Misidentification of Escherichia albertii strains by ... · by using two commercial identification...
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1,* 2 1 1 1 Ivo Sedláček, Linda Grillová, Jitka Černohlávková, Eva Kroupová, Pavel Švec 1 Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic. 2 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. * Corresponding author: [email protected] Conclusions Our results confirmed that E. albertii, a newly emerging enteric human pathogen, is an enteric bacterium inhabiting seals, which implies possibility of zoonoses. The biotyping misidentified E. albertii isolates as atypical E. coli or E. coli inactive. These data clearly showed inconvenience of ENTEROtest 24 and Biolog GN2 kits to distinguish E. albertii from E. coli due to missing data of E. albertii in databases. Automated ribotyping are useful discriminative techniques in case of a doubtful biochemical identification and E. albertii suspicion. Sequencing of 16S rDNA or the whole cell protein analysis have been shown as essential techniques applicable for the reliable identification of E. albertii unless the biochemical data are included in commercial identification systems. Acknowledgements This work was supported by the project CZ.1.07/2.3.00/20.0183 and Czech Polar project LM2010009. References Huys, G., Cnockaert, M., Janda, J.M. and Swings, J. (2003). Escherichia albertii sp. nov., a diarrhoeagenic species isolated from stool specimens of Bangladeshi children. International Journal of Systematic and Evolutionary Microbiology 53: 807-810. Oaks, J.L., Besser, T.E., Walk, S.T., Gordon, D.M., Beckmen, K.B., Burek, K.A., Haldorson, G.J., Bradway, D.S., Ouellette, L., Rurangirwa, F.R., Davis, M.A., Dobbin, G. and Whittam, T.S. (2010): Escherichia albertii in wild and domestic birds. Emerging Infectious Diseases, 16: 638-646. Ooka, T., Seto, K., Kawano, K., Kobayashi, H., Etoh, Y., Ichihara, S., Kaneko, A., Isobe, J., Yamaguchi, K., Horikawa, K., Gomes, T.A.T., Linden, A., Bardiau, M., Mainil, J.G., Beutin, L., Ogura, Y. and Hayashi, T. (2012): Clinical significance of Escherichia albertii. Emerging Infectious Diseases, 18: 488-492. Sedláček, I., Grillová, L., Kroupová, E., Černohlávková, J., Šmajs, D. (2013). Isolation of huiman pathogen Escherichia albertii from faeces of seals (Leptonychotes weddelli) in James Ross Island, Antarctica. Czech Polar Reports, 3: 173-183. Material and methods A group of nine Escherichia strains isolated from randomly collected faeces of seals (Leptonychotes weddeli) in James Ross Island, Antarctica was analysed. The bacteria were identified to the species level by using two commercial identification systems, ENTEROtest 24 and by Biolog GN2 Micro Plate kits. Moreover, several conventional tube and plate tests (e.g. catalase, oxidase, OF test, gas from glucose, motility, beta glucuronidase etc.) were additionally carried out to confirm the genus determination. Automated ribotyping performed using the RiboPrinter system (DuPont Qualicon) and rep-PCR fingerprinting with the (GTG) primer were used for a more detailed characterization of the isolates. 5 T T Reference strains E. albertii and E. coli, including type strains (CCM 7160 and CCM 5172 ) were obtained from the Czech Collection of Microorganisms (http://www.sci.muni.cz/ccm/). Results The biotyping with the commercial kit ENTEROtest 24 identified six strains as E. coli and remaining 3 strains (P4652, P4653 and P4740) as atypical E. coli. Using Biolog GN2 kit all 3 strains previously identified as atypical E. coli were assigned as E. coli inactive (Table 1, Fig. 1, Fig. 2). In contrast to the E. coli species description, the isolates P4652, P4653 and P4740 revealed negative test results for motility, beta glucuronidase production and fermentation of sorbitol and melibiose. Genotyping based on an automated ribotyping confirmed identification of six isolates as E. coli species but remaining three isolates P4652, P4653 and P4740 were not identified within the RiboPrinter Identification database. Cluster analysis of the ribotype patterns divided tested Escherichia spp. isolates into two clearly separate groups (Fig. 3). The three unidentified isolates P4652, P4653 and P4740 revealed almost identical ribotypes clearly separated from the remaining analysed Escherichia strains but closely similar to a ribotype of the E. albertii type strain. Further genotypic characterization using the rep-PCR fingerprinting (Fig. 4) revealed visually identical band patterns separating analysed strains into a few clusters. All three isolates P4652, P4653 and P4740 were separated from the remaining analysed Escherichia isolates and reference strains in full correspondence with the automatic ribotyping results. Sequencing of 16S rDNA, supplemented with bacteriocin detection and whole cell protein analysis revealed species-specific clusters and classified three above mentioned strains (P4652, P4653 and P4740) as true members of Escherichia albertii (Sedláček et al., 2013). Misidentification of Escherichia albertii strains by ENTEROtest 24 and Biolog GN2 Micro Plate Introduction and Purpose Escherichia spp. are very important enteric bacteria and five valid species are recognized in the genus Escherichia at present. Among them, some strains of Escherichia coli and majority of Escherichia albertii are recognized as important human's pathogens. The species E. albertii was described eleven years ago from diarrheal diseases (Huys et al., 2003) and afterwards was found as a probable emerging major human enteric pathogen because many E. albertii strains might have been misidentified as enterohemorrhagic (EHEC) or enteropathogenic (EPEC) E. coli Czech Collection of Microorganisms http://www.sci.muni.cz/ccm/ Fig 1. Identification result of strain P4652 obtained with ENTEROtest 24. Fig 2. Identification result of strain P4652 obtained with Biolog GN2 Plate. Fig 3. Dendrogram based on cluster analysis of ribotypes revealed by analysed and reference Escherichia spp. strains. Fig 4. Dendrogram based on cluster analysis of ( GTG) -PCR fingerprints revealed by analysed and 5 reference Escherichia spp. strains. Table 1. Identification results obtained using ENTEROtest 24 and Biolog GN2 Plate. CCM 4517 CCM 4787 CCM 7395 CCM 7929 P4656 P4657 P4652 P4740 P4653 T CCM 7160 P4665 P4722 P4664 T CCM 5172 P4733 CCM 4035 E. coli E. coli E. coli E. coli E. coli E. coli E. albertii E. albertii E. albertii E. albertii E. coli E. coli E. coli E. coli E. coli E. fergusonii 20 80 90 100 30 40 50 60 Similarity (%) 70 Plate Type: GN2 Strain Type : GN-ENT Strain Number : P 4652 Strain Name : Other : Data Input Mode : Reader 590/750 Filters Used : 6 / 5 Threshold Mode : Automatic: Color: 33/125 Number +/b/- Reactions : 26 / 8 / 62 Database To Search : MicroLog Data Base(s) Searched : C:\Program Files\Biolog\ML3_42_05\Databases\GN601.KID Key : <X>: positive; <X-: mismatched positive; X: negative; X+: mismatched negative {X}: borderline; -X: less than A1 well Color 1 2 3 4 5 6 7 8 9 10 11 12 A 0 2 < 200> 16 11 31 < 209> < 351> 0 < 364> -1 17 B -5 < 236> < 271> < 156> -2 < 209> 0 0 8 < 268> < 226> < 301> C -2 { 45} -3 3 -13 10 -10 < 274> 0 -1 < 219> 3 D { 108} -4 -1 0 < 275> -7 < 289> -6 11 9 18 1 E 0 -4 29 24 -2 < 209> -13 { 100} -14 4 -13 { 115} F < 201> -1 { 49} 4 1 -7 { 91} { 106} < 144> 1 { 112} 10 G 0 1 -2 14 -11 11 -15 < 586> 24 10 -5 3 H -3 < 161> < 229> < 135> -3 5 0 -1 < 284> < 186> < 259> < 337> => Species ID: Escherichia coli inactive <= Species PROB SIM DIST TYPE =>1 ) Escherichia coli inactive 89 0.665 3.84 GN-ENT 2 ) Escherichia coli 7 0.049 4.69 GN-ENT 3 ) Citrobacter farmeri 2 0.015 5.08 GN-ENT 4 ) Salmonella gp 6 (indica) 1 0.004 5.52 GN-ENT 5 ) Citrobacter braakii 0 0.003 5.67 GN-ENT 6 ) Shigella sonnei 0 0.001 5.86 GN-ENT 7 ) Escherichia fergusonii 0 0.001 6.19 GN-ENT 8 ) Hafnia alvei 0 0.000 6.47 GN-ENT 9 ) Citrobacter amalonaticus 0 0.000 6.56 GN-ENT 10 ) Escherichia coli (USP5-7085) 0 0.000 6.74 GN-ENT Other ) similarity, % 100 95 90 85 80 75 1 .0 0 1 .5 0 2 .0 0 2 .5 0 3.00 3 .50 5.00 6.00 7 .0 0 8.00 10.00 1 5.00 20 .0 0 30 .0 0 4 4 0. . 0 0 0 0 (Kb) P4652 P4653 P4740 T CCM 7160 P4656 P4657 P4664 P4665 P4722 CCM 4225 T CCM 5172 P4733 E. albertii E. coli ENTEROtest 24 Biolog GN2 plate P4652 E. coli atypical E. coli inactive P4653 E. coli atypical E. coli inactive P4656 E. coli E. coli P4657 E. coli E. coli P4664 E. coli Citrobacter sp. P4665 E. coli Citrobacter freundii P4722 E. coli E. coli P4733 E. coli E. coli P4740 E. coli atypical E. coli inactive Identification results Strain number
Transcript of Misidentification of Escherichia albertii strains by ... · by using two commercial identification...
1,* 2 1 1 1Ivo Sedláček, Linda Grillová, Jitka Černohlávková, Eva Kroupová, Pavel Švec
1Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
2Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
*Corresponding author: [email protected]
Conclusions
Our results confirmed that E. albertii, a newly emerging enteric human pathogen, is an enteric bacterium inhabiting seals, which implies possibility of zoonoses. The biotyping misidentified E. albertii isolates as atypical E. coli or E. coli inactive.
These data clearly showed inconvenience of ENTEROtest 24 and Biolog GN2 kits to distinguish E. albertii from E. coli due to missing data of E. albertii in databases.
Automated ribotyping are useful discriminative techniques in case of a doubtful biochemical identification and E. albertii suspicion.
Sequencing of 16S rDNA or the whole cell protein analysis have been shown as essential techniques applicable for the reliable identification of E. albertii unless the biochemical data are included in commercial identification systems.
Acknowledgements
This work was supported by the project CZ.1.07/2.3.00/20.0183 and Czech Polar project LM2010009.
References
Huys, G., Cnockaert, M., Janda, J.M. and Swings, J. (2003). Escherichia albertii sp. nov., a diarrhoeagenic species isolated from stool specimens of Bangladeshi children. International Journal of Systematic and Evolutionary Microbiology 53: 807-810.
Oaks, J.L., Besser, T.E., Walk, S.T., Gordon, D.M., Beckmen, K.B., Burek, K.A., Haldorson, G.J., Bradway, D.S., Ouellette, L., Rurangirwa, F.R., Davis, M.A., Dobbin, G. and Whittam, T.S. (2010): Escherichia albertii in wild and domestic birds. Emerging Infectious Diseases, 16: 638-646.
Ooka, T., Seto, K., Kawano, K., Kobayashi, H., Etoh, Y., Ichihara, S., Kaneko, A., Isobe, J., Yamaguchi, K., Horikawa, K., Gomes, T.A.T., Linden, A., Bardiau, M., Mainil, J.G., Beutin, L., Ogura, Y. and Hayashi, T. (2012): Clinical significance of Escherichia albertii. Emerging Infectious Diseases, 18: 488-492.
Sedláček, I., Grillová, L., Kroupová, E., Černohlávková, J., Šmajs, D. (2013). Isolation of huiman pathogen Escherichia albertii from faeces of seals (Leptonychotes weddelli) in James Ross Island, Antarctica. Czech Polar Reports, 3: 173-183.
Material and methods
A group of nine Escherichia strains isolated from randomly collected faeces of seals (Leptonychotes weddeli) in James Ross Island, Antarctica was analysed. The bacteria were identified to the species level by using two commercial identification systems, ENTEROtest 24 and by Biolog GN2 Micro Plate kits. Moreover, several conventional tube and plate tests (e.g. catalase, oxidase, OF test, gas from glucose, motility, beta glucuronidase etc.) were additionally carried out to confirm the genus determination. Automated ribotyping performed using the RiboPrinter system (DuPont Qualicon) and rep-PCR fingerprinting with the (GTG) primer were used for a more detailed characterization of the isolates. 5
T TReference strains E. albertii and E. coli, including type strains (CCM 7160 and CCM 5172 ) were obtained from the Czech Collection of Microorganisms (http://www.sci.muni.cz/ccm/).
Results
The biotyping with the commercial kit ENTEROtest 24 identified six strains as E. coli and remaining 3 strains (P4652, P4653 and P4740) as atypical E. coli. Using Biolog GN2 kit all 3 strains previously identified as atypical E. coli were assigned as E. coli inactive (Table 1, Fig. 1, Fig. 2). In contrast to the E. coli species description, the isolates P4652, P4653 and P4740 revealed negative test results for motility, beta glucuronidase production and fermentation of sorbitol and melibiose.
Genotyping based on an automated ribotyping confirmed identification of six isolates as E. coli species but remaining three isolates P4652, P4653 and P4740 were not identified within the RiboPrinter Identification database. Cluster analysis of the ribotype patterns divided tested Escherichia spp. isolates into two clearly separate groups (Fig. 3). The three unidentified isolates P4652, P4653 and P4740 revealed almost identical ribotypes clearly separated from the remaining analysed Escherichia strains but closely similar to a ribotype of the E. albertii type strain.
Further genotypic characterization using the rep-PCR fingerprinting (Fig. 4) revealed visually identical band patterns separating analysed strains into a few clusters. All three isolates P4652, P4653 and P4740 were separated from the remaining analysed Escherichia isolates and reference strains in full correspondence with the automatic ribotyping results.
Sequencing of 16S rDNA, supplemented with bacteriocin detection and whole cell protein analysis revealed species-specific clusters and classified three above mentioned strains (P4652, P4653 and P4740) as true members of Escherichia albertii (Sedláček et al., 2013).
Misidentification of Escherichia albertii strains by ENTEROtest 24 and Biolog GN2 Micro Plate
Introduction and Purpose
Escherichia spp. are very important enteric bacteria and five valid species are recognized in the genus Escherichia at present. Among them, some strains of Escherichia coli and majority of Escherichia albertii are recognized as important human's pathogens. The species E. albertii was described eleven years ago from diarrheal diseases (Huys et al., 2003) and afterwards was found as a probable emerging major human enteric pathogen because many E. albertii strains might have been misidentified as enterohemorrhagic (EHEC) or enteropathogenic (EPEC) E. coli
Czech Collection of Microorganismshttp://www.sci.muni.cz/ccm/
Fig 1. Identification result of strain P4652 obtained with ENTEROtest 24.
Fig 2. Identification result of strain P4652 obtained with Biolog GN2 Plate.
Fig 3. Dendrogram based on cluster analysis of ribotypes revealed by analysed and reference Escherichia spp. strains.
Fig 4. Dendrogram based on cluster analysis of (GTG) -PCR fingerprints revealed by analysed and 5
reference Escherichia spp. strains.
Table 1. Identification results obtained using ENTEROtest 24 and Biolog GN2 Plate.
CCM 4517
CCM 4787
CCM 7395
CCM 7929
P4656
P4657
P4652
P4740
P4653TCCM 7160
P4665
P4722
P4664TCCM 5172
P4733
CCM 4035
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. albertii
E. albertii
E. albertii
E. albertii
E. coli
E. coli
E. coli
E. coli
E. coli
E. fergusonii
20 80 90 10030 40 50 60
Similarity (%)
70
Plate
Type: GN2
Strain
Type : GN-ENT
Strain
Number : P 4652
Strain
Name :
Other :
Data Input Mode : Reader
590/750
Filters Used : 6 / 5
Threshold
Mode : Automatic: Color: 33/125
Number
+/b/-
Reactions : 26 / 8 / 62
Database
To Search : MicroLog
Data Base(s) Searched : C:\Program
Files\Biolog\ML3_42_05\Databases\GN601.KID
Key : <X>: positive; <X-: mismatched
positive: <X>: positive; <X-: mismatched
positive; X: negative; X+: mismatched
negative
{X}: borderline; -X: less than A1
well
Color 1 2 3 4 5 6 7 8 9
10
11
12
A
0
2 <
200>
16
11
31 <
209> <
351>
0 <
364>
-1
17
B
-5 <
236> <
271> <
156>
-2 <
209>
0
0
8 <
268> <
226> <
301>
C
-2 {
45}
-3
3
-13
10
-10 <
274>
0
-1 <
219>
3
D {
108}
-4
-1
0 <
275>
-7 <
289>
-6
11
9
18
1
E
0
-4
29
24
-2 <
209>
-13 {
100}
-14
4
-13 {
115}
F <
201>
-1 {
49}
4
1
-7 {
91} {
106} <
144>
1 {
112}
10
G
0
1
-2
14
-11
11
-15 <
586>
24
10
-5
3
H
-3 <
161> <
229> <
135>
-3
5
0
-1 <
284> <
186> <
259> <
337>
=> Species ID: Escherichia coli
inactive
<=
Species PROB SIM DIST TYPE
=>1
) Escherichia coli
inactive
89
0.665
3.84 GN-ENT
2
) Escherichia coli 7
0.049
4.69 GN-ENT
3
) Citrobacter farmeri 2
0.015
5.08 GN-ENT
4
) Salmonella
gp
6
(indica) 1
0.004
5.52 GN-ENT
5
) Citrobacter
braakii 0
0.003
5.67 GN-ENT
6
) Shigella sonnei 0
0.001
5.86 GN-ENT
7
) Escherichia fergusonii 0
0.001
6.19 GN-ENT
8
) Hafnia
alvei 0
0.000
6.47 GN-ENT
9
) Citrobacter
amalonaticus 0
0.000
6.56 GN-ENT
10
) Escherichia coli
(USP5-7085) 0
0.000
6.74 GN-ENT
Other
)
similarity, %
10
0
95
90
85
80
75
1.0
0
1.5
0
2.0
0
2.5
0
3.0
0
3.5
0
5.0
0
6.0
0
7.0
0
8.0
0
10.
00
15.0
0
20
.00
30
.00
44
0.
.
0
0
0
0
(Kb
)
P4652
P4653
P4740TCCM 7160
P4656
P4657
P4664
P4665
P4722
CCM 4225TCCM 5172
P4733
E. albertii
E. coli
ENTEROtest 24 Biolog GN2 plate
P4652 E. coli atypical E. coli inactive
P4653 E. coli atypical E. coli inactive
P4656 E. coli E. coli
P4657 E. coli E. coli
P4664 E. coli Citrobacter sp.
P4665 E. coli Citrobacter freundii
P4722 E. coli E. coli
P4733 E. coli E. coli
P4740 E. coli atypical E. coli inactive
Identification resultsStrain
number
