Journal of Microbiological Methods 98 (2014) 64–66
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
A rapid differentiation method for enteroinvasive Escherichia coli Swarmistha Devi Aribam, Jiro Hirota, Masahiro Kusumoto, Tomoyuki Harada, Kazumasa Shiraiwa, Yohsuke Ogawa, Yoshihiro Shimoji, Masahiro Eguchi ⁎ National Institute of Animal Health, NARO Tsukuba, Ibaraki 305-0856, Japan
a r t i c l e
i n f o
Article history: Received 19 November 2013 Accepted 22 November 2013 Available online 9 January 2014 Keywords: Enteroinvasive Escherichia coli Rapid detection Gentamicin invasion assay
a b s t r a c t Enteroinvasive Escherichia coli (EIEC) comprise 21 major serotypes defined by the presence of O and H antigens, and diagnosis depends on determining its invasive potential. Using HEp-2 cells infected with an EIEC strain, we developed a simple growth-dependent assay that differentiated EIEC strain from non-invasive strains 6 h after infection. © 2014 Elsevier B.V. All rights reserved.
Enteroinvasive Escherichia coli (EIEC) are unique amongst E. coli in that they have some biochemical properties of E. coli and a mode of infection similar to that of Shigella (Lan et al., 2001; Sansonetti et al., 1983; van den Beld and Reubsaet, 2012). They cause dysentery by a mechanism that involves the epithelial invasion of the intestine. Historically, the invasive potential of EIEC has been used for differentiating EIEC from other non-invasive E. coli strains via the Sereny test, which uses laboratory animals, or by specific tests such as real-time PCR or DNA microarray techniques that can identify invasion-associated proteins or genes (Beutin et al., 1997; Croxen et al., 2013). Most medical laboratories use conventional culture and microscopy diagnostic methods for the identification (de Boer et al., 2010; O'Leary et al., 2009). These methods are laborious and normally require 3 to 4 days to obtain the final result (de Boer et al., 2010). In this study, we developed an invasion assay that shortened the processing time, was simple to perform, and obtained results within approximately 6 h. The intracellular survival and replication of bacteria in eukaryotic cells after invasion have been widely studied with the gentamycin invasion assay, wherein extracellular bacteria are killed by gentamycin, while the intracellular bacteria are protected due to the inability of gentamycin to permeate the cells (Elsinghorst, 1994). Using this principle, we analyzed the growth of an invasive bacterial population within HEp-2 cells in order to develop a strategy for differentiating EIEC from other E. coli strains. We used three pathogenic strains in the experiments, EIEC O164:H− (GTC14235), enterohemorrhagic E. coli (EHEC) O157:H− (982243; Ogura et al., 2007), and enterotoxigenic E. coli (ETEC) O149:H− (E0092), and used the nonpathogenic E. coli DH5α strain as a control. With the exception of EIEC, the strains are
⁎ Corresponding author. Tel./fax: +81 29 838 7790. E-mail address: [email protected] (M. Eguchi). 0167-7012/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.11.012
noninvasive in nature. The bacteria were maintained in Luria Bertani (LB) broth at 37 °C without shaking. HEp-2 cells were utilized for the eukaryotic cell line and maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and supplemented with penicillin and streptomycin. For the study, 96-well tissue culture plates were seeded with 1 × 105 HEp-2 cells per well and grown to confluency overnight at 37 °C in a humidified 5% CO2 incubator. To determine the optimal multiplicity of infection (MOI) for EIEC, we infected HEp-2 cells with EIEC O164:H− and DH5α cells at MOI 50, MOI 10, and MOI 5 and analyzed their growth at different time points postinfection, with the DH5α cells acting as a control. The bacteria were pelleted at the bottom of the wells of a 96-well plate by centrifugation at 185 ×g for 10 min, and infected at 37 °C for 60 min. The HEp-2 cell pellets were washed with PBS, and the bacteria that remained extracellular were killed by incubation in 200 μL of medium supplemented with gentamycin (100 μg/mL) for 60 min at 37 °C. The adherent HEp-2 cells were washed with PBS and lysed with 30 μL of 1% Triton X-100 in PBS for 5 min. LB broth (200 μL) was added to each well, and the bacterial growth was measured with a microplate reader (PerkinElmer Inc., USA) at 595 nm at time points ranging from 0 to 22 h (Fig. 1A). The bacterial replication rate is expressed as the mean absorbance (A595) from 4 wells for each strain. The replication rate of the EIEC strain after infection at MOI 50 was significantly higher than that of the DH5α cells, and the increased replication was observable as early as 6 h postinfection (p = 0.0368, student t test). At this time, the mean A595 values for the EIEC strain at MOI 50, MOI 10, and MOI 5 were 0.084 ± 0.021, 0.063 ± 0.017, and 0.061 ± 0.006, respectively, indicating that 50 was the optimal MOI. The number of colony forming units (CFUs) of the invading EIEC cells was investigated by infecting HEp-2 cells with EIEC or DH5α cells at MOI 50. The CFU for the EIEC strain was 2.5 × 103 ± 0.898 × 103, much higher than that of DH5α (3.4 × 102 ± 1.1 × 102, Fig. 1B). To visualize
S.D. Aribam et al. / Journal of Microbiological Methods 98 (2014) 64–66
0.4
0.4
0.3
0.3
p < 0.0001
0.2
0.1
0.0
0.0 5
10
15
20
0.4 p < 0.0001
10
15
20
25
0
h
C
0.2
0.0 5
h
B
p < 0.0001
0.3
0.1 0
25
MOI 5
0.5
0.2
0.1 0
MOI 10
0.5
A595
MOI 50
A595
A595
A 0.5
65
5
10
15
20
25
h
EIEC O164
DH5
3
4 10
Mean CFU
p = 0.0159
3 103 2 103 1 103
5 H
EI
EC
D
O
16 4
0
Fig. 1. (A) Determination of the multiplicity of infection (MOI) for replicating enteroinvasive Escherichia coli (EIEC) in HEp-2 cells. Measurements are for the EIEC O164:H− (circle) and DH5α (square) cells after infection and treatment with gentamycin, and for untreated HEp-2 cells (inverted triangles). The mean ± standard error is shown for 4 wells for each strain. Two-way ANNOVA for bacterial growth from 0 h to 22 h was analyzed. (B) Bacterial invasion number measured as colony forming units (CFUs). The mean ± standard error is shown for 3 wells. A student t test was used to compare the CFU of each group. (C) Giemsa staining for bacteria internalized within eukaryotic HEp-2 cells. Polygonal-shaped Hep-2 cells with dark blue nuclei and scant cytoplasm are seen. Giemsa-stained EIEC cells appear as rods (arrows). Scale bar: 10 μm.
the invasion within HEp-2, we stained the cells with Giemsa for 30 min after incubation with gentamycin by washing the HEp-2 cells with PBS, drying, and fixation with 100% methanol. Microscopic observation showed that the EIEC invaded the HEp-2 cells (Fig. 1C). Together, these three simple experiments indicated that a rapid differentiation strategy could be designed using the principles of the gentamycin invasion assay. To assess the specificity of this method, we performed the growth analysis assay using the pathogenic EHEC O157 and ETEC O149 strains and found that after 6 h, the EIEC strain grew to significantly higher numbers than did the other strains (Fig. 2). This demonstrated that
p = 0.0078 p = 0.0062 p = 0.0231 0.20
A595
0.15
0.10
Acknowledgment We thank Dr Tetsuya Hayashi (University of Miyazaki) for providing the E. coli 982243 strain. We gratefully acknowledge the financial support provided by a grant from the National Institute of Animal Health 2012.
0.05
ed
References
ec t
5 H U
ni
nf
D
14 9 O ET
EC
O EC EH
EC
O
15
16
7
4
0.00
EI
the method described here can be used to differentiate EIEC from other noninvasive diarrheagenic E. coli (DEC). The differentiation of DEC is laborious, time-consuming, and costly (Fujioka et al., 2009). However, we have described an invasion assay that is simple, reliable, and cost-effective, and it provides results in as little as 6 h. This would make mass inspection of samples possible, ruling out the omission of EIEC from routine laboratory examinations for enteric pathogens. The virulence of EIEC is dependent on its ability to invade and multiply within the intestinal cells (Taylor et al., 1988). Because EIEC represents a group of DEC whose diagnosis often poses a problem, its prevalence is underestimated (Nataro and Kaper, 1998; Taylor et al., 1988). Therefore, developing a rapid and reliable differentiation method is essential. This in vitro infection study mimicked the conditions where EIEC invades and replicates within intestinal cells, leading to infection. The time factor and reliability of a diagnostic method used for making a treatment decision are crucial for curing a particular ailment, with the reliability being paramount. The technique developed in this study is based on the same principle as one of the most extensively used laboratory tests, the gentamycin invasion assay, and proved to be both economical and specific for intracellular pathogens.
−
−
Fig. 2. Bacterial invasion and replication analysis for the EIEC O164:H , EHEC O157:H , ETEC O149:H−, and DH5α strains 6 h after infecting HEp-2 cells and treating with gentamycin, and uninfected control. The mean ± standard error is shown for 4 wells. A student t test was used to compare the bacterial growth at 6 h.
Beutin, L., Gleier, K., Kontny, I., Echeverria, P., Scheutz, F., 1997. Origin and characteristics of enteroinvasive strains of Escherichia coli (EIEC) isolated in Germany. Epidemiol. Infect. 118, 199–205. Croxen, M.A., Law, R.J., Scholz, R., Keeney, K.M., Wlodarska, M., Finlay, B.B., 2013. Recent advances in understanding enteric pathogenic Escherichia coli. Clin. Microbiol. Rev. 26, 822–880.
66
S.D. Aribam et al. / Journal of Microbiological Methods 98 (2014) 64–66
de Boer, R.F., Ott, A., Kesztyüs, B., Kooistra-Smid, A.M.D., 2010. Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach. J. Clin. Microbiol. 48, 4140–4146. Elsinghorst, E.A., 1994. Measurement of invasion by gentamycin resistance. Methods Enzymol. 236, 405–420. Fujioka, M., Kasai, K., Miura, T., Sato, T., Otomo, Y., 2009. Rapid diagnostic method for the detection of diarrheagenic Escherichia coli by multiplex PCR. Jpn. J. Infect. Dis. 62, 476–480. Lan, R., Lumb, B., Ryan, D., Reeves, P.R., 2001. Molecular evolution of large virulence plasmid in Shigella clones and enteroinvasive Escherichia coli. Infect. Immun. 69, 6303–6309. Nataro, J.P., Kaper, J.B., 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11, 142–201. O'Leary, J., Corcoran, D., Lucey, B., 2009. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J. Clin. Microbiol. 47, 3449–3453.
Ogura, Y., Ooka, T., Asadulghani, Terajima, J.T., Nougayréde, J.-P., Kurokawa, K., Tashiro, K., Tobe, T., Nakayama, K., Kuhara, S., Oswald, E., Watanabe, H., Hayashi, T., 2007. Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes. Genome Biol. 8, R138. Sansonetti, P.J., d'Hauteville, H., Écobichon, C., Pourcel, C., 1983. Molecular comparison of virulence plasmids Shigella and enteroinvasive Escherichia coli. Ann. Inst. Pasteur Microbiol. 134, 295–318. Taylor, D.N., Echeverria, P., Sethabutr, O., Pitarangsi, C., Leksomboon, U., Blacklow, N.R., Rowe, B., Gross, R., Cross, J., 1988. Clinical and microbiologic features of Shigella and enteroinvasive Escherichia coli infections detected by DNA hybridization. J. Clin. Microbiol. 26, 1362–1366. van den Beld, M.J.C., Reubsaet, F.A.G., 2012. Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli. Eur. J. Clin. Microbiol. Infect. Dis. 31, 899–904.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
A rapid differentiation method for enteroinvasive Escherichia coli Swarmistha Devi Aribam, Jiro Hirota, Masahiro Kusumoto, Tomoyuki Harada, Kazumasa Shiraiwa, Yohsuke Ogawa, Yoshihiro Shimoji, Masahiro Eguchi ⁎ National Institute of Animal Health, NARO Tsukuba, Ibaraki 305-0856, Japan
a r t i c l e
i n f o
Article history: Received 19 November 2013 Accepted 22 November 2013 Available online 9 January 2014 Keywords: Enteroinvasive Escherichia coli Rapid detection Gentamicin invasion assay
a b s t r a c t Enteroinvasive Escherichia coli (EIEC) comprise 21 major serotypes defined by the presence of O and H antigens, and diagnosis depends on determining its invasive potential. Using HEp-2 cells infected with an EIEC strain, we developed a simple growth-dependent assay that differentiated EIEC strain from non-invasive strains 6 h after infection. © 2014 Elsevier B.V. All rights reserved.
Enteroinvasive Escherichia coli (EIEC) are unique amongst E. coli in that they have some biochemical properties of E. coli and a mode of infection similar to that of Shigella (Lan et al., 2001; Sansonetti et al., 1983; van den Beld and Reubsaet, 2012). They cause dysentery by a mechanism that involves the epithelial invasion of the intestine. Historically, the invasive potential of EIEC has been used for differentiating EIEC from other non-invasive E. coli strains via the Sereny test, which uses laboratory animals, or by specific tests such as real-time PCR or DNA microarray techniques that can identify invasion-associated proteins or genes (Beutin et al., 1997; Croxen et al., 2013). Most medical laboratories use conventional culture and microscopy diagnostic methods for the identification (de Boer et al., 2010; O'Leary et al., 2009). These methods are laborious and normally require 3 to 4 days to obtain the final result (de Boer et al., 2010). In this study, we developed an invasion assay that shortened the processing time, was simple to perform, and obtained results within approximately 6 h. The intracellular survival and replication of bacteria in eukaryotic cells after invasion have been widely studied with the gentamycin invasion assay, wherein extracellular bacteria are killed by gentamycin, while the intracellular bacteria are protected due to the inability of gentamycin to permeate the cells (Elsinghorst, 1994). Using this principle, we analyzed the growth of an invasive bacterial population within HEp-2 cells in order to develop a strategy for differentiating EIEC from other E. coli strains. We used three pathogenic strains in the experiments, EIEC O164:H− (GTC14235), enterohemorrhagic E. coli (EHEC) O157:H− (982243; Ogura et al., 2007), and enterotoxigenic E. coli (ETEC) O149:H− (E0092), and used the nonpathogenic E. coli DH5α strain as a control. With the exception of EIEC, the strains are
⁎ Corresponding author. Tel./fax: +81 29 838 7790. E-mail address: [email protected] (M. Eguchi). 0167-7012/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.11.012
noninvasive in nature. The bacteria were maintained in Luria Bertani (LB) broth at 37 °C without shaking. HEp-2 cells were utilized for the eukaryotic cell line and maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and supplemented with penicillin and streptomycin. For the study, 96-well tissue culture plates were seeded with 1 × 105 HEp-2 cells per well and grown to confluency overnight at 37 °C in a humidified 5% CO2 incubator. To determine the optimal multiplicity of infection (MOI) for EIEC, we infected HEp-2 cells with EIEC O164:H− and DH5α cells at MOI 50, MOI 10, and MOI 5 and analyzed their growth at different time points postinfection, with the DH5α cells acting as a control. The bacteria were pelleted at the bottom of the wells of a 96-well plate by centrifugation at 185 ×g for 10 min, and infected at 37 °C for 60 min. The HEp-2 cell pellets were washed with PBS, and the bacteria that remained extracellular were killed by incubation in 200 μL of medium supplemented with gentamycin (100 μg/mL) for 60 min at 37 °C. The adherent HEp-2 cells were washed with PBS and lysed with 30 μL of 1% Triton X-100 in PBS for 5 min. LB broth (200 μL) was added to each well, and the bacterial growth was measured with a microplate reader (PerkinElmer Inc., USA) at 595 nm at time points ranging from 0 to 22 h (Fig. 1A). The bacterial replication rate is expressed as the mean absorbance (A595) from 4 wells for each strain. The replication rate of the EIEC strain after infection at MOI 50 was significantly higher than that of the DH5α cells, and the increased replication was observable as early as 6 h postinfection (p = 0.0368, student t test). At this time, the mean A595 values for the EIEC strain at MOI 50, MOI 10, and MOI 5 were 0.084 ± 0.021, 0.063 ± 0.017, and 0.061 ± 0.006, respectively, indicating that 50 was the optimal MOI. The number of colony forming units (CFUs) of the invading EIEC cells was investigated by infecting HEp-2 cells with EIEC or DH5α cells at MOI 50. The CFU for the EIEC strain was 2.5 × 103 ± 0.898 × 103, much higher than that of DH5α (3.4 × 102 ± 1.1 × 102, Fig. 1B). To visualize
S.D. Aribam et al. / Journal of Microbiological Methods 98 (2014) 64–66
0.4
0.4
0.3
0.3
p < 0.0001
0.2
0.1
0.0
0.0 5
10
15
20
0.4 p < 0.0001
10
15
20
25
0
h
C
0.2
0.0 5
h
B
p < 0.0001
0.3
0.1 0
25
MOI 5
0.5
0.2
0.1 0
MOI 10
0.5
A595
MOI 50
A595
A595
A 0.5
65
5
10
15
20
25
h
EIEC O164
DH5
3
4 10
Mean CFU
p = 0.0159
3 103 2 103 1 103
5 H
EI
EC
D
O
16 4
0
Fig. 1. (A) Determination of the multiplicity of infection (MOI) for replicating enteroinvasive Escherichia coli (EIEC) in HEp-2 cells. Measurements are for the EIEC O164:H− (circle) and DH5α (square) cells after infection and treatment with gentamycin, and for untreated HEp-2 cells (inverted triangles). The mean ± standard error is shown for 4 wells for each strain. Two-way ANNOVA for bacterial growth from 0 h to 22 h was analyzed. (B) Bacterial invasion number measured as colony forming units (CFUs). The mean ± standard error is shown for 3 wells. A student t test was used to compare the CFU of each group. (C) Giemsa staining for bacteria internalized within eukaryotic HEp-2 cells. Polygonal-shaped Hep-2 cells with dark blue nuclei and scant cytoplasm are seen. Giemsa-stained EIEC cells appear as rods (arrows). Scale bar: 10 μm.
the invasion within HEp-2, we stained the cells with Giemsa for 30 min after incubation with gentamycin by washing the HEp-2 cells with PBS, drying, and fixation with 100% methanol. Microscopic observation showed that the EIEC invaded the HEp-2 cells (Fig. 1C). Together, these three simple experiments indicated that a rapid differentiation strategy could be designed using the principles of the gentamycin invasion assay. To assess the specificity of this method, we performed the growth analysis assay using the pathogenic EHEC O157 and ETEC O149 strains and found that after 6 h, the EIEC strain grew to significantly higher numbers than did the other strains (Fig. 2). This demonstrated that
p = 0.0078 p = 0.0062 p = 0.0231 0.20
A595
0.15
0.10
Acknowledgment We thank Dr Tetsuya Hayashi (University of Miyazaki) for providing the E. coli 982243 strain. We gratefully acknowledge the financial support provided by a grant from the National Institute of Animal Health 2012.
0.05
ed
References
ec t
5 H U
ni
nf
D
14 9 O ET
EC
O EC EH
EC
O
15
16
7
4
0.00
EI
the method described here can be used to differentiate EIEC from other noninvasive diarrheagenic E. coli (DEC). The differentiation of DEC is laborious, time-consuming, and costly (Fujioka et al., 2009). However, we have described an invasion assay that is simple, reliable, and cost-effective, and it provides results in as little as 6 h. This would make mass inspection of samples possible, ruling out the omission of EIEC from routine laboratory examinations for enteric pathogens. The virulence of EIEC is dependent on its ability to invade and multiply within the intestinal cells (Taylor et al., 1988). Because EIEC represents a group of DEC whose diagnosis often poses a problem, its prevalence is underestimated (Nataro and Kaper, 1998; Taylor et al., 1988). Therefore, developing a rapid and reliable differentiation method is essential. This in vitro infection study mimicked the conditions where EIEC invades and replicates within intestinal cells, leading to infection. The time factor and reliability of a diagnostic method used for making a treatment decision are crucial for curing a particular ailment, with the reliability being paramount. The technique developed in this study is based on the same principle as one of the most extensively used laboratory tests, the gentamycin invasion assay, and proved to be both economical and specific for intracellular pathogens.
−
−
Fig. 2. Bacterial invasion and replication analysis for the EIEC O164:H , EHEC O157:H , ETEC O149:H−, and DH5α strains 6 h after infecting HEp-2 cells and treating with gentamycin, and uninfected control. The mean ± standard error is shown for 4 wells. A student t test was used to compare the bacterial growth at 6 h.
Beutin, L., Gleier, K., Kontny, I., Echeverria, P., Scheutz, F., 1997. Origin and characteristics of enteroinvasive strains of Escherichia coli (EIEC) isolated in Germany. Epidemiol. Infect. 118, 199–205. Croxen, M.A., Law, R.J., Scholz, R., Keeney, K.M., Wlodarska, M., Finlay, B.B., 2013. Recent advances in understanding enteric pathogenic Escherichia coli. Clin. Microbiol. Rev. 26, 822–880.
66
S.D. Aribam et al. / Journal of Microbiological Methods 98 (2014) 64–66
de Boer, R.F., Ott, A., Kesztyüs, B., Kooistra-Smid, A.M.D., 2010. Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach. J. Clin. Microbiol. 48, 4140–4146. Elsinghorst, E.A., 1994. Measurement of invasion by gentamycin resistance. Methods Enzymol. 236, 405–420. Fujioka, M., Kasai, K., Miura, T., Sato, T., Otomo, Y., 2009. Rapid diagnostic method for the detection of diarrheagenic Escherichia coli by multiplex PCR. Jpn. J. Infect. Dis. 62, 476–480. Lan, R., Lumb, B., Ryan, D., Reeves, P.R., 2001. Molecular evolution of large virulence plasmid in Shigella clones and enteroinvasive Escherichia coli. Infect. Immun. 69, 6303–6309. Nataro, J.P., Kaper, J.B., 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11, 142–201. O'Leary, J., Corcoran, D., Lucey, B., 2009. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J. Clin. Microbiol. 47, 3449–3453.
Ogura, Y., Ooka, T., Asadulghani, Terajima, J.T., Nougayréde, J.-P., Kurokawa, K., Tashiro, K., Tobe, T., Nakayama, K., Kuhara, S., Oswald, E., Watanabe, H., Hayashi, T., 2007. Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes. Genome Biol. 8, R138. Sansonetti, P.J., d'Hauteville, H., Écobichon, C., Pourcel, C., 1983. Molecular comparison of virulence plasmids Shigella and enteroinvasive Escherichia coli. Ann. Inst. Pasteur Microbiol. 134, 295–318. Taylor, D.N., Echeverria, P., Sethabutr, O., Pitarangsi, C., Leksomboon, U., Blacklow, N.R., Rowe, B., Gross, R., Cross, J., 1988. Clinical and microbiologic features of Shigella and enteroinvasive Escherichia coli infections detected by DNA hybridization. J. Clin. Microbiol. 26, 1362–1366. van den Beld, M.J.C., Reubsaet, F.A.G., 2012. Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli. Eur. J. Clin. Microbiol. Infect. Dis. 31, 899–904.
