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Detection and Identification of Citrobacter sedlakii in the Czech Republic
Zbl. Bakt. 285, 389-396 (1997)
© Gustav Fischer Verlag, Jena
Detection and Identification of Citrobacter sedlakii in the Czech Republic EVA ALDOV A, JIiU SCHINDLER, t JIiU SOUREK, ALEXANDER NEMEC, and PAVLA URBAsKOV A National Institute of Public Health, Prague Srobarova 48, 10042 Received August 28, 1995 . Revision received March 25,1996 . Accepted April 26, 1996
Summary The biochemical characters of eight strains identified as Citrobacter sedlakii were inves tigated with the aid of six methods (tube tests, API 50 CH, API 20 E, MICROSCAN, BIO LOG, and CRYSTAL). All the strains were well defined on the basis of biochemcial prop erties investigated with the aid of laboratory-prepared tube tests. The results obtained by the identification kits could not be correctly interpreted. Commercial diagnostic kits should include the reference data necessary for the identification of new Citrobacter species. In the 8th edition of Bergey's Manual, the genus Citrobacter is subdivided into three species: C. freundii, C. diversus, and C. amalonaticus (6). Farmer and Kelly recognize in addition C. amalonaticus biogroup 1 which differs from C. amalonaticus in that it ferments raffinose (3). Brenner et al. classify the genus Citrobacter by 11 genomospe cies (1) wherein genomospecies is explained as "a species defined by DNA relatedness" Wayne et aI., 1987 (ref. 1). The designation Citrobacter sedlakii was assigned to geno mospecies 8. Shortly thereafter, a simple guide was published for the identification of species belonging to the genus Citrobacter, allowing to recognize new Citrobacter spe cies on the basis of a limited set of tests (5). Prior to the description of new Citrobacter species (1), such strains had to be includ ed under C. freundii, the only one which displayed a great variability in biochemical properties whereas the remaining three species gave unequivocal reactions in most bio chemical tests. The exact characterization of new Citrobacter species including C. sed lakii facilitates their identification. The aim of the study has been to describe 8 strains of Citrobacter sedlakii (geno mospecies 8) from our collection and to compare several commercial kits. Materials and Methods
Strains. The origin of the eight strains is given in Table 1. Six strains were isolated from stool specimens, two from diarrhoeic patients, the rest from preventive examinations car-
390
E. Aldova et al.
ried out routinely in hospitalized patients. Strain F 205 was isolated in southern Yemen as the prevailing finding in the stools from a patient suffering from chronic hemorrhagic diar rhoea. Tests for Entamoeba histolytiea, E. coli 0157, shigellae and salmonellae were nega tive; cultivation for detection of Campylobaeter jejuni was not performed. Strain F 204 was isolated from urine, strain F 203 from surface water, and F 208 from the pulp of a water melon cantaloup suspected as the source of a diarrhoeic affection. Type strains of all eleven genomospecies were examined along with the eight strains under study (Table 2).
Table 1. Origin of strains identified as Citrobaeter sedlakii NIPH NRL
CNCTC CDC No. No.
Place of occurence
Year
Material Diagnosis
F201 17590
5315
8337-93
Pardubice
1973
stool
Bronchopneumonia
F202 18376
5313
8333-93
C. Budejovice
1974
stool
Not stated
F203 18593
5314
8334-93
Plzen
1974
water
F204 22949
5034
C. Budejovice
1980
UrIne
Proteinuria
F205 28749
5035
Jemen-Aden
1989
stool
Hemorrhagic diarrhoea
5036
Ostrava
1989
stool
Diarrhoea
5037
B. Bystrica
1992
stool
Bronchitis
1989
Melon pulp
F206 28968 F207 30015 F208 28660
5158
Jemen-Aden
Table 2. List of Type strains: 11 genomospecies of the genus Citrobacter Genomospecies 1
2 3 4 5 6 7 8 9
10 11
Name
No.ATCC
No. CDC Atlanta
C.freundii C. koserii C. amalonatieus c.farmeri c.youngae C. braakii C. werkmanii C. sedlakii unnamed unnamed unnamed
8090 27156 25405 51112 29935 51113 51114 51115 51116 51117 51118
621-64 7613-67 9020-77 2991-81 460-61 80-58 876-58 4696-86 1843-73 4693-86 2970-59
Detection and Identification of Citrobacter sedlakii
391
Biochemical tests. Biochemical characters were tested with several assay and interpreta tion systems: a set of 47 conventional test-tube tests, the BIOLOG® kit (Biolog, USA), CRYSTAL BBL®(Becton Dickinson, USA), API 20 E and API 50 CH (Bio Merieux, France), and MICROSCAN (Baxter Diagnostic, USA). Test tube reactions were read upon incubation at 37 DC for seven days. The Voges Proskauer test, methyl red test, and nitrate reduction were carried out according to Cowan and Steel (2). The lysine decarboxylase, ornithine decarboxylase, and arginine dehydrolase tests were read after three days and positivity was verified with Nessler's reagent (2). Dia gnostic kits were implemented according to manufacturers' instructions and evaluated with the aid of their respective software. Susceptibility testing. MICs were determined by the broth micromethod (4) in 0.1 mL volumes of cation-adjusted Mueller-Hinton broth (Unipath Oxoid, United Kingdom). Anti microbial agents were obtained as laboratory powders of known potency from their respec tive manufacturers. For MIC determinations, suspensions equivalent to that of 0.5 Mc Far land standard were prepared by suspending growth from nutrient agar plates in 2 mL sa line. Suspensions were further diluted 1: 10 to obtain a final inoculum of 104 du/well. Mi crodilution plates were inoculated with an automatic MIC-2000 pin inoculator (Dynatech Laboratories, Inc., Alexandria, VA) and incubated overnight in ambient air at 37 DC. Stan dard quality control strains (4) were included in each run. MICs were determined as the lowest concentration of each antibiotic which inhibited visible growth. Numerical analysis. Data stored in a data base medium were processed with STATISTI CA software (StatSoft, 1994). As criterion of similarity the correlation coefficient (l-Pear son r) was chosen. The method (UPGMA) was used for cluster analysis (Fig. 1).
Results
1. Morphology of colonies. On incubation at 37 DC for 18 hours, most strains iden tified as C. sedlakii grew in small, smooth, translucent colonies of about 1 mm in dia meter. Also the type strain of genomospecies 8 (CDC 4696-85, ATCC 51115) had the same morphology. The colonies of most strains had a soggy consistence, only strain F 203 grew in colonies sticking to the agar surface. 2. Biochemical characteristics. In most strains of C. sedlak ii, the biochemical char acteristics found in tube tests were practically the same. They corresponded to the bio chemical profile of C. sedlakii strains described in the paper by Brenner et al. (1). The results of tests obtained with strains of C. sedlakii including the type strain were very similar in both the BIOLOG and CRYSTAL kit systems and differed markedly from type strains of other species. In the remaining systems, the phenotypical discrimination of the group of C. sedlakii strains studied was small (API 20 E), or a certain strain dif fered considerably from the other C. sedlakii strains (F 207 in tube tests and API 20 E, F 205 in API 50 CH, F 202 in MICROSCAN). Identical results of biochemical tests by systems were as follows: Conventional ar ray of 47 tube tests -F 202 and F 203; F 205 and F 206; CRYSTAL -F 205 and F 206; F 203 and F 208; API 50 CH -F 201 and F 206; F 202, F 203 and F 208; API 20 E -F 201, F 202 and CDC 4696-86; F 203, F 204, F 206 and F 208; MICRO SCAN -F 201 and CDC 4696-86, F 203, F 204, F 206 and F 207; F 205 and F 208 (Table 3). Strains differing markedly from other C. sedlakii strains as well as from the type strain in the respective system were: Set of 47 conventional tube tests -F 204 was the only non-motile one, F 207 was anaerogenic and nitrate-negative; API 50 CH -F 205 (negative fermentation tests with D-arabinose, dulcitol, L-fructose, and 2-ketogluco-
392
E. Aldova et al. b)
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Fig. 1. Phenograms according to phenotypical characteristic in C. sedlakii and type strains of the genus Citrobacter. Input data are results of a) an array of 47 conventional tube tests (Farmer et aI., 1991), and commercial kits b) API 50 CH, c) BIOLOG and d) CRYSTAL. As criterion of similarity, the correlation coefficient (l-Pearson r) was chosen. The un weighted pair group method with averages (UPGMA) was applied for cluster analysis. The C. youngae type strain was biochemically inactive in the CRYSTAL system, and the C. ama lonacticus type strain gave results of very low intensity in the BIOLOG system. Therefore, they are not included in the respective phenograms. GS = genomospecies.
Detection and Identification of Citrobacter sedlakii
393
Table 3. Strains identical in different identification kits System
Identical results
47 conventionel tube tests
F 202 and F 203, F 205 and F 206
API 50 CH
F20l, F202, F203 and F206, F207 and F208
API20E
F201, F202 and CDC 4699-86, F203, F204, F206 and F208
CRYSTAL
F 203 and F 208, F 205 and F 206
MICROSCAN
F 201 and CDC 4696-86, F 203, F 204, F 206 and F 207, Fl05 and F 208
nate; positive tests for fermentation of amygdalin and hydrolysis of aesculin); API 20 E -F 207 (negative for arginine dehydrolase and reduction of nitrates); MICROSCAN -F 202 (negative for malonate utilization). Biochemical characteristics of type strains. Type strains of eleven genomospecies dif fered from the group of eight strains under study in all of the diagnostic systems ap plied (tube tests, API 50, BIOLOG, and CRYSTAL). The C. amalonaticus type strain (CDC 9020-77) in the BIOLOG system gave results of very low intensity that could not be interpreted. The C. youngae type strain (CDC 460-61) was inactive biochemi cally in the CRYSTAL, API 20 E, and MICROSCAN systems. The Citrobacter sp. 11 type strain (CDC 2970-59) was inactive biochemically in the API 20 E and MICRO SCAN systems. Identification: All eight strains of C. sedlakii corresponded in their characteristics exactly to those presented in the guide given by O'Hara et al. (5). The 8 C. sedlakii strains were identified in each diagnostic system as follows: Conventional tube tests (3) - Citrobacter sp. (8 strains), BIOLOG kit - C. freundii (7 strains), unidentified (1), CRYSTAL kit - Citrobacter sp. (1), unidentified (7), MI CROSCAN kit - C. amalonaticus (1), Citrobacter sp. (4), E. agglomerans (2), uniden tified (1), API 20 E kit - Citrobacter sp. (6), unidentified (2) (Table 4). 3. Clustering according to biochemical characters. A cluster analysis was performed for the assessment of similarity in biochemical properties; dendrograms of similarity according to each of the identification systems are presented in Figure 1. Set of 47 conventional tube tests: A distinct cluster of six strains and the C. sedlakii type strain at the D (dissimilarity value) =0.085 level. The remaining two strains, F 204 and F 207, were separated at the D = 0.13 and D = 0.17 levels, respectively. The API 50 CH kit (49 tests): A distinct cluster of seven strains and the C. sedlakii type strain was formed at D = 0.06. Strain F 205 and type strains of other species sep arated at the D = 0.23 level. The BIOLOe kit (95 tests): A distinct cluster of all C. sedlakii strains at the D = 0.16 level. Type strains of other species of the genus Citrobacter separated from them at the D =0.27 level. The C. amalonaticus type strain (CDC 9020-77) has not been included. The CRYSTAL kit (32 tests): A distinct cluster of all C. sedlakii strains at D = 0.15. Type strains of other species of the genus Citrobacter separated from them at D = 0.24. The biochemically inactive type strain of C. youngae (CDC 460-61) is not included.
394
E. Aldova et al.
Table 4. Unidentified strains System
Identification
Strains
47 Test tube tests
Citrobacter species
8/8*
Citrobacter freundii
7/8
Citrobacter species
Not identified
1/8 7/8
Citrobacter amalonaticus Citrobacter species E. agglomerans
1/8 5/8 2/8
Citrobacter species
6/8 2/8
BIOLOG CRYSTAL MICROSCAN
API 20 E
Not identified
Not identified
118
* Denominator stands for the total number of strains.
The API 20 E kit (22 tests): At the D =0.14 level, there was a cluster of seven of the strains under study, the C. sedlakii type strain, C. koserii, and C. amalonaticus. Strain F 207 was separated from them at D =0.26, the other type strains, at the D =0.36 level. The MICROSCAN kit (24 tests): At the D = 0.11 level, there was a cluster of sev en strains under study and the C. sedlakii type strain. Strain F 205 and the type strains of other species separated at the D =0.21 level. 4. Antibiotic susceptibility. All strains of C. sedlakii were resistant to ampicillin, amoxicillin/clavulanate, ticarcillin, azlocillin, cefazolin and cefuroxime according to NCCLS criteria. Two strains (F 201, F 204) were resistant to cefoxitin (MIC 32 mg/L), to chloramphenicol (MIC 16 mg/L) and to tetracycline (MIC 4 mg/L). Strain F 204 iso lated from urine, was resistant also to trimethoprimlsulfamethoxazole (MIC 16 mg/L) and to oxolinic acid (MIC 16 mg/L). All strains were susceptible to all aminoglyco sides (kanamycin, gentamicin, tobramycin, netilmicin, amikacin), cefotaxime, ceftazi dime, cefepime, aztreonam and ciprofloxacin. Discussion Already in 1987, Sourek et al. (8,9) have encountered strains similar to C. divers us but differing by the fermentation of melibiose and growth in KCN, and drew atten tion to them at the 2nd Conference on Taxonomy and Automatic Identification (1988). Three strains sent by Sourek to CDC, Atlanta, were verified as genomospecies 8. In a relatively rich collection of the Citrobacter spp. strains, another five similar iso lates were found. Except for the description of their biochemical characters by conven tional tube tests which for some isolates differed from the strains described by Bren ner et al. (1) authors wanted to check which of the used commercial kits would be able to identify this newly described species. The examination of strains classified as C. sedlakii by six diagnostic systems has shown that the systems BIOLOG and CRYSTAL discriminated the strains as a separ ate cluster.
Detection and Identification of Citrobacter sedlakii
395
From the point of view of interpretation the results were homogeneous. The systems tested, except MICROSCAN, classified all strains correctly to the genus level. Species identification was naturally oriented by taxons included in the reference data. The identification of Citrobaeter species is to be considered as fitting more closely because it identifies the genus and not a species which is unknown to the system. For correct identification, the diagnostic systems need to supplement their reference data with the taxon of Citrabaeter sedlakii. Strains identified as C. sedlakii were in conformity with the biochemical profile of the type strain, with two exceptions: Strain F 204 differed from the other seven strains and the genomospecies 8 type strain in that it was non-motile and grew in mucous col onies. Strain F 207 did not reduce nitrate and was anaerogenic. All six strains of C. sedlakii described by Brenner et al. (1) were positive in both these tests. Strains of C. sedlakii which had been identical in their biochemical characteristics according to one system, differed in another one. Strain F 205 that was identical with other C. sedlakii strains in tube tests and the BIOLOG and CRYSTAL system, ap peared to be markedly different in the API 50 CH system. Strain F 207 differed in the API 20 E system and the test tube tests (anaerogenic and nitrate-negative). Strain F 202 differed in the MICROSCAN system. All of the C. sedlakii strains were resistant to ampicillin, ticarcillin and cefazolin. Most strains of C. freundii are resistant to the 1st generation of cephalosporins (ce phalotin and cefazolin), in contrast to C. diversus (7, 3). Thus, strains of C. sedlakii seem to be closer to C. freundii in their antibiotic susceptibility pattern. Six of our strains came from clinical material. Noteworthy was strain F 205 which had been isolated as the prevalent finding in an old man suffering from recurring chronic hemorrhagic diarrhea with no other pathogen found (cultivation for Campyla baeter was not performed). Most probably, the clinical significance of C. sedlakii is not different from other citrobacters but its proper evaluation requires more strains to be studied. Acknowledgement. The authors are indebted to Dr. D.]. Brenner of CDC Atlanta for kindly identifying older strains and for supplying type strains of all genomospecies of the genus Citrobacter. This study has been carried out with a financial support of IGA MZ CR, assignment No. 1628-3.
References
1. Brenner, D.J., P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageran, and C. F. Riddle: Classification of Citro bacteria by DNA hybridization: designation of Citrobac ter farmeri sp. nov., Citrobacter youngae sp. nov., Citrabacter braakii, sp. nov., Citrobac ter werkmanii, sp. nov., Citrobacter sedlakii sp. nov., and three unnamed Citrobacter ge nomospecies. J. Syst. Bacteriol. 43 (1993) 645-658 2. Cowan, S. T.: Cowan and Steel's Manual for the identification of medical bacteria. 2nd ed. Cambridge University Press 1974 3. Farmer III, J.J. and M. T. Kelly: Enterobacteriaceae. In: A. Balows, w.]. Hausler, Jr., K. L. Herrmann, H. O. Isenberg, and H.]. Shadomy (ed).: Manual of Clinical Microbiol ogy, 5th ed. Amer. Soc. Microbiology, Washington, D. C. 1991 4. National Committee for Clinical Laboratory Standards. Methods for dilution antimicro bial susceptibility tests for bacteria that grow aerobically - 3rd ed. Document M7-A3. National Committee for Clinical Laboratory Standards, Villanova, Pa. 1993 26
Zbl. Bakt. 28513
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E. Aldova et a!.
5. O'Hara, C. M., S. B. Roman, and]. M. Miller: Ability of commercial identification sy stems to identify newly recognized species of Citrobacter. J. Clin. Microbio!' 33 (1995) 242-245 6. Sakazaki, R.: Genus IV. Citrobacter Werkman and Gillen 1932, 173, pp. 458-461. In: N. R. Krieg and]. C. Holt (ed.): Bergey's Manual of Systematic Bacteriology, Vo!' 1, Wil liams and Wilkins, Baltimore/MD 21202, USA 1984 7. Southern, P. M. and M. K. Bagby: Antimicrobial susceptibility patterns (antibiograms) as an aid in differentiating Citrobacter species. Am. J. Clin. Patho!' 67 (1977) 187-189 8. Sourek,]. and E. Aldova: Importance of serological tests in the diagnosis of Citrobacter diversus and Citrobacter amalonaticus. System. App!. Microbio!. 11 (1988) 60-66 9. Sourek,]., J. Schindler, and E. Aldova: Biochemical characters of hydrogensulfide-nega tive Citrobacters. Abstracts, 2nd Conference on Taxonomy and Automatic Identification of Bacteria, Prague, Czechoslovakia, June 29-July 3 1987
Dr. Eva Aldova, National Institute of Public Health, Srobarova 48, 10042 Praha 10, Czech Republic
© Gustav Fischer Verlag, Jena
Detection and Identification of Citrobacter sedlakii in the Czech Republic EVA ALDOV A, JIiU SCHINDLER, t JIiU SOUREK, ALEXANDER NEMEC, and PAVLA URBAsKOV A National Institute of Public Health, Prague Srobarova 48, 10042 Received August 28, 1995 . Revision received March 25,1996 . Accepted April 26, 1996
Summary The biochemical characters of eight strains identified as Citrobacter sedlakii were inves tigated with the aid of six methods (tube tests, API 50 CH, API 20 E, MICROSCAN, BIO LOG, and CRYSTAL). All the strains were well defined on the basis of biochemcial prop erties investigated with the aid of laboratory-prepared tube tests. The results obtained by the identification kits could not be correctly interpreted. Commercial diagnostic kits should include the reference data necessary for the identification of new Citrobacter species. In the 8th edition of Bergey's Manual, the genus Citrobacter is subdivided into three species: C. freundii, C. diversus, and C. amalonaticus (6). Farmer and Kelly recognize in addition C. amalonaticus biogroup 1 which differs from C. amalonaticus in that it ferments raffinose (3). Brenner et al. classify the genus Citrobacter by 11 genomospe cies (1) wherein genomospecies is explained as "a species defined by DNA relatedness" Wayne et aI., 1987 (ref. 1). The designation Citrobacter sedlakii was assigned to geno mospecies 8. Shortly thereafter, a simple guide was published for the identification of species belonging to the genus Citrobacter, allowing to recognize new Citrobacter spe cies on the basis of a limited set of tests (5). Prior to the description of new Citrobacter species (1), such strains had to be includ ed under C. freundii, the only one which displayed a great variability in biochemical properties whereas the remaining three species gave unequivocal reactions in most bio chemical tests. The exact characterization of new Citrobacter species including C. sed lakii facilitates their identification. The aim of the study has been to describe 8 strains of Citrobacter sedlakii (geno mospecies 8) from our collection and to compare several commercial kits. Materials and Methods
Strains. The origin of the eight strains is given in Table 1. Six strains were isolated from stool specimens, two from diarrhoeic patients, the rest from preventive examinations car-
390
E. Aldova et al.
ried out routinely in hospitalized patients. Strain F 205 was isolated in southern Yemen as the prevailing finding in the stools from a patient suffering from chronic hemorrhagic diar rhoea. Tests for Entamoeba histolytiea, E. coli 0157, shigellae and salmonellae were nega tive; cultivation for detection of Campylobaeter jejuni was not performed. Strain F 204 was isolated from urine, strain F 203 from surface water, and F 208 from the pulp of a water melon cantaloup suspected as the source of a diarrhoeic affection. Type strains of all eleven genomospecies were examined along with the eight strains under study (Table 2).
Table 1. Origin of strains identified as Citrobaeter sedlakii NIPH NRL
CNCTC CDC No. No.
Place of occurence
Year
Material Diagnosis
F201 17590
5315
8337-93
Pardubice
1973
stool
Bronchopneumonia
F202 18376
5313
8333-93
C. Budejovice
1974
stool
Not stated
F203 18593
5314
8334-93
Plzen
1974
water
F204 22949
5034
C. Budejovice
1980
UrIne
Proteinuria
F205 28749
5035
Jemen-Aden
1989
stool
Hemorrhagic diarrhoea
5036
Ostrava
1989
stool
Diarrhoea
5037
B. Bystrica
1992
stool
Bronchitis
1989
Melon pulp
F206 28968 F207 30015 F208 28660
5158
Jemen-Aden
Table 2. List of Type strains: 11 genomospecies of the genus Citrobacter Genomospecies 1
2 3 4 5 6 7 8 9
10 11
Name
No.ATCC
No. CDC Atlanta
C.freundii C. koserii C. amalonatieus c.farmeri c.youngae C. braakii C. werkmanii C. sedlakii unnamed unnamed unnamed
8090 27156 25405 51112 29935 51113 51114 51115 51116 51117 51118
621-64 7613-67 9020-77 2991-81 460-61 80-58 876-58 4696-86 1843-73 4693-86 2970-59
Detection and Identification of Citrobacter sedlakii
391
Biochemical tests. Biochemical characters were tested with several assay and interpreta tion systems: a set of 47 conventional test-tube tests, the BIOLOG® kit (Biolog, USA), CRYSTAL BBL®(Becton Dickinson, USA), API 20 E and API 50 CH (Bio Merieux, France), and MICROSCAN (Baxter Diagnostic, USA). Test tube reactions were read upon incubation at 37 DC for seven days. The Voges Proskauer test, methyl red test, and nitrate reduction were carried out according to Cowan and Steel (2). The lysine decarboxylase, ornithine decarboxylase, and arginine dehydrolase tests were read after three days and positivity was verified with Nessler's reagent (2). Dia gnostic kits were implemented according to manufacturers' instructions and evaluated with the aid of their respective software. Susceptibility testing. MICs were determined by the broth micromethod (4) in 0.1 mL volumes of cation-adjusted Mueller-Hinton broth (Unipath Oxoid, United Kingdom). Anti microbial agents were obtained as laboratory powders of known potency from their respec tive manufacturers. For MIC determinations, suspensions equivalent to that of 0.5 Mc Far land standard were prepared by suspending growth from nutrient agar plates in 2 mL sa line. Suspensions were further diluted 1: 10 to obtain a final inoculum of 104 du/well. Mi crodilution plates were inoculated with an automatic MIC-2000 pin inoculator (Dynatech Laboratories, Inc., Alexandria, VA) and incubated overnight in ambient air at 37 DC. Stan dard quality control strains (4) were included in each run. MICs were determined as the lowest concentration of each antibiotic which inhibited visible growth. Numerical analysis. Data stored in a data base medium were processed with STATISTI CA software (StatSoft, 1994). As criterion of similarity the correlation coefficient (l-Pear son r) was chosen. The method (UPGMA) was used for cluster analysis (Fig. 1).
Results
1. Morphology of colonies. On incubation at 37 DC for 18 hours, most strains iden tified as C. sedlakii grew in small, smooth, translucent colonies of about 1 mm in dia meter. Also the type strain of genomospecies 8 (CDC 4696-85, ATCC 51115) had the same morphology. The colonies of most strains had a soggy consistence, only strain F 203 grew in colonies sticking to the agar surface. 2. Biochemical characteristics. In most strains of C. sedlak ii, the biochemical char acteristics found in tube tests were practically the same. They corresponded to the bio chemical profile of C. sedlakii strains described in the paper by Brenner et al. (1). The results of tests obtained with strains of C. sedlakii including the type strain were very similar in both the BIOLOG and CRYSTAL kit systems and differed markedly from type strains of other species. In the remaining systems, the phenotypical discrimination of the group of C. sedlakii strains studied was small (API 20 E), or a certain strain dif fered considerably from the other C. sedlakii strains (F 207 in tube tests and API 20 E, F 205 in API 50 CH, F 202 in MICROSCAN). Identical results of biochemical tests by systems were as follows: Conventional ar ray of 47 tube tests -F 202 and F 203; F 205 and F 206; CRYSTAL -F 205 and F 206; F 203 and F 208; API 50 CH -F 201 and F 206; F 202, F 203 and F 208; API 20 E -F 201, F 202 and CDC 4696-86; F 203, F 204, F 206 and F 208; MICRO SCAN -F 201 and CDC 4696-86, F 203, F 204, F 206 and F 207; F 205 and F 208 (Table 3). Strains differing markedly from other C. sedlakii strains as well as from the type strain in the respective system were: Set of 47 conventional tube tests -F 204 was the only non-motile one, F 207 was anaerogenic and nitrate-negative; API 50 CH -F 205 (negative fermentation tests with D-arabinose, dulcitol, L-fructose, and 2-ketogluco-
392
E. Aldova et al. b)
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Fig. 1. Phenograms according to phenotypical characteristic in C. sedlakii and type strains of the genus Citrobacter. Input data are results of a) an array of 47 conventional tube tests (Farmer et aI., 1991), and commercial kits b) API 50 CH, c) BIOLOG and d) CRYSTAL. As criterion of similarity, the correlation coefficient (l-Pearson r) was chosen. The un weighted pair group method with averages (UPGMA) was applied for cluster analysis. The C. youngae type strain was biochemically inactive in the CRYSTAL system, and the C. ama lonacticus type strain gave results of very low intensity in the BIOLOG system. Therefore, they are not included in the respective phenograms. GS = genomospecies.
Detection and Identification of Citrobacter sedlakii
393
Table 3. Strains identical in different identification kits System
Identical results
47 conventionel tube tests
F 202 and F 203, F 205 and F 206
API 50 CH
F20l, F202, F203 and F206, F207 and F208
API20E
F201, F202 and CDC 4699-86, F203, F204, F206 and F208
CRYSTAL
F 203 and F 208, F 205 and F 206
MICROSCAN
F 201 and CDC 4696-86, F 203, F 204, F 206 and F 207, Fl05 and F 208
nate; positive tests for fermentation of amygdalin and hydrolysis of aesculin); API 20 E -F 207 (negative for arginine dehydrolase and reduction of nitrates); MICROSCAN -F 202 (negative for malonate utilization). Biochemical characteristics of type strains. Type strains of eleven genomospecies dif fered from the group of eight strains under study in all of the diagnostic systems ap plied (tube tests, API 50, BIOLOG, and CRYSTAL). The C. amalonaticus type strain (CDC 9020-77) in the BIOLOG system gave results of very low intensity that could not be interpreted. The C. youngae type strain (CDC 460-61) was inactive biochemi cally in the CRYSTAL, API 20 E, and MICROSCAN systems. The Citrobacter sp. 11 type strain (CDC 2970-59) was inactive biochemically in the API 20 E and MICRO SCAN systems. Identification: All eight strains of C. sedlakii corresponded in their characteristics exactly to those presented in the guide given by O'Hara et al. (5). The 8 C. sedlakii strains were identified in each diagnostic system as follows: Conventional tube tests (3) - Citrobacter sp. (8 strains), BIOLOG kit - C. freundii (7 strains), unidentified (1), CRYSTAL kit - Citrobacter sp. (1), unidentified (7), MI CROSCAN kit - C. amalonaticus (1), Citrobacter sp. (4), E. agglomerans (2), uniden tified (1), API 20 E kit - Citrobacter sp. (6), unidentified (2) (Table 4). 3. Clustering according to biochemical characters. A cluster analysis was performed for the assessment of similarity in biochemical properties; dendrograms of similarity according to each of the identification systems are presented in Figure 1. Set of 47 conventional tube tests: A distinct cluster of six strains and the C. sedlakii type strain at the D (dissimilarity value) =0.085 level. The remaining two strains, F 204 and F 207, were separated at the D = 0.13 and D = 0.17 levels, respectively. The API 50 CH kit (49 tests): A distinct cluster of seven strains and the C. sedlakii type strain was formed at D = 0.06. Strain F 205 and type strains of other species sep arated at the D = 0.23 level. The BIOLOe kit (95 tests): A distinct cluster of all C. sedlakii strains at the D = 0.16 level. Type strains of other species of the genus Citrobacter separated from them at the D =0.27 level. The C. amalonaticus type strain (CDC 9020-77) has not been included. The CRYSTAL kit (32 tests): A distinct cluster of all C. sedlakii strains at D = 0.15. Type strains of other species of the genus Citrobacter separated from them at D = 0.24. The biochemically inactive type strain of C. youngae (CDC 460-61) is not included.
394
E. Aldova et al.
Table 4. Unidentified strains System
Identification
Strains
47 Test tube tests
Citrobacter species
8/8*
Citrobacter freundii
7/8
Citrobacter species
Not identified
1/8 7/8
Citrobacter amalonaticus Citrobacter species E. agglomerans
1/8 5/8 2/8
Citrobacter species
6/8 2/8
BIOLOG CRYSTAL MICROSCAN
API 20 E
Not identified
Not identified
118
* Denominator stands for the total number of strains.
The API 20 E kit (22 tests): At the D =0.14 level, there was a cluster of seven of the strains under study, the C. sedlakii type strain, C. koserii, and C. amalonaticus. Strain F 207 was separated from them at D =0.26, the other type strains, at the D =0.36 level. The MICROSCAN kit (24 tests): At the D = 0.11 level, there was a cluster of sev en strains under study and the C. sedlakii type strain. Strain F 205 and the type strains of other species separated at the D =0.21 level. 4. Antibiotic susceptibility. All strains of C. sedlakii were resistant to ampicillin, amoxicillin/clavulanate, ticarcillin, azlocillin, cefazolin and cefuroxime according to NCCLS criteria. Two strains (F 201, F 204) were resistant to cefoxitin (MIC 32 mg/L), to chloramphenicol (MIC 16 mg/L) and to tetracycline (MIC 4 mg/L). Strain F 204 iso lated from urine, was resistant also to trimethoprimlsulfamethoxazole (MIC 16 mg/L) and to oxolinic acid (MIC 16 mg/L). All strains were susceptible to all aminoglyco sides (kanamycin, gentamicin, tobramycin, netilmicin, amikacin), cefotaxime, ceftazi dime, cefepime, aztreonam and ciprofloxacin. Discussion Already in 1987, Sourek et al. (8,9) have encountered strains similar to C. divers us but differing by the fermentation of melibiose and growth in KCN, and drew atten tion to them at the 2nd Conference on Taxonomy and Automatic Identification (1988). Three strains sent by Sourek to CDC, Atlanta, were verified as genomospecies 8. In a relatively rich collection of the Citrobacter spp. strains, another five similar iso lates were found. Except for the description of their biochemical characters by conven tional tube tests which for some isolates differed from the strains described by Bren ner et al. (1) authors wanted to check which of the used commercial kits would be able to identify this newly described species. The examination of strains classified as C. sedlakii by six diagnostic systems has shown that the systems BIOLOG and CRYSTAL discriminated the strains as a separ ate cluster.
Detection and Identification of Citrobacter sedlakii
395
From the point of view of interpretation the results were homogeneous. The systems tested, except MICROSCAN, classified all strains correctly to the genus level. Species identification was naturally oriented by taxons included in the reference data. The identification of Citrobaeter species is to be considered as fitting more closely because it identifies the genus and not a species which is unknown to the system. For correct identification, the diagnostic systems need to supplement their reference data with the taxon of Citrabaeter sedlakii. Strains identified as C. sedlakii were in conformity with the biochemical profile of the type strain, with two exceptions: Strain F 204 differed from the other seven strains and the genomospecies 8 type strain in that it was non-motile and grew in mucous col onies. Strain F 207 did not reduce nitrate and was anaerogenic. All six strains of C. sedlakii described by Brenner et al. (1) were positive in both these tests. Strains of C. sedlakii which had been identical in their biochemical characteristics according to one system, differed in another one. Strain F 205 that was identical with other C. sedlakii strains in tube tests and the BIOLOG and CRYSTAL system, ap peared to be markedly different in the API 50 CH system. Strain F 207 differed in the API 20 E system and the test tube tests (anaerogenic and nitrate-negative). Strain F 202 differed in the MICROSCAN system. All of the C. sedlakii strains were resistant to ampicillin, ticarcillin and cefazolin. Most strains of C. freundii are resistant to the 1st generation of cephalosporins (ce phalotin and cefazolin), in contrast to C. diversus (7, 3). Thus, strains of C. sedlakii seem to be closer to C. freundii in their antibiotic susceptibility pattern. Six of our strains came from clinical material. Noteworthy was strain F 205 which had been isolated as the prevalent finding in an old man suffering from recurring chronic hemorrhagic diarrhea with no other pathogen found (cultivation for Campyla baeter was not performed). Most probably, the clinical significance of C. sedlakii is not different from other citrobacters but its proper evaluation requires more strains to be studied. Acknowledgement. The authors are indebted to Dr. D.]. Brenner of CDC Atlanta for kindly identifying older strains and for supplying type strains of all genomospecies of the genus Citrobacter. This study has been carried out with a financial support of IGA MZ CR, assignment No. 1628-3.
References
1. Brenner, D.J., P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageran, and C. F. Riddle: Classification of Citro bacteria by DNA hybridization: designation of Citrobac ter farmeri sp. nov., Citrobacter youngae sp. nov., Citrabacter braakii, sp. nov., Citrobac ter werkmanii, sp. nov., Citrobacter sedlakii sp. nov., and three unnamed Citrobacter ge nomospecies. J. Syst. Bacteriol. 43 (1993) 645-658 2. Cowan, S. T.: Cowan and Steel's Manual for the identification of medical bacteria. 2nd ed. Cambridge University Press 1974 3. Farmer III, J.J. and M. T. Kelly: Enterobacteriaceae. In: A. Balows, w.]. Hausler, Jr., K. L. Herrmann, H. O. Isenberg, and H.]. Shadomy (ed).: Manual of Clinical Microbiol ogy, 5th ed. Amer. Soc. Microbiology, Washington, D. C. 1991 4. National Committee for Clinical Laboratory Standards. Methods for dilution antimicro bial susceptibility tests for bacteria that grow aerobically - 3rd ed. Document M7-A3. National Committee for Clinical Laboratory Standards, Villanova, Pa. 1993 26
Zbl. Bakt. 28513
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5. O'Hara, C. M., S. B. Roman, and]. M. Miller: Ability of commercial identification sy stems to identify newly recognized species of Citrobacter. J. Clin. Microbio!' 33 (1995) 242-245 6. Sakazaki, R.: Genus IV. Citrobacter Werkman and Gillen 1932, 173, pp. 458-461. In: N. R. Krieg and]. C. Holt (ed.): Bergey's Manual of Systematic Bacteriology, Vo!' 1, Wil liams and Wilkins, Baltimore/MD 21202, USA 1984 7. Southern, P. M. and M. K. Bagby: Antimicrobial susceptibility patterns (antibiograms) as an aid in differentiating Citrobacter species. Am. J. Clin. Patho!' 67 (1977) 187-189 8. Sourek,]. and E. Aldova: Importance of serological tests in the diagnosis of Citrobacter diversus and Citrobacter amalonaticus. System. App!. Microbio!. 11 (1988) 60-66 9. Sourek,]., J. Schindler, and E. Aldova: Biochemical characters of hydrogensulfide-nega tive Citrobacters. Abstracts, 2nd Conference on Taxonomy and Automatic Identification of Bacteria, Prague, Czechoslovakia, June 29-July 3 1987
Dr. Eva Aldova, National Institute of Public Health, Srobarova 48, 10042 Praha 10, Czech Republic