Abstract
In a population-based study of diarrhea in rural, northern Egypt, 60 Shigella flexneri strains were identified, of which 10 could not be definitively serotyped. Serological analysis with commercial reagents suggested that they were serotype 1, but the strains failed to react with subserotype 1a- or 1b-specific antibodies. All 10 strains reacted with MASF 1c, a monoclonal antibody specific for a provisional S. flexneri subserotype, 1c, first identified in Bangladesh and not previously detected outside of that region. Our results show that S. flexneri subserotype 1c is not unique to Bangladesh and that the inability to detect it may reflect both the limited use of suitable screening methods and the rarity of this subserotype.
Shigella spp. are a major bacterial cause of diarrhea, and their sole reservoir appears to be humans (reviewed in reference 5). Serological analysis of Shigella has long been used to characterize isolates for epidemiological and bacteriological purposes (1, 8, 9). Within this genus there are four recognized serogroups (species): A (S. dysenteriae), B (S. flexneri), C (S. boydii), and D (S. sonnei) (1, 6). S. flexneri is further subdivided into eight serotypes—1, 2, 3, 4, 5, 6, X, and Y—with eleven subserotypes—1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, and 5b (4, 7). This typing scheme exploits the polysaccharide O antigen found on the cellular surface (12–14).
In a community-based study conducted from 1991 to 1994 that investigated the bacterial causes of diarrhea in rural, northern Egypt, 60 S. flexneri strains were identified (11). Rectal or fecal swabs collected from individuals with diarrhea were inoculated into Cary-Blair transport medium and stored at 4°C for no more than 4 days prior to laboratory analysis. Specimens were cultured on Shigella-Salmonella, Hektoen-Enteric, xylose lysine desoxycholate (XLD), and MacConkey agars (Difco Laboratories, Detroit, Mich.). S. flexneri was preliminarily identified by Gram stain, colony morphology, lactose fermentation, and motility, as well as by results of general biochemical tests (6). All primary isolates appeared as translucent white colonies on MacConkey agar that were nonmotile and nonlactose fermenting. They showed alkaline-acid (K/A) reactions on either triple sugar-iron agar or Kliger’s iron agar and variable indole degradation, and were negative for both lysine and ornithine decarboxylation. Confirmatory identifications were performed with API-20E test kits used according to the instructions of the manufacturer (Bio-Merieux Vitek Inc., Hazelwood, Mo.). Serogroups and serotypes were determined by visual inspection of slide agglutination assays, with commercial antisera used as described by the manufacturer (Difco Laboratories) (10). Briefly, strains were subcultured on tryptic soy agar (Difco) and tested for agglutination on glass slides. Slides were divided into two sections with a wax pencil. A drop (∼20 μl) of 0.85% NaCl solution was placed in one section for use as a negative control, and a drop of the appropriate antiserum was placed in the other section. By using a sterile inoculation loop, a portion of the culture was emulsified with the NaCl solution. This process was repeated with the section of the slide containing the antiserum. The slide was then gently rocked, and relative agglutination was scored after 60 s according to the following scale: ++++, 100% agglutination of the cells; +++, ≥75% agglutination; ++, ≥50% agglutination; +, <50% agglutination; −, no agglutination detected. A strain was considered positive if its relative agglutination was +++ or greater.
Of the 60 S. flexneri strains identified, 10 could not be definitively serotyped with commercial reagents. These serologically atypical strains displayed conflicting agglutination patterns, reacting strongly with serotype 1-specific antisera but also weakly with serotype 4-specific antisera. These equivocal results may reflect the limitations of available commercial antibody reagents to reliably detect the full diversity of serologic variants of S. flexneri (8, 10).
In light of these contradictory results a panel of 10 mouse and rat monoclonal antibodies specific for the different type- and group-specific O-antigenic determinants of S. flexneri lipopolysaccharide was used in the slide agglutination assay for the further serological analysis of these 10 strains (Table 1) (2–4). Each strain reacted strongly with the serogroup B-specific antibody, MASF B, confirming that all were S. flexneri. Each strain reacted strongly with the serotype 4-specific antibody, MASF IV-2, but not with the type 1-specific antibody, MASF I, suggesting that all were serotype 4. However, none of the 10 strains agglutinated with the MASF reagents in a pattern specific for subserotypes 4a and 4b or for subserotypes 1a and 1b. Since a similar pattern of reactivity had been reported for the S. flexneri strain Y400 of the provisional subserotype, 1c (4, 4a, 15), we tested the 10 S. flexneri strains for reactivity with MASF 1c, a monoclonal antibody specific for this provisional subserotype (4a). All of these strains reacted strongly with the MASF 1c antibody (Table 2).
TABLE 1.
Key for S. flexneri subserotyping using the MASF panela
| Serotype | Type antigen specific
|
Group antigen specific
|
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| I | II | IV-2 | V | VI | B | Y-5 | 6 | 7,8 | IV-1 | |
| 1a | + | − | − | − | − | + | +/− | − | − | − |
| 1b | + | − | − | − | − | + | − | + | − | − |
| 1c | − | − | +/− | − | − | + | − | − | − | +/− |
| 2a | − | + | − | − | − | + | + | − | − | − |
| 2b | − | + | − | − | − | + | − | − | + | − |
| 3a | − | − | − | − | − | + | − | + | + | − |
| 3b | − | − | − | − | − | + | + | + | − | − |
| 4a | − | − | + | − | − | + | +/− | − | − | +/− |
| 4b | − | − | + | − | − | + | − | + | − | − |
| 5a | − | − | − | + | − | + | + | − | − | − |
| 5b | − | − | − | + | − | + | − | − | + | − |
| 6 | − | − | − | − | + | + | − | − | − | +/− |
| X | − | − | − | − | − | + | − | − | + | +/− |
| Y | − | − | − | − | − | + | + | − | − | + |
+, positive reaction; +/−, variable reaction (antigen may or may not be detected); −, negative reaction (no antigen detected).
TABLE 2.
Serological characteristics of 10 atypical S. flexneri strains from Egypt tested with the MASF panela
| Strain | Type antigen specific
|
Group antigen specific
|
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| I | II | IV-2 | V | VI | B | Y-5 | 6 | 7,8 | IV-1 | |
| ALX0559 | − | − | ++++ | − | − | ++++ | − | − | − | ++ |
| ALX1592 | − | − | ++++ | − | − | +++ | − | − | − | + |
| ALX1869 | − | − | ++++ | − | − | ++++ | − | − | − | − |
| ALX1929 | − | − | ++++ | − | − | ++++ | − | − | − | ++ |
| ALX1930 | − | − | ++++ | − | − | ++++ | − | − | − | ++ |
| ALX1938 | − | − | ++++ | − | − | ++++ | − | − | − | ++ |
| ALX2463 | − | − | +++ | − | − | +++ | − | − | − | − |
| ALX2539 | − | − | +++ | − | − | +++ | − | − | − | − |
| ALX3216 | − | − | ++++ | − | − | ++++ | − | − | − | ++ |
| ALX3326 | − | − | +++ | − | − | +++ | − | − | − | + |
++++, all of the cells agglutinated; +++, ≥75% of the cells agglutinated; ++, ≥50% of the cells agglutinated; +, <50% of the cells agglutinated; −, no agglutination detected.
Based on our results, these 10 strains belong to the provisional 1c subserotype, first described in Bangladesh (4, 15) in the late 1980s and not previously detected outside of that region. While the clinical and epidemiological significance of this observation remains unclear, our results indicate that this subserotype is not restricted to Bangladesh. We suspect that the failure to detect S. flexneri subserotype 1c in other regions may reflect both the limited use of appropriate screening methods and the rarity of this subserotype. In light of our findings, we recommend that reference laboratories performing serological analysis of S. flexneri consider expanding current screening methods to include detection of subserotype 1c, particularly for those isolates which give equivocal results.
Acknowledgments
This research was supported by the U.S. Naval Medical Research and Development Command (work unit no. 00101.EDX.3413 and 00101.HDX.3414).
REFERENCES
- 1.Boyd J S K. The antigenic structure of the mannitol fermenting group of dysentery bacilli. J Hyg. 1938;38:477–499. doi: 10.1017/s0022172400011335. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Carlin N I A, Lindberg A A. Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type I and type III:6,7,8 antigens, group 6 antigen, and a core epitope. Infect Immun. 1986;53:103–109. doi: 10.1128/iai.53.1.103-109.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Carlin N I A, Lindberg A A. Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3,4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 strains. Infect Immun. 1987;55:1412–1420. doi: 10.1128/iai.55.6.1412-1420.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Carlin N I A, Rahman M, Sack D A, Zaman A, Kay B, Lindberg A A. Use of monoclonal antibodies to type Shigella flexneri in Bangladesh. J Clin Microbiol. 1989;27:1163–1166. doi: 10.1128/jcm.27.6.1163-1166.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4a.Carlin, N. I. A. Unpublished results.
- 5.DuPont H L. Shigella. Infect Dis Clin N Am. 1988;2:599–605. [PubMed] [Google Scholar]
- 6.Ewing W H. Edwards and Ewing’s identification of Enterobacteriaceae. New York, N.Y: Elsevier Science Publishing Co., Inc.; 1986. pp. 169–181. [Google Scholar]
- 7.Ewing W H, Carpenter K P. Recommended designations for the subserotypes of Shigella flexneri. Int J Syst Bacteriol. 1966;16:145–149. [Google Scholar]
- 8.Grinberg L D. Fimbrial antigens of S. flexneri bacteria and the methods of their serological study. I J Hyg Epidemiol Microbiol Immunol. 1967;11:451–458. [PubMed] [Google Scholar]
- 9.Grinberg L D, Gordon M A. Further study of fimbrial antigens in Enterobacteriaceae. III. Distribution of the fimbrial phase in the genus Shigella. J Hyg Epidemiol Microbiol Immunol. 1971;15:293–297. [PubMed] [Google Scholar]
- 10.Lefebvre J, Gosselin F, Ismail J, Lorange M, Lior H, Woodward D. Evaluation of commercial antisera for Shigella serogrouping. J Clin Microbiol. 1995;33:1997–2001. doi: 10.1128/jcm.33.8.1997-2001.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Oyofo B A, El-Etr S H, Wasfy M O, Peruski L F, Jr, Kay B A, Mansour M, Campbell J R, Svennerholm A-M, Churilla A M, Murphy J R. Colonization factors of enterotoxigenic E. coli (ETEC) from residents of northern Egypt. Microbiol Res. 1995;150:1–8. doi: 10.1016/S0944-5013(11)80027-3. [DOI] [PubMed] [Google Scholar]
- 12.Seltmann G. Antigen structure of Shigellae. Chemical basis of Shigella flexneri antigens. Z Allg Mikrobiol. 1972;12:497–520. doi: 10.1002/jobm.3630120610. [DOI] [PubMed] [Google Scholar]
- 13.Simmons D A R. Immunochemistry of Shigella flexneri O-antigens: a study of structural and genetic aspects of the biosynthesis of cell-surface antigens. Bacteriol Rev. 1971;35:117–148. doi: 10.1128/br.35.2.117-148.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Simmons D A, Romanowska E. Structure and biology of Shigella flexneri O antigens. J Med Microbiol. 1987;23:289–302. doi: 10.1099/00222615-23-4-289. [DOI] [PubMed] [Google Scholar]
- 15.Wehler T, Carlin N I. Structural and immunochemical studies of the lipopolysaccharide from a new provisional serotype of Shigella flexneri. Eur J Biochem. 1988;176:471–476. doi: 10.1111/j.1432-1033.1988.tb14304.x. [DOI] [PubMed] [Google Scholar]