Survey of plasmid profiles of Shigella species isolated in Malaysia during

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1 World Journal of Microbiology & Biotechnology (2005) 21: Ó Springer 2005 DOI /s Survey of plasmid profiles of Shigella species isolated in Malaysia during C.H. Hoe 1, R.M. Yassin 2, Y.T. Koh 2 and K.L. Thong 1, * 1 Microbiology Division, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia 2 Bacteriological Unit, Institute for Medical Research, Kuala Lumpur, Malaysia *Author for correspondence: Tel.: , Fax: , thongkl@um.edu.my Received 2 March 2004; accepted 13 July 2004 Keywords: Antimicrobial susceptibility, Malaysia, multi-drug resistance, plasmid profile, Shigella Summary Plasmid profiling was used to characterize 219 strains of Shigella species isolated from sporadic cases of shigellosis in Malaysia during the period Heterogeneous plasmid patterns were observed in all Shigella spp. There was a correlation between plasmid patterns and serotypes of S. flexneri, S. dysenteriae and S. sonnei. Five common small plasmids (>20.0 kb) were observed in S. flexneri 1b and 2a, whereas six common small plasmids were found in serotype 3a. Some of these plasmids appeared to maintain their existence stably in each individual serotype. Plasmids of size and 4.20 kb were present only in S. flexneri 2a isolates, whereas the 4.40 kb plasmid was unique for serotype 3a. Large (>150 kb) or mid-range plasmid ( kb) was not observed from any S. flexneri 1b isolates. Eighty-nine percent of S. flexneri of various serotypes harboured the plasmid of 3.20 kb. All S. dysenteriae type 2 isolates harboured the 9.00 kb plasmid, while four common small plasmids were found in S. sonnei isolates. The 2.10 kb plasmid was only seen in S. sonnei. Streptomycin resistance in S. dysenteriae type 2 and multi-drug resistance in S. sonnei may be associated with the 9.00 and 14.8 kb plasmids, respectively. Plasmid profiling provided a further discrimination beyond serotyping and a useful alternative genotypic marker for differentiation of Shigella species. To the best of our knowledge, this is the first report on the plasmid prevalence of the Malaysian Shigella species. Introduction Shigellosis or bacillary dysentery is an acute diarrhoeal disease caused by members of the bacterial genus Shigella, which is composed of four species; S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. It is one of the major causes of morbidity and mortality in children with diarrhoea in developing countries (Chiou et al. 2001). Worldwide, the annual number of Shigella episodes was estimated to be million, of which million were in developing countries and 1.5 million in industrialized countries. Shigellosis is responsible for about 1.1 million deaths per year throughout the world, and two-thirds of the patients are children under 5 years of age (Kotloff et al. 1999). Shigellosis is endemic in Malaysia. From 1978 to 1997, 26,320 stool specimens were collected by the University of Malaya Medical Center (UMMC) from children aged less than 16 years with clinical diarrhoea. Among these, 386 isolates tested were positive for Shigella species, representing 1.4% of the 26,320 total stool specimens and 13% of 2986 isolates positive for bacterial pathogens (Lee & Puthucheary 2002). The actual incidence of shigellosis in this country could be higher, as the disease is self-limiting in adults and the affected individuals would usually go to different general practitioner clinics for medical care or employ self-medication. Thus, the vast majority of the cases are not notified. In recent years, traditional typing techniques based on phenotypic characteristics e.g. biotyping, serotyping, antibiotic susceptibility, are increasingly challenged by the use of DNA-based techniques. Approaches at molecular level have been used to assess the relatedness of closely related bacterial isolates. Plasmid profiling was found to be useful for the characterization or differentiation of bacterial strains harbouring plasmids, including Shigella (Chiou et al. 2001; Gebre-Yohannes & Drasar 1997; Litwin et al. 1991; Liu et al. 1995). Plasmid profiling is rapid, easy to perform and cost effective. Thus, the aim of this study was to evaluate this method for subtyping of our Malaysian Shigella spp. To the best of our knowledge, this is the first study to report the plasmid profile of a large collection of Malaysian Shigella spp.

2 272 C.H. Hoe et al. Material and methods Bacterial strains A total of 219 clinical isolates of Shigella spp., isolated during from sporadic cases of endemic shigellosis in different parts of Malaysia, were studied. Of the 219 clinical isolates, 146 (66.7%) were S. flexneri, 9 (4.1%) were S. dysenteriae, 4 (1.8%) were S. boydii, and 60 (27.4%) were S. sonnei. S. flexneri was represented by 9 serotypes: 1a (n ¼ 1), 1b (n ¼ 25), 2a (n ¼ 72), 3a (n ¼ 40), 3b (n ¼ 1), 4a (n ¼ 3), 4b (n ¼ 1), 6 (n ¼ 1), X (n ¼ 1), and Y (n ¼ 1). All S. dysenteriae isolates were of serotype 2, while each of the four isolates of S. boydii was of a different serotype (serotype 1, 2, 5, and 6). The Shigella spp. were identified by biochemical and serological tests (Mast Diagnostics, UK) at Institute for Medical Research, Kuala Lumpur, Malaysia. Repeated sub-culturing was avoided. Bacterial isolates were cultured in Luria Broth (LB) and stored in final concentration of 25% glycerol at )80 C. Figure 1. Plasmid profiles of representative strains of Shigella spp. Lanes 1 10, S. flexneri serotype 1a, 1b, 2a, 3a, 3b, 4a, 4b, 6, Y and X, respectively; lane 11, S. dysenteriae type 2; lanes 12-14, S. boydii type 1, 2, 5, respectively; lane 15, S. sonnei; lane 16, S. flexneri 2b ATCC 12022; lane 17, S. sonnei ATCC 11060; lane 18, E. coli 39R861; lane 19, E. coli V517. Antimicrobial susceptibility test Susceptibility was determined by the disk diffusion method (Bauer et al. 1966). The antimicrobial disks were obtained from Oxoid (UK), and the six antimicrobial agents tested included: ampicillin (AMP, 10 lg), tetracycline (TET, 30 lg), chloramphenicol (CHL, 30 lg), streptomycin (STR, 10 lg), kanamycin (KAN, 30 lg), and trimethoprim-sulfamethoxazole (SXT, 25 lg). Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC were used as quality control organisms. Zones of inhibition were recorded in mm and interpreted as sensitive, intermediate, or resistant according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS, 2000). Plasmid profile analysis Plasmid DNAs were extracted from 1.5 ml of overnight cell cultures, according to the method described by Olsen (1990) with some modifications. Approximately 2 ll of lysozyme (10 mg/ml) was added into lysis buffer prior to the 35 min of incubation. Extracted DNA was dissolved in 50 ll of pre-heated Tris-EDTA buffer (60 C). Plasmid DNAs were then separated by electrophoresis in 0.8% horizontal agarose gel at a constant voltage of 100 V for 6 h in cold 0.5 Tris-borate buffer. Plasmid extraction of all Shigella strains was repeated twice to determine the reproducibility. Only bright bands were used in the analysis and faint bands were interpreted as relaxed forms of brighter bands. Reference plasmids carried in E. coli 39R861 and E. coli V517 were used as molecular weight markers. Plasmid sizes were calculated by using Gel-Compar II software (Applied Maths, Kortrijk, Belgium) (Figure 1). Results Antimicrobial susceptibility Test Resistance rates of Shigella species to various antimicrobial agents are shown in Table 1. Twenty-eight percent of the S. flexneri isolates were susceptible to all six antimicrobial agents, whereas the remaining isolates were resistant to one to six antimicrobial agents. Fifty percent of the isolates were susceptible to all six antimicrobial agents, whereas the other 50% were resistant to one to four antimicrobial agents. Plasmid profile analysis All Shigella isolates were typeable and reproducible plasmid patterns were obtained when the analysis was repeated twice (data not shown). The isolates were divided into different groups based on the presence of small common plasmids in each serotype of Shigella spp.. The isolates in each group were then sub-divided into different profiles or patterns, according to the presence or absence of the less common plasmids and unique plasmids (Tables 2 4). Overall, heterogeneous plasmid patterns were found among the Malaysian Shigella spp. as the majority of the patterns were represented by only one isolate. Analysis of plasmid DNA showed that all Shigella spp. harboured 1 to 11 plasmids, ranging in size from 1.35 to 230 kb. A 3.20 kb plasmid was observed in 89% S. flexneri isolates. This plasmid was not found in other Shigella species (Table 2). S. flexneri 1b isolates displayed very different plasmid profiles compared to the other S. flexneri serotypes in this study. No large

3 Plasmid profiling of Shigella species 273 Table 1. Antimicrobial resistance rates of Shigella species. Antimicrobial agent Percent Shigella resistance S. flexneri (n = 146) S. dysenteriae (n =9) S. boydii a (n =4) S. sonnei (n = 60) Ampicillin Kanamycin Streptomycin Tetracycline Chloramphenicol Trimethoprim-sulfamethoxazole a Resistance rate of S. boydii was contributed by S. boydii type 1 only. All other serotypes of S. boydii isolates showed full sensitivity to all antimicrobial agents tested. (>150 kb) or mid-range ( kb) plasmid above the chromosomal band was observed. Plasmids of 4.50, 3.20, 2.65, 2.25, and 1.55 kb were commonly found in this serotype. The 1.55 and 2.65 kb plasmids were observed in all S. flexneri 1b isolates, while the 4.50 kb plasmid was found in 96% of the isolates (Table 2). Five common small plasmids were observed in S. flexneri 2a: 11.4 (67.1%), 9.00 (76.4%), 5.00 (86.1%), 4.20 (89%), and 3.20 kb (98.6%). Both plasmids, 11.4 kb and 4.20 kb were unique for S. flexneri 2a and were not found in other serotypes of Shigella species. Other less common plasmids observed were the 230 kb (17.8%), and 163 kb (38.4%) plasmids. Occasionally, unique plasmids of different sizes, ranging from 2.70 to 215 kb were also observed. The large 230 kb was absent from the S. flexneri 2a strains isolated in 1994 but present in 9.1% (1 of 11) of strains isolated in 1996 (Table 2). Six of the most common small plasmids found in S. flexneri 3a isolates were the 7.40, 6.50, 4.85, 4.40, 3.50, and 3.20 kb plasmids, with prevalence rates ranging from 42.5 to 92.5%. The 4.40 kb plasmid was only observed in S. flexneri 3a isolates. A mid-range plasmid of 90.5 kb was observed in 45% of the isolates. A greater diversity of plasmids was observed in S. flexneri 3a compared to that of S. flexneri 2a. Fourty-three plasmids of different sizes were found among the 40 strains of S. flexneri 3a. Unique plasmids ranging from 1.50 to 200 kb in size were observed. Similarly, the 230 kb plasmid was only present in the strains isolated after 1996 (Table 2). The 60 isolates of S. sonnei were subtyped into 10 groups comprising of 53 profiles, with 44 different plasmids of different sizes ranging from 1.55 to 230 kb (Table 3). The 163 kb plasmid, which was found in S. flexneri and S. dysenteriae, was absent in S. sonnei. Four of the most common small plasmids found were 10.4, 8.20, 2.65, and 2.10 kb, with the prevalence ranging from 31.7 to 60%. Most of the isolates (60%) harboured the 2.10 kb plasmid, which was absent in other Shigella serotypes (Table 3). All the nine S. dysenteriae type 2 isolates were subtyped into 4 groups with a predominant 9.00 kb plasmid (Table 4). This plasmid was also prevalent in S. flexneri 2a isolates but less frequent in S. flexneri 1b (2 of 25), and serotype 3a (5 of 39). Each of the S. boydii isolates had a distinct plasmid profile, comprising of 3 7 plasmids, ranging in size from 1.90 to 216 kb. Discussion Few published data are available on the association of the plasmid patterns of Shigella species with their serotypes. Haider et al. (1985) reported that 2.0 MDa (3.0 kb) and 2.7 MDa (4.1 kb) plasmids were found in 94 and 97% of 125 S. flexneri strains isolated in Dhaka, Bangladesh, respectively. S. flexneri 2a isolates only harboured two of these plasmids, whereas plasmids of various other sizes were found in the other serotypes. Litwin et al. (1991) reported similar finding in which a number of small plasmids ranging in size from 1.50 to 7.00 kb, were commonly seen in S. flexneri serotype 1, 2 and 4, including the 4.10 and 3.20 kb plasmid which were observed in 54 and 77% of the isolates, respectively. In this study, we also found several small plasmids common to S. flexneri 1b, 2a, and 3a. 44.0% of S. flexneri 1b isolates had plasmid profile F1.1b.P6. All 25 of the S. flexneri 1b isolates harboured the 1.55 (1.0 MDa) and 2.65 kb (1.75 MDa) plasmids, whereas 96% harboured the 4.50 kb (3.0 MDa) plasmid. Each of the plasmids, 3.20 (2.1 MDa) and 2.25 kb (1.5 MDa), was present in 84% of the isolates. These five plasmids were observed in both the susceptible and resistant strains. Thus, these plasmids might be considered as the core plasmids of serotype 1b, and might be useful in identification of the isolates. A similar result was reported by Talukder et al. (2003), in which plasmids of 2.8, 2.1, 1.8, and 1.0 MDa were found in all the 144 strains of S. flexneri serotype 1 isolated in Bangladesh. Interestingly, no large or mid-range plasmid was observed in the S. flexneri 1b isolates in this study. In contrast to our finding, Talukder et al. (2003), found that all the S. flexneri 1a and 1b strains and about 88% of the 1c strains in their study harboured the 140 MDa plasmid. Therefore, this unique characteristic could be useful to differentiate our Malaysian strains of S. flexneri 1b from isolates of other countries.

4 274 C.H. Hoe et al. Table 2. Plasmid profiles of Shigella flexneri. Group Common small plasmids (kb) Pattern Other plasmids present (kb) No. Strain S. flexneri 1b F1.1b P1 10.9, P2 9.00, P P P P6 11 F2.1b P7 2 F3.1b P8 2 F4.1b P F5.1b P F6.1b P11 1 S. flexneri 2a F1.2a P1 a 230, 163, 136, P2 230, 163, 159, P3 230, 163, P4 230, P P P7 163, P P P P P P P P15 18 F2.2a P16 1 F3.2a P17 230, P18 1 F4.2a P P20 3 F5.2a P21 230, 163, 80.0, 72.0, P22 230, 163, 80.0, P23 230, P24 230, P P P P28 136, P P30 2 F6.2a P F7.2a 3.20 P32 163, P P S. flexneri 3a F1.3a P1 230, 163, P2 230, 90.5, P3 230, 90.5, P4 230, P5 200, 120, P6 200, P7 163, 130, F2.3a P8 230, 163, P9 163, 90.5, 69.0, 11.3, 8.60, P10 163, F3.3a P11 163, F4.3a P12 163, F5.3a P13 163, F6.3a P14 230, 163, 86.5, F7.3a P15 230, 163, 90.5, 9.00, 5.30, 4.30, P16 163, 106, P17 159, P

5 Plasmid profiling of Shigella species 275 Table 2. (Continued.) Group Common small plasmids (kb) Pattern Other plasmids present (kb) No. Strain F8.3a P F9.3a P20 163, F10.3a P , F11.3a P22 230,163, 9.80, 9.00, P23 163, 66.0, 9.80, 9.15, 9.00, P24 163, 9.00, P25 159, F12.3a P26 163, 130, F13.3a P27 173, 95.5, 8.30, 6.70, 5.00, , 2.05, 1.50 F14.3a P28 163, 90.5, 80.0, F15.3a 3.20 P29 230, 163, 6.25, P30 230, 159, 6.25, P31 230, P32 200, 130, P33 163, 16.5, 13.0, 5.80, 5.30, P34 159, a The 230 kb plasmid shown in the table was not included into plasmid profiling due to its instability on long storage and subculture. S. flexneri 2a is the predominant serotype encountered in developing countries (60%) (WHO 2003), including Malaysia (Kotloff et al. 1999). In our study, S. flexneri 2a constituted about 33% of the total Shigella isolates. Plasmid profile analysis showed that five small plasmids ranging in size from 3.20 to 11.4 kb were commonly seen in S. flexneri 2a isolates. The 3.20 kb plasmid was observed in all the isolates, whereas the 4.20 and 5.00 kb plasmids were found in 88.9 and 86.1% of the isolates, respectively. These three plasmids were also found in both the susceptible and resistant strains and therefore, could be considered as the core plasmids of serotype 2a. The 11.4 and 4.20 kb plasmids were not observed in other serotypes of Shigella species. This finding suggests that these two plasmids could be used as potential extrachromosomal markers in identification of S. flexneri 2a isolates. Although Kotloff et al. (1999) cited that S. flexneri 3a is the third most prevalent serotype of S. flexneri isolated in developing countries, there were very few published reports on this serotype. In this study, six common small plasmids were observed among 40 strains of S. flexneri 3a isolates. About 92.3% of the isolates harboured the 3.20 kb plasmid, whereas the prevalence of the five other common plasmids was relatively low (42.5 to 52.5%). Apparently, the existence of these plasmids was not stable. The 4.40 kb plasmid was only present in S. flexneri 3a (47.5%). Nevertheless, due to its low prevalence the usefulness of this plasmid as an extra-chromosomal marker is limited. The low prevalence of these common plasmids, along with the presence of numerous plasmids of different sizes in the S. flexneri 3a isolates suggested that these strains have multiple unrelated origins. Most of the S. flexneri isolates in this study harboured the 3.20 kb plasmid (89%). Several previous studies also showed the high prevalence of this 3.20 kb plasmid in different serotypes of S. flexneri. The presence of the 3.20 kb plasmid in different serotypes of S. flexneri isolates from widely separated geographic regions such as Malaysia, Bangladesh (Talukder et al. 2003), Arizona (Litwin et al. 1991), and Taiwan (Chiou et al. 2001) suggests that it may carry important housekeeping genes that are important to the survival of this species. A correlation between resistant phenotypes with plasmids was described in a number of previous studies (Talukder et al. 2002, 2003), in which resistance to tetracycline, ampicillin, and trimethoprim-sulfamethoxazole in S. flexneri was associated with mid-range plasmids. However, such an association was not observed in this study. Twenty eight percent of S. flexneri 1b isolates were resistant to multiple antibiotics, including tetracycline, ampicillin, and trimethoprimsulfamethoxazole. As mentioned earlier, all isolates of serotype 1b harboured only small plasmids. Multi-drug resistant strains were also observed in other serotypes of S. flexneri isolates, with and without mid-range plasmids. Genes conferring antibiotic resistance traits could be located on chromosomal DNA or other small plasmids. This assumption is supported by the study of Casalino et al. (1994), who observed that genes for resistance to ampicillin, chloramphenicol, spectinomycin, and tetracycline formed a linkage group located on the chromosome of the strains of all serotypes of S. flexneri isolated in Somalia. However, further study is needed in order to confirm this assumption. Plasmid profiling appeared to be insufficient to characterize multiple antibiotic resistance in our Malaysian strains of S. flexneri. Hence, other molecular subtyping techniques such as PFGE, PCR based technologies, are currently being applied to further characterize these S. flexneri strains. S. dysenteriae type 2, also known as Schmitz bacillus, is rarely encountered worldwide. Nevertheless, it could be the predominant serotype of S. dysenteriae circulating in Malaysia, as more than 93% of the S. dysenteriae

6 276 C.H. Hoe et al. Table 3. Plasmid profiles of Shigella sonnei. Group Common small plasmids (kb) Pattern Other plasmids present (kb) No. strain S P1 117, 107, 11.5, 6.40, P2 83.0, 14.8, 11.5, P3 14.8, 11.5, S P4 a 230,155, 14.8, 11.5, P5 230, 88.0, 14.8, 11.5, P6 14.8, S P7 14.8, 11.5, P8 14.8, 3.60, 2.55, S P9 11.5, P P , 5.00, P , 2.50, P13 1 S P14 230, 129, 88.0, 13.3, 11.5, P15 129, 101, P , P S P18 230, 14.8, P , P P21 1 S P S P23 129, 80.0, S P24 230, 101, P25 200, 133, P26 102, P P P , P , P P , 2.50, P P , 5.00, 4.50, 2.50, P , 2.50, P P , P38 1 S10 P39 230, 129, 97.0, 14.4, P40 230, 129, 11.9, 11.2, 8.50, 7.30, P41 230, P42 230, P43 135, 11.9, P44 133, P45 129, 97.0, 5.00, P46 129, 11.9, P47 129, P48 129, 8.50, 4.80, 4.50, P49 129, P50 129, P P P , 2.50, a The 230 kb plasmid shown in the table was not included into plasmid profiling due to its instability on long storage and subculture. isolates submitted to the Institute for Medical Research, Malaysia during were serotype 2. Each of the S. dysenteriae type 2 isolates had a distinct plasmid pattern, with the 9.00 kb plasmid as its core plasmid. All these strains were isolated in Ipoh, Malaysia, and were resistant to streptomycin only. As resistance to streptomycin in S. dysenteriae is either chromosomal or smaller plasmid-mediated (Gebre-Yohannes & Drasar, 1990), there is a possibility that this resistance is linked to the 9.00 kb plasmid. The uniformity in antimicrobial resistance pattern, and geographical distribution, along with the presence of the 9.00 kb core plasmid might suggest a common source of infection. Numerous plasmids of different sizes were observed in 60 strains of S. sonnei isolates. Four of the most common plasmids (10.4, 8.20, 2.65, and 2.10 kb) were

7 Plasmid profiling of Shigella species 277 Table 4. Plasmid profiles of Shigella dysenteriae type 2. Group Common small plasmids (kb) Pattern Other plasmids present (kb) No. Strain D P1 a 230, P P P P5 1 D P6 216, 163, D P7 230, P D P9 163, 5.80, a The 230 kb plasmid shown in the table was not included into plasmid profiling due to its instability on long storage and subulture. observed at relatively low prevalence (31.7 to 60%). The 2.10 kb plasmid, which was found in 60% of S. sonnei isolates, was not observed in other Shigella species. Although previous studies (Lee et al. 2003) reported the association of multi-drug resistance in S. sonnei with a mid-range plasmid of 110 kb, the Malaysian multi-drug resistant (trimethoprim-sulfamethoxazole, streptomycin, tetracycline) strains of S. sonnei may be associated with the 14.8 kb small plasmid, as all the isolates that possessed this resistant phenotype harboured this plasmid. However, this plasmid was absent from other S. sonnei isolates that had additional resistance to ampicillin. The genes associated with virulence in Shigella are found on a large 230 kb plasmid, often referred as virulence plasmid or invasive plasmid. The size of the virulence plasmid ranged from 180 kb (S. sonnei) and 210 to 240 kb (S. flexneri) (Sasakawa 1995). It was noted that the 230 kb virulence plasmid was absent from all the Shigella strains isolated in 1994, and had a very low prevalence rate in strains isolated in 1996 compared to the later years ( ). This indicated that the 230 kb plasmid is unstable on long storage or subculture, as suggested by Litwin et al. (1991). Thus, this plasmid was not included in the plasmid profiling. The detailed analysis of this plasmid is beyond the scope of this study and will be further investigated in future. Our study showed that plasmid profiling is a valuable typing technique in subtyping Shigella species. Heterogeneous plasmid patterns were seen in different serotypes of Shigella in this study. In this instance, plasmid profiling offered a refinement beyond serotyping. Technically, plasmid profile analysis is one of the oldest DNA-based methods and can be efficiently performed with basic electrophoretic equipment. In conclusion, our study has demonstrated that plasmid profiling is useful in differentiating isolates of Shigella in our population. We found the correlation between the serotypes and plasmid patterns in Shigella species, which may be useful for the detection of the epidemic strains. Unique plasmid characteristic observed in S. flexneri 1b isolates was a good indicator in differentiating our Malaysian strains of serotype 1b isolates from those of other countries. Several plasmids in this study were found to have potential to be used as extrachromosomal markers for identification of new serotypes and for differentiation of existing serotypes as well. To the best of our knowledge, this is the first report on plasmid analysis of Malaysian strains of Shigella species, and the data generated will be useful for the future surveillance of this genus. Acknowledgement This work was supported by the IRPA grants and from Ministry of Science, Technology and Environment, Malaysia, Vote-F F0107/2003B and PASCA from University of Malaya. References Bauer, A.W., Kirby, W.M., Sherris, J.C. & Turek, N Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 45, Casalino, M., Nicoletti, M., Salvia, A., Colonna, B., Pazzani, C., Calconi, A., Mohamud, K.A. & Maimone. F Characterization of endemic Shigella flexneri strains in Somalia: antimicrobial resistance, plasmid profiles, and serotype correlation. Journal of Clinical Microbiology 32, Chiou, C.S., Hsu, W.B., Wei, H.L. & Chen, J.H Molecular epidemiology of a Shigella flexneri outbreak in a mountainous township in Taiwan, Republic of China. Journal of Clinical Microbiology 40, Gebre-Yohannes, A. & Drasar, B.S Plasmid profiles of antibiotic-resistant Shigella dysenteriae types 2, 3, 4, 6 and 7 isolates in Ethiopia during Epidemiology and Infection 105, Gebre-Yohannes, A. & Drasar, B.S Plasmid profiles of drug resistant Shigella boydii types 1 5, 8, 10, from Ethopia ( ). Epidemiology and Infection 119, Haider, K., Huq, M.I., Samadi, A.R. & Ahmad, K Plasmid characterization of Shigella spp. isolated from children with shigellosis and asymptomatic excretors. Journal of Antimicrobial Chemotherapy 16, Kotloff, K.L., Winickoff, J.P., Ivanoff, B., Clemens, J.D., Swerdlow, D.L., Sansonetti, P.J., Adak, G.K. & Levine, M.M Global burden of Shigella infections: Implications for vaccine development and implementation of control strategies. Bulletin of the World Health Organization 77, Lee, T.M., Chang, C.Y., Chang, L.L., Chen, W.M., Wanf, T.K. & Chang, S.F One predominant type of genetically closely related Shigella sonnei prevalent in four sequential outbreaks in school children. Diagnostic Microbiology and Infectious Disease 45,

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