogenes with the API 813-4. 20 Strep system. J7 Clin Microbiol 1988;26: 1865-6. http://jcp.bmj.com/ on September 27, 2021 by guest. Protected copyright.