May 11, 1987 - Eighty-one Escherichia coli strains belonging to all known invasive O serogroups were ... Enteroinvasive Escherichia coli (EIEC), like Shigella.
JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1987, p. 2025-2027 0095-1137/87/102025-03$02.00/0 Copyright © 1987, American Society for Microbiology
Vol. 25, No. 10
DNA Probes for Identification of Enteroinvasive Escherichia coli TANIA A. T. GOMES,' M. REGINA F. TOLEDO,' LUIZ R. TRABULSI,l* PATRICK K. WOOD,2 AND J. GLENN MORRIS, JR.2 Department of Microbiology, Immunology, and Parasitology, Escola Paulista de Medicina, 04023 Sao Paulo, Brazil,l and Division of Geographic Medicine, Department of Medicine, and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 212012 Received 11 May 1987/Accepted 1 July 1987
Eighty-one Escherichia coli strains belonging to all known invasive O serogroups were tested with two distinct invasiveness probes (pMR17 and pSF55). Ali 54 Sereny test-positive strains and 5 strains that lost Sereny positivity during storage hybridized with both probes. Probe-positive strains carried a 120- to 140-megadalton plasmid, did not produce lysine decarboxylase, and, with the exception of certain serotypes, were nonmotile. Motile strains of serotype 0144:H25 were for the first time characterized as invasive by hybridization with the probes.
Enteroinvasive Escherichia coli (EIEC), like Shigella spp., causes dysentery due to the ability to penetrate, multiply within, and destroy the enterocytes of the colonic mucosa (4, 6). The penetration step in the invasion process is encoded by genes located on a 120- to 140-megadalton (MDa) plasmid (5, 12, 13) designated pInv (R. M. Silva, Ph.D. thesis, Escola Paulista de Medicina, Sao Paulo, Brazil, 1983). EIEC strains can be identified with relative accuracy by biochemical and serological methods (21). However, strains identified in this way must still be screened in the Sereny test (production of keratoconjunctivitis in the guinea pig [14]) to confirm that they are, indeed, virulent. Furthermore, the Sereny test is currently the only method available to detect EIEC strains which do not belong to known EIEC O serogroups. Hybridization techniques utilizing DNA probes have been shown to be useful in the detection of pathogenic E. coli (10, 11, 18) and appear to be practical for use in clinical and research laboratories. For detection of EIEC and Shigella spp., two different probes (pMR17 and pSF55) have been constructed; they identify distinct regions on pInv (22). pMR17 is a recombinant plasmid that carries a 17-kilobase EcoRI fragment from the pInv of a Shigellaflexneri serotype 5 strain (2, 15); pSF55 is a recombinant plasmid bearing a 2.5-kilobase HindIII fragment isolated from the pInv of an EIEC strain (17). In a recent study that included a limited number of EIEC strains, both probes were evaluated under identical laboratory conditions, and a highly significant association between the presence of pInv and hybridization with the probes was demonstrated (22). The current study was undertaken to further evaluate the usefulness of these two DNA probes in the identification of EIEC and to compare probe results with previously described serological and phenotypic markers for invasive strains. We studied 81 E. coli strains representing all known EIEC serogroups. These strains were from serogroups 028ac (nine strains), 029 (nine strains), 0112ac (three strains), 0124 (nine strains), 0136 (nine strains), 0143 (six strains), 0144 (seven strains), 0152 (nine strains), 0164 (nine strains), and 0167 (six strains) and from E. coli Sao Paulo 2 (five strains). Most of these strains were isolated from human diarrheic stools. Forty-eight strains had been isolated in the Microbi*
ology Laboratory at Escola Paulista de Medicina, Sao Paulo, Brazil; the remaining strains were provided by V. Prado (Chile), M. Toucas (France), M. Chashi and R. Sakazaki (Japan), and the Centers for Disease Control. Strains were identified or confirmed as E. coli by biochemical assays employing EPM (20) and MILi (19) media, and O and H antigens were typed by the method of Edwards and Ewing
(3).
Invasiveness was confirmed by screening strains for their ability to cause keratoconjunctivitis in guinea pigs; tests were performed as originally described by Sereny (14). Plasmids were extracted by an alkaline plasmid extraction technique (1) and sized by using marker plasmids of known molecular weights. As previously described (22), DNA probes were isolated by digestion with appropriate restriction enzymes, preparative gel electrophoresis, and electroelution and were labeled by nick translation with ra-32P]dATP (7). Strains were screened by colony hybridization for the presence of gene sequences homologous with those of the DNA probes; filters were prepared and hybridizations were conducted under high-stringency conditions as described by Moseley et al. (10). Among the 81 strains studied, 60 were Sereny positive at the time of isolation, 13 were Sereny negative, and 8 had not been previously tested in the Sereny test. Of the 60 strains that were originally Sereny positive, 6 were negative when retested; therefore, only 54 strains were Sereny positive when the present study started. Results are summarized in Table 1. All 54 Sereny-positive strains hybridized with both probes. These strains were nonmotile (with the exception of two 0124:H30 strains) and lysine decarboxylase negative and carried a plasmid of 120 to 140 MDa. Five of the six strains that had become Sereny negative prior to this study reacted with both probes and carried a 120- to 140-MDa plasmid. The remaining strain was probe negative and did not carry a large plasmid. All six of these strains were lysine decarboxylase negative. Two of the six strains (both serotype 0124:H30) were motile. It is already known that strains that become Sereny negative during storage may sometimes retain their ability to invade HeLa cells (16). We assayed our five Serenynegative, probe-positive strains for HeLa cell invasion. Methods were as described by Marques et al. (8), except that the infection and multiplication periods were 2 and 3 h,
Corresponding author. 2025
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NOTES
J. CLIN. MICROBIOL.
TABLE 1. Sereny test results, presence of plnv, and hybridization of 81 E. coli strains belonging to invasive O groups with pMR17 and pSF55 probes Sereny test result
+ + (-)a
-
Unknown a
No. of strains studied
54 6 13 8
O serogroups represented
All 28ac, 124 28ac, 29, 124, 136, 143, 152, 164 112ac, 136, 143, 144, 152, 164
No. of strains
With
Probe
pInv
positive
54 5 0
54 5 0
2
2
Strains originally Sereny positive that became negative prior to this study.
respectively. All five strains retained their in vitro ability to invade HeLa cells, although in two cases, the frequency of cell invasion was lower than seen with other invasive strains. When strains were plated on Congo red agar (9), stock cultures of all five strains had mixed populations containing colonies that were able to absorb the dye (Pcr+) and colonies that were nonabsorbent (Pcr-). As the genes responsible for dye absorption are likely to be correlated with invasiveness (9), we decided to purify one Pcr+ and one Pcr- colony of each of the five strains and compare them for invasive characteristics (Table 2). Except for one strain (C800912 Jap), all Pcr+ colonies had their positivity in the Sereny test restored, while the Pcrderivatives remained Sereny negative. All Pcr+ colonies were HeLa cell positive, while all but one Pcr- colony lost the ability to invade HeLa cells. pInv was present in Pcrcolonies, although in two instances, plasmid size was decreased, suggesting that there had been internal deletions in the plasmid. Except for strain 2835-81 CDC Pcr- (which carried a plasmid that had undergone a reduction in size), all Pcr- colonies retained reactivity with the 2.5-kilobase probe. Of the 13 originally Sereny-negative strains, 12 were motile or produced lysine decarboxylase or both (Table 1). The remaining strain (isolated from water) was nonmotile and lysine decarboxylase negative. None of these strains hybridized with either probe. Among the eight strains that had not been screened initially in the Sereny test, two reacted with the probes (Table 1). These two strains belonged to serotype 0144:H25 and were motile and lysine decarboxylase negative. By assaying these two strains in guinea pigs, we demonstrated that one strain was able to produce keratoconjunctivitis; the other strain, although Sereny negative, invaded HeLa cells. Both strains carried a plasmid of about 120 MDa. The remaining six strains were all lysine decarboxylase positive and motile (except for one) and did not carry a large plasmid or hybridize with the probes. Our results confirm the high sensitivity and specificity of the invasiveness probes for detecting EIEC strains. These DNA probes have been reported to give occasional falsepositive results (i.e., there are strains that are probe positive but Sereny negative), particularly when used to screen strains that have been stored for some time in the laboratory (22). Our data indicate that in most instances these probepositive, Sereny-negative "strains" contain a mixed population of cells, including some cells that retain Sereny positivity. Demonstration of a positive Sereny reaction requires some type of selection process (such as plating on Congo red agar) to identify those cells that retain the ability
TABLE 2. Characteristics of Pcr+ and Pcr- derivatives of five enteroinvasive E. coli strains that became Sereny negative during storage Response in: HybridiStrain
2835-81 CDC 2835-81 CDC C800912Jap C800912 Jap T4-66 SP T4-66 SP 1-72 lP 1-72 IP T5-68 SP T5-68 SP
Presence of plInvb
Sereny
HeLa cell test
nation with zaton it pSF55
+ -
+ +'
+ -
+ -
+
+
+
+
+ -
+ + + +
+ + + + -
+ +
+ + + +
+ +
+
Pcre
+ +
-
+
+ +
test
a Pcr, Phenotype strains on Congo red agar; -, no uptake of dye. b Molecular size of 120 to 140 MDa. 'Plasmids demonstrated a decrease in size.
-
+ +
+, uptake of Congo red dye;
to cause keratoconjunctivitis. At the same time, it is also clear that cells which carry pInv (and which hybridize with the DNA probes) can lose their Sereny positivity, with or without loss of the ability to invade HeLa cells; presumably, this is due to spontaneous loss of critical plasmid or chromosomal gene sequences (13). As shown in other studies and as demonstrated by our data, EIEC can be identified with some degree of precision by serotyping and screening strains for lysine decarboxylase activity and motility (21) or by a combination of these tests and plasmid analysis (15, 16). However, these techniques are limited in that they do not permit identification of EIEC outside of known invasive serogroups or of strains that do not fit previously identified phenotypic patterns. In this study, for example, by using the DNA probes, we identified two strains of serogroup 0144:H25 that had invasive properties; this is the first time that motile strains from serogroups other than 0124 have been shown to be invasive. DNA probes also have an advantage in that they can be used to screen E. coli strains (and possibly stool samples [18]) directly without the need for other laboratory tests. These observations suggest that the invasiveness probes provide the best means currently available for the identification of EIEC strains. This work was supported by grants from Financiadora de Estudos e Projetos and the Pan-American Health Organization. Plasmid pSF55 was generously provided by Pamela Small and Stanley Falkow and pMR17 was provided by Orntipa Sethabutr and Peter Echeverria. We thank Anita Wright for technical assistance with the project. LITERATURE CITED 1. Birnboim, H. C., and H. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2. Boileau, C. R., H. M. d'Hauteville, and P. J. Sansonetti. 1984. DNA hybridization technique to detect Shigella species and enteroinvasive Escherichia coli. J. Clin. Microbiol. 20:959-961. 3. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneap-
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15. Sethabutr, O., P. Echeverria, D. N. Taylor, T. Pal, and B. Rowe. 1985. DNA hybridization in the identification of enteroinvasive Escherichia coli and Shigella in children with dysentery, p. 350-356. In S. Tzipori (ed.), Infectious diarrhoea in the young. Excerpta Medica, Amsterdam. 16. Silva, R. M., M. R. F. Toledo, and L. R. Trabulsi. 1982. Correlation of invasiveness with plasmid in enteroinvasive strains of Escherichia coli. J. Infect. Dis. 146:706. 17. Small, P. L. C., and S. Falkow. 1986. Development of a DNA probe for the virulence plasmid of Shigella spp. and enteroinvasive Escherichia coli, p. 121-124. In L. Leive (ed.), Microbiology-1986. American Society for Microbiology, Washington, D.C. 18. Taylor, D. N., P. Echeverria, T. Pal, O. Sethabutr, S. Saiborisuth, S. Sricharmorn, B. Rowe, and J. Cross. 1986. The role of Shigella spp., enteroinvasive Escherichia coli, and other enteropathogens as causes of childhood dysentery in Thailand. J. Infect. Dis. 153:1132-1138. 19. Toledo, M. R. F., C. F. Fontes, and L. R. Trabulsi. 1982. MlLium meio para a realizacao dos testes de motilidade, indol e lisina descarboxilase. Rev. Microbiol. 13:230-235. 20. Toledo, M. R. F., and C. F. Fontes, L. R. Trabulsi. 1982. EPMmodificacao do meio de Rugai e Araujo para a relizacao simultanea dos testes de producao de gas a partir da glico se, H2S, urease e triptofano desaminase. Rev. Microbiol. 13:309315. 21. Toledo, M. R. F., and L. R. Trabuisi. 1983. Correlation between biochemical and serological characteristics of Escherichia coli and results of the Serény test. J. Clin. Microbiol. 17:419-421. 22. Wood, P. K., J. G. Morris, Jr., P. L. C. Small, O. Sethabutr, M. R. F. Toledo, L. Trabulsi, and J. B. Kaper. 1986. Comparison of DNA probes and the Sereny test for identification of invasive Shigella and Escherichia coli strains. J. Clin. Microbiol. 24:498-500.
