Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2003 Jul;41(7):3078–3088. doi: 10.1128/JCM.41.7.3078-3088.2003

Molecular Epidemiology of Shigella in a Taiwan Township during 1996 to 2000

Jiann-Hwa Chen 1,*, Chien-Shun Chiou 2, Pei-Chun Chen 1,†, Tung-Liang Liao 2, Tsai-Ling Liao 2, Jen-Ming Li 1,‡, Wen-Bin Hsu 1
PMCID: PMC165219  PMID: 12843047

Abstract

A previously identified Shigella flexneri serotype 2a strain was responsible for an outbreak of shigellosis in a Taiwan township in August 1996. In order to find the relationship between this outbreak strain and subsequent Shigella infections in the area, 59, 47, 35, and 20 Shigella isolates recovered in 1997, 1998, 1999, and 2000, respectively, were collected and typed by serological and pulsed-field gel electrophoresis (PFGE) techniques. Of these 161 isolates, 139 isolates were S. flexneri serotype 2a, and one-third of them (47 isolates) exhibited the outbreak pattern. The remaining 92 S. flexneri serotype 2a isolates displayed 49 different NotI-PFGE patterns. Forty-five patterns were closely related to the outbreak pattern, with deletions of three specific NotI fragments occurring with high frequency. While the outbreak strain remained the main cause of shigellosis after the outbreak, the continuous emergence of closely related though poorly transmissible strains from the outbreak strain contributed to the observed annual decrease of shigellosis in the area.

Shigellosis is an acute gastroenteritis caused by Shigella species, including Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei. It is one of the major causes of diarrheal diseases worldwide. In Taiwan, about 250 to 500 cases of shigellosis were reported annually from 1995 to 2000, with an average annual incidence rate of 1 to 3 cases per 100,000 persons (4). Infections are usually caused by S. flexneri and S. sonnei, with the former mostly found in mountainous central Taiwan and the latter found in western industrial Taiwan. Infections caused by S. dysenteriae and S. boydii are rare in Taiwan and are observed only in imported cases.

An outbreak of shigellosis occurred in the Renai Township in central Taiwan in August 1996. The outbreak lasted 3 months, and 10 villages from the township were affected. During the outbreak, 37 Shigella isolates were collected from Renai and neighboring townships, and all were shown to be S. flexneri serotype 2a. By genotyping with NotI- and XbaI-pulsed-field gel electrophoresis (PFGE), we further demonstrated that this serotype was endemic in the area and that a single strain with a distinct PFGE pattern and a person-to-person mode of transmission was responsible for the outbreak (2).

After the outbreak, however, the number of shigellosis cases still occurring in the Renai Township was considered high. The purpose of this study is to understand whether the high incidence had any relation to the outbreak in 1996 and, in particular, to the outbreak strain. We collected 161 Shigella isolates that had been recovered from Renai and neighboring townships from 1997 to 2000 and typed them by serological and PFGE methods. Since NotI-PFGE could differentiate almost all the strains that were previously differentiated by using NotI- and XbaI-PFGE with the exception of isolate SH4799 (2), we chose to use only NotI-PFGE for this study. While the results indicate that the outbreak strain continued to be the main strain causing shigellosis during the 4-year period, the percentage of outbreak strain isolates among S. flexneri serotype 2a isolates recovered in the year decreased annually. Many closely related strains emerged during this period but were shown to not transmit as well as the outbreak strain.

MATERIALS AND METHODS

Bacterial isolates.

During 1997 through to 2000, stool specimens from patients with either diarrhea or dysentery (bloody diarrhea) in Nantou County were collected and screened for Shigella species by conventional biochemical methods (17) in two local hospitals (Puli Christian Hospital and Puli Veteran Hospital) and our laboratories. Shigella species recovered were serotyped by a slide agglutination test with commercial polyclonal antiserum (Denka Seiken Co. Ltd., Tokyo, Japan). The isolates were cultured in Luria broth and were stored in 15% glycerol at −70°C. Shigella isolates recovered from Renai and neighboring townships (Fig. 1) were used in the present study. Some of the isolates recovered in 1996 were used as reference strains. Characteristics of S. flexneri serotype 2a isolates from 1996 to 2000 are described in Table 1, whereas those of non-S. flexneri serotype 2a isolates will be provided upon request. These non-S. flexneri serotype 2a isolates were S. sonnei (SH7150, SH7156, SH8255, SH8289, SH8542, SH9397, SH10657, SH11620, SH14974), S. boydii (SH7571); S. flexneri serotypes 1a (SH18504), 1b (SH19453, SH19455, SH19700), 3a (SH20928, SH20904, SH20907), 3b (SH5619, SH5621), y (SH7396); and nontypeable S. flexneri (SH16934, SH19406).

FIG. 1.

FIG. 1.

Open in a new tab

Distribution of isolates of the outbreak strain and closely related strains. Numbers of isolates of the outbreak strain recovered in a specific village or township in 1996, 1997, 1998, 1999, and 2000 are indicated from left to right in parentheses next to the village or township, whereas numbers of isolates of the closely related strains are indicated in brackets.

TABLE 1.

Characteristics of 180 S. flexneri serotype 2a isolates recovered from Renai and the neighboring townships during 1996 to 2000a

Isolate Isolation date (mo-day-yr) Source of isolation
PFGE types and subtypes Relationship among members of the same epidemiological groups
Township Village
SH1105 1-3-1996 Puli A1
SH2182b 8-12-1996 Renai Tsunyang A
SH3896 8-18-1996 Renai Chingying A
SH2214b 8-19-1996 Renai Tsunyang A
SH2229 8-20-1996 Renai Chingying A
SH2276 8-23-1996 Renai Tatung B
SH2302 8-27-1996 Renai Tsunyang A
SH2286 8-29-1996 Renai Fahsiang A
SH2291 8-31-1996 Renai Chingying A
SH2308c,d 9-4-1996 Renai Tsunyang A A2 has addition of band 21 from A
SH2557c 9-4-1996 Renai Tsunyang A
SH2590c 9-4-1996 Renai Tsunyang A2
SH2343b 9-5-1996 Renai Tsunyang A
SH2371e 9-5-1996 Renai Chinai A
SH2372e,f 9-5-1996 Renai Chinai A
SH2374f 9-5-1996 Renai Chinai A
SH2576 9-5-1996 Renai Tsunyang A3
SH2558 9-7-1996 Puli A4
SH2594 9-9-1996 Renai Hotso A4
SH2585c 9-10-1996 Renai Nanfeng A
SH3151 9-12-1996 Hsini A5
SH3160 9-12-1996 Renai Tsunyang A
SH2683 9-13-1996 Renai Chinai A6
SH3010 9-13-1996 Renai Tsunyang A3
SH3162 9-13-1996 Renai Nanfeng A7
SH3006g 9-13-1996 Renai Tsunyang A3
SH2955g 9-14-1996 Renai Tsunyang A3
SH2953 9-14-1996 Renai Nanfeng A8
SH3094d 9-14-1996 Renai Chingying A
SH3364 9-19-1996 Hsini A4
SH4029 9-25-1996 Renai Wanfeng A
SH4232 10-12-1996 Renai Lihsing A
SH4217 10-14-1996 Renai Lihsing A
SH4347h 10-18-1996 Renai Fahsiang A
SH4377i 10-23-1996 Renai Chinai A
SH4332 10-24-1996 Renai Lihsing A
SH4418i 10-31-1996 Renai Huchu A
SH4798h 10-31-1996 Renai Fahsiang A
SH4520 11-14-1996 Puli A
SH4799 12-30-1996 Renai Fahsiang A
SH4785 12-31-1996 Puli A
SH4795 1-7-1997 Puli A
SH4797j 1-8-1997 Puli A
SH4834j 1-14-1997 Puli A
SH4903k 1-23-1997 Renai Chinai A9
SH4916k 1-30-1997 Renai Chinai A
SH4966 2-5-1997 Renai Chingying A
SH4968 2-11-1997 Puli A10
SH4991 2-20-1997 Hsini C
SH5268 3-19-1997 Puli A11
SH5518 3-21-1997 Puli A
SH5598 3-31-1997 Puli A
SH5620 4-8-1997 Yuchr A12
SH5977l 5-6-1997 Renai Chungcheng A11
SH5978 5-6-1997 Puli A
SH6337m 5-13-1997 Renai Chungcheng A
SH7032 5-22-1997 Renai Chinai A
SH7056m 5-23-1997 Renai Chungcheng A
SH7080l 5-23-1997 Renai Chungcheng A
SH7149 5-29-1997 Renai Chingying A4
SH7227 6-6-1997 Renai Chingying A13
SH7228 6-6-1997 Renai Chinai A
SH7291 6-13-1997 Hsini A4
SH7292 6-13-1997 Renai Hotso A
SH7344 6-17-1997 Renai Tsunyang A
SH7372 6-19-1997 Renai Chungcheng A14
SH7408 6-27-1997 Renai Chinai A15
SH7565 7-8-1997 Renai Chinai A16
SH7734 7-21-1997 Renai Wanfeng A
SH7773 7-25-1997 Renai Tsunyang A
SH7594 8-6-1997 Renai Chinai A
SH7826 8-6-1997 Renai Chinai D
SH7845 8-7-1997 Renai Hotso E
SH7932 8-20-1997 Renai Chungcheng A17
SH8188 9-9-1997 Puli A
SH8189 9-9-1997 Renai Chingying A18
SH8254 9-12-1997 Renai Chingying A
SH8357 9-22-1997 Renai Chungcheng A19
SH8500 9-26-1997 Renai Chingying A20
SH8670 10-1-1997 Renai Lihsing A21
SH8732 10-1-1997 Renai Tatung A4
SH8653 10-9-1997 Renai Tatung A18
SH8922 11-1-1997 Renai Chungcheng A22
SH8925 11-1-1997 Renai Huchu A
SH8923n,o 11-1-1997 Renai Huchu A A21 has deletion of band 32 from A, All has deletion of band 19 from A21
SH8924n 11-1-1997 Renai Huchu A21
SH8957n,p 11-2-1997 Renai Huchu A11
SH8981 11-2-1997 Puli A23
SH9154n,p 12-1-1997 Renai Huchu A24 A25 has difference of 5 bands from A24
SH9155n,o 12-1-1997 Renai Huchu A25
SH9658q 2-4-1998 Hsini A26 A27 has addition of band 16 and deletion of band 14 from A26
SH9672q 2-6-1998 Hsini A26
SH9677q 2-6-1998 Hsini A27
SH9729 2-11-1998 Renai Wanfeng A12
SH9981 2-15-1998 Renai Chinai A11
SH10004r 2-24-1998 Renai Chinai A11
SH10008r 3-27-1998 Renai Chinai A
SH10200 3-27-1998 Puli F
SH10245s 4-7-1998 Renai Chingying A
SH10262s 4-10-1998 Puli A
SH10279 4-16-1998 Renai Wanfeng A28
SH10326 4-24-1998 Puli A29
SH10492t 5-11-1998 Renai Tsunyang A
SH10592t 5-20-1998 Renai Tsunyang A
SH10823 6-3-1998 Renai Fahsiang A
SH10862 6-10-1998 Renai Nanfeng A30
SH10900 6-16-1998 Puli A
SH11119u,v 7-22-1998 Renai Nanfeng A A30 has addition of band 31 and deletion of band 32 from A
SH11143u 7-24-1998 Renai Nanfeng A30
SH11174 7-28-1998 Puli A31
SH11201u,v 8-5-1998 Renai Nanfeng A
SH11209 8-6-1998 Renai Chungcheng A32
SH11221 8-11-1998 Renai Tsunyang A4
SH11229 8-13-1998 Renai Lihsing A
SH11350 8-19-1998 Renai Tatung A23
SH11356 8-20-1998 Renai Nanfeng A30
SH11365 8-25-1998 Renai Nanfeng A33
SH11575 8-25-1998 Renai Chingying A23
SH11430w 9-1-1998 Renai Chingying A23
SH11443w 9-2-1998 Renai Chingying A23
SH11491 9-5-1998 Yuchr A3
SH11492 9-6-1998 Puli A
SH11543w 9-9-1998 Renai Chingying A
SH11662x 9-18-1998 Renai Hsinhseng A23 A34 has addition of band 24 from A23
SH11646x 9-20-1998 Renai Hsinhseng A34
SH11661 9-20-1998 Renai Chingying A35
SH11668 9-20-1998 Kuoshing A
SH11710 9-20-1998 Renai Tsunyang A23
SH11684 9-25-1998 Renai Hotso A36
SH11787 9-30-1998 Renai Hotso A37
SH11788 10-1-1998 Renai Tsunyang A23
SH12581 10-29-1998 Renai Chingying A38
SH13917 11-21-1998 Renai Hsinhseng A
SH14092 12-2-1998 Puli A23
SH15112 1-22-1999 Puli A4
SH15111y 1-24-1999 Hsini A39
SH15279y 2-8-1999 Hsini A39
SH15744 6-8-1999 Hsini A40
SH15779 6-20-1999 Puli A41
SH15778 6-21-1999 Puli A
SH16000 7-8-1999 Renai Tsunyang A
SH16026 7-11-1999 Renai Hotso A23
SH16027z 7-11-1999 Hsini A42
SH16053z 7-14-1999 Hsini A42
SH16286z 7-21-1999 Hsini A42
SH16492a1 7-21-1999 Puli A
SH16475 7-25-1999 Renai Chingying A43
SH16566 8-5-1999 Renai Chingying A
SH16632 8-16-1999 Puli A23
SH16633a1 8-16-1999 Puli A
SH16776 9-8-1999 Hsini A44
SH16893b1 10-1-1999 Renai Nanfeng A23
SH17015b1 10-5-1999 Renai Nanfeng A23
SH17081b1 10-9-1999 Renai Nanfeng A23
SH17247c1,d1,e1 10-9-1999 Renai Chingying A
SH17155 10-10-1999 Renai Nanfeng A30
SH17279f1 10-12-1999 Renai Nanfeng A45 A23 has difference of 5 bands from A45
SH17289f1 10-12-1999 Renai Nanfeng A23
SH17274 10-13-1999 Puli A
SH17384d1 10-15-1999 Renai Hotso A46
SH17656c1,e1 10-15-1999 Renai Chingying A4
SH17669e1 10-15-1999 Renai Chingying A4
SH17802 10-18-1999 Renai Chingying A
SH17843 10-20-1999 Renai Chingying A47
SH18145 11-1-1999 Renai Fahsiang A
SH18237 11-15-1999 Renai Hotso A4
SH18509 12-31-1999 Renai Tsunyang A21
SH18661 2-3-2000 Puli A48
SH18682 2-11-2000 Hsini A42
SH18742 2-18-2000 Renai Tatung A49
SH19325 5-29-2000 Renai Huchu A
SH19699g1 7-1-2000 Renai Hotso A4 A3 has addition of band 20 and deletion of band 19 from A4
SH19698g1 7-1-2000 Renai Hotso A3
SH19723g1 7-2-2000 Renai Hotso A4
SH20107 8-2-2000 Hsini A42
SH20185 8-10-2000 Renai Chingying A
SH20693 8-16-2000 Hsini A42
SH20764 8-16-2000 Hsini A42
SH20974 12-7-2000 Hsini A50
D21072 12-22-2000 Renai Chingying A51
Open in a new tab
a

Twelve epidemiological groups are boxed. The superscripts b, e, j, l, m, n, q, r, z, a1, and c1 indicate family members; c, g, and i, indicate close relatives and friends; d and d1 indicate classmates; f, h, k, b1, e1, f1, and g1 indicate neighbors; o, p, t, v, and x indicate same persons; s, u, w and y indicate contacts.

Epidemiological data.

Epidemiological data from the patients were obtained from standardized case report forms filled in by the county public health authorities. The reports included basic patient information, such as date of onset, sex, age, residency, symptoms, medical treatment, and travel history.

PFGE of bacterial isolates.

Genomic DNA of Shigella species recovered from 1997 to 2000 were digested with NotI and were analyzed by PFGE, as in our previous study with the 1996 isolates (2). Yeast chromosomal DNA (New England BioLabs, Inc., Beverly, Mass.) was used as a size standard. DNA bands on the gel were visualized by ethidium bromide staining and UV and were photographed.

PFGE pattern analysis.

PFGE patterns were analyzed by visual inspection of the photographs of the strained gels. An index isolate was included in each photograph for comparison of isolates in separate photographs. In our previous study we identified a single strain with a distinct NotI-PFGE pattern as the cause of the 1996 outbreak (2). Since isolates with the outbreak pattern were still the most prevalent among isolates recovered from 1997 to 2000, the classification described by Tenover et al. (21) was also used for interpretation of isolates recovered from 1996 to 2000 and their PFGE patterns. Difference in one to three bands is considered to represent a single genetic event, and difference in four to six bands is considered to represent two genetic events. Isolates with the same NotI-PFGE pattern as the 1996 outbreak strain (2) were designated the outbreak strain and were the most prevalent in the present study. The PFGE pattern is reported as type A. Isolates with PFGE patterns different from the outbreak pattern in one to three bands or four to six bands are considered probably or possibly related to the outbreak strain. Their patterns are defined as subtypes of type A and are designated type A1, A2, etc. Isolates with PFGE patterns different from the outbreak pattern in more than six bands are considered unrelated to the outbreak strain, and their patterns are designated type B, C, etc.

Estimation of similarity among isolates and construction of a dendrogram.

Genetic similarities between pairs of patterns were calculated by Nei and Li's F statistic (18). A matrix of F values for all pairs of patterns was prepared and was used for construction of a dendrogram by the NTSYS-PC software (Numerical Taxonomy and Multivariate Analysis System, version 1.50) from Applied Biostatistics, Inc. (Setauket, N.Y.).

RESULTS

Shigella species recovered in Nantou County from 1995 to 2000.

Bacteria of Shigella species were recovered from patients in Nantou County from 1997 to 2000 for confirmation of shigellosis, and the numbers of Shigella species recovered monthly are listed in Table 2. The numbers for 1995 and 1996 (2) are also listed and analyzed together. From 1995 to 2000, 8, 41, 60, 57, 35, and 20 Shigella isolates were recovered yearly. Of the total of 221 isolates, 210 isolates (95%) were from Renai and its neighboring townships (Hsini, Yuchr, Puli, and Guoshing townships; see Fig. 1), 1 isolate was from Nantou city, and 10 isolates were from the Jushan Township. Of the total, 185 isolates (84%) were S. flexneri serotype 2a, and the remaining 36 isolates (16%) were S. sonnei (20 isolates), S. boydii (1 isolate), S. flexneri serotypes 1a (1 isolate), 1b (3 isolates), 3a (4 isolates), 3b (2 isolates), and y (3 isolates), and nontypeable S. flexneri (2 isolates).

TABLE 2.

Numbers of Shigella spp. isolated monthly from townships of Nantou County from 1995 to 2000

Month No. of Shigella isolates recovered in indicated township or city in the yeara
1995 (n = 8)
1996 (n = 41)
1997 (n = 60)
1998 (n = 57)
1999 (n = 35)
2000 (n = 20)
Renai Hsini Yuchr Renai Puli Hsini Renai Puli Hsini Yuchr Nantou Renai Puli Hsini Yuchr Guoshing Jushan Renai Puli Hsini Renai Puli Hsini
January 1 2 3 1 1
February 1 1 1 3 3 1 1 1 1
March 3 1 1
April 1 1 (3b) 1 (3b) 1 2 2
May 6 1 2 (2:Ss) 2 1 (Ss) 1
June 1 (y) 7 (1:y) 1 2 1 2 1 1 (N) 2 (2:1b)
July 3 1 (Sb) 2 1 3 1 3 3 1 (1b)
August 2 1 8 4 1 (Ss) 8 1 2 1 3
September 1 (3a) 19 1 2 6 (2:Ss) 1 9 2 (1:Ss) 1 1 1
October 7 3 1 (Ss) 2 6 (6:Ss) 13 (1:N) 1
November 1 5 1 1 4 (4:Ss) 2 3 (3:3a)
December 1 (y) 1 1 1 3 (1:Ss) 1 1 (Ss) 2 (1:1a) 1 1
Total 4 3 1 35 4 2 41 13 4 1 1 32 9 3 2 1 10 21 7 7 11 4 5
Open in a new tab
a

S. flexneri isolates with serotypes other than 2a are indicated with 3a, 3b, Y, 1a, 1b, and N (nontypable) in parentheses; species of S. boydii and S. sonnei are also indicated in parentheses with Sb and Ss, respectively.

All of the 185 S. flexneri serotype 2a isolates recovered were from Renai and neighboring townships. Of them, 5, 41, 49, 44, 33, and 13 isolates were recovered yearly from 1995 to 2000. Two outbreaks of S. flexneri serotype 2a occurred in Renai Township during the 6-year period with sporadic cases before, between, and after the two outbreaks. The first outbreak occurred from August to October 1996, and the second occurred in October 1999. Thirty-four and 12 S. flexneri serotype 2a isolates were recovered in the two outbreaks, respectively. There were more sporadic cases between and after the two outbreaks than before (Table 2).

PFGE of the S. flexneri serotype 2a isolates.

NotI-PFGE of the 161 S. flexneri serotype 2a isolates recovered in the area from 1997 to 2000 was performed, and 50 NotI-PFGE patterns were identified, including one pattern identical to the NotI-PFGE pattern previously identified with the outbreak strain (2). A representative photograph showing 17 NotI-PFGE patterns from 19 S. flexneri serotype 2a isolates is shown in Fig. 2A. By using 0 or 1 to represent the absence or presence of the individual NotI bands, respectively, the 50 NotI-PFGE patterns were coded in binary format and are shown in Table 3. For comparison, seven additional nonoutbreak NotI-PFGE patterns identified previously with the 1996 S. flexneri serotype 2a isolates were also binary coded and are shown in Table 3. By using this process, a total of 57 patterns and 39 NotI fragments were identified from the isolates recovered from 1996 to 2000. The classification described by Tenover et al. (21) was used to designate the 57 patterns, and a main type (type A), 51 subtypes of type A (types A1 to A51), and 5 types unrelated to type A (types B to F) were identified. Patterns for each of the S. flexneri serotype 2a isolates recovered from 1996 to 2000 are listed in Table 1. A dendrogram depicting the genetic similarity of the 51 subtypes and the 6 types was constructed and is shown in Fig. 2B. It shows genetic similarities of greater than 80% among type A and the 51 subtypes. Except for type A and 11 subtypes, only one isolate was recovered for each of these types and subtypes. The numbers of isolates of type A and the 11 subtypes recovered each year from 1996 to 2000 are also shown in Fig. 2B.

FIG. 2.

FIG. 2.

FIG. 2.

Open in a new tab

(A) Photograph of NotI-PFGE patterns of 19 S. flexneri serotype 2a strains and 8 non-S. flexneri serotype 2a strains. Arrows indicate bands 19, 31, and 32. An asterisk indicates the yeast chromosomes used as molecular size standards with sizes of selected chromosomes indicated in kilobases. (B) Dendrogram of types A, B, C, D, E, and F and 51 subtypes (A1 to A51) of type A. For the type and subtypes with more than one isolates recovered from 1996 to 2000, numbers of isolates are indicated.

TABLE 3.

Binary-coded PFGE patterns of type A, B, C, D, E and F, and the 51 subtypes of type A (A1 to A51)

Type or subtype Binary code for PFGE band numbera
Class
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
A 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0
A1 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 0 1 0 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 I
A2 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 1 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 VI
A3 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 IV
A4 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 I
A5 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 1 1 0 1 0 0 0 1 0 1 0 1 0 IV
A6 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 0 0 1 0 1 0 1 0 1 0 1 0 I
A7 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 0 1 1 0 I
A8 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 1 I
A9 0 0 1 0 0 1 0 0 0 1 0 0 1 1 0 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 VI
A10 0 0 1 0 0 1 0 0 1 1 0 0 0 1 1 0 1 1 0 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 0 IV
A11 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 0 IV
A12 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 III
A13 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 0 0 1 1 0 0 1 0 1 0 1 0 0 0 1 0 1 0 1 0 V
A14 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 1 VI
A15 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 1 1 0 0 1 0 1 0 1 1 0 1 0 0 1 0 1 1 IV
A16 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 1 1 0 0 1 0 1 0 1 0 0 1 1 0 1 0 1 0 V
A17 0 0 1 0 0 1 0 0 0 1 1 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 0 II
A18 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 1 1 0 0 1 0 1 0 1 0 0 0 1 0 1 0 1 0 V
A19 0 0 0 1 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 0 0 1 1 0 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 IV
A20 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 0 1 1 1 0 1 1 0 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 I
A21 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 0 II
A22 0 0 1 0 0 1 0 0 0 1 1 0 0 1 0 0 1 1 0 0 0 1 1 0 0 1 0 1 0 1 0 0 0 1 0 1 0 1 0 V
A23 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 III
A24 0 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 1 0 1 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 VI
A25 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 0 IV
A26 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 0 0 1 1 0 0 1 0 1 0 1 0 0 0 1 0 1 1 1 0 V
A27 0 0 1 0 0 1 0 0 0 1 0 0 0 0 1 1 1 1 0 0 0 1 1 0 0 1 0 1 0 1 0 0 0 1 0 1 1 1 0 V
A28 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 1 1 1 1 0 II
A29 0 0 1 0 0 0 1 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 VI
A30 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 0 1 1 0 1 0 1 0 II
A31 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 0 0 1 1 0 1 0 I
A32 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 1 1 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 1 II
A33 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 1 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 0 II
A34 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 1 1 1 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 III
A35 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 III
A36 0 0 1 0 0 1 0 0 0 1 0 0 1 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 VI
A37 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 1 0 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 III
A38 0 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 VI
A39 0 0 1 0 0 1 0 0 0 0 1 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 I
A40 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 I
A41 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 I
A42 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 1 1 0 I
A43 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 0 1 1 0 1 1 1 0 1 0 1 0 I
A44 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 0 0 1 1 1 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 III
A45 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 0 1 1 0 1 1 1 0 II
A46 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 1 0 1 0 0 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 III
A47 0 0 1 0 0 1 0 0 0 0 1 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 VI
A48 0 0 1 0 0 1 0 0 0 1 0 1 0 1 1 0 0 1 1 0 0 1 1 0 0 1 0 1 0 1 0 0 0 1 0 1 0 1 0 IV
A49 0 0 1 0 0 1 0 0 0 1 0 0 1 1 0 0 1 1 1 0 1 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 I
A50 0 0 1 0 0 0 0 0 0 1 0 1 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 I
A51 0 0 1 0 0 0 1 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 1 0 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 I
B 1 0 0 0 0 0 0 0 0 1 0 1 0 1 1 1 0 1 0 0 0 1 1 0 0 1 0 0 1 1 0 0 0 1 0 1 0 1 0
C 0 0 1 0 0 1 0 0 0 1 0 0 0 1 1 0 1 0 1 1 0 1 1 0 1 1 0 1 0 1 0 1 1 0 0 0 1 1 0
D 0 0 0 1 0 1 0 1 0 1 0 0 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 0
E 0 1 0 0 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 0 0 1 1 0 0 1 0 1 0 1 0 0 0 1 0 0 1 1 0
F 0 0 1 0 1 0 1 0 0 0 0 0 0 0 1 0 1 1 1 0 1 1 1 0 0 1 0 1 0 1 1 1 0 1 1 0 0 1 0
Open in a new tab
a

Codes different from the code of the same band in the type A pattern are in bold face.

Isolates of type A pattern (the outbreak pattern) were recovered each year and accounted for 41% (74 out of 180) of the S. flexneri serotype 2a isolates recovered from 1996 to 2000. Numbers of the type A isolates recovered decreased annually. Percentages of the type A isolates among S. flexneri serotype 2a isolates recovered in the year also decreased annually, i.e., 66% (27 out of 41) in 1996, 45% (22 out of 49) in 1997, 35% (14 out of 44) in 1998, 27% (9 out of 33) in 1999, and 15% (2 out of 13) in 2000.

Isolates of the 51 subtypes (A1 to A51) and the other 5 types (B to F) accounted for the remaining 59% (106 out of 180) of the S. flexneri serotype 2a isolates recovered from 1996 to 2000. Compared with the number of type A isolates, the numbers of isolates recovered for each subtype and the other types are low. Only one isolate was recovered for 40 of the 51 subtypes and all of the other 5 types during the 5-year period, while 2 to 15 isolates were recovered for the other 11 subtypes (Fig. 2B). Only isolates of 8 subtypes were recovered in more than one year. This includes isolates of subtype A4 that were recovered in each year of the 5 years studied, isolates of subtypes A3 and A23 in 3 years, and isolates of subtypes A11, A12, A21, A30, and A42 in 2 years (Fig. 2B). The yearly distributions of isolates of type A and the 51 subtypes in villages of the Renai Township and in four neighboring townships are depicted in Fig. 1. This figure shows that 10 villages in Renai Township and Puli Township had isolates of type A and its subtypes recovered from 1996 to 2000. Locations that had the most isolates of type A recovered, such as Chingying, Tsunyang, and Chinai villages and the Puli Township, also had the most isolates of the subtypes recovered.

Relationship among members of the same epidemiological groups.

Many isolates of type A and the subtypes were recovered from family members, neighbors, classmates, or contacts within a couple of days. They are considered epidemiologically related. We arbitrarily defined those isolates that were epidemiologically related and were recovered within 1 to 3 days as the same epidemiological group. This yielded 12 groups from the 180 S. flexneri serotype 2a isolates recovered from 1996 to 2000, which are boxed and described in Table 1. For four groups (SH2371, SH2372, and SH2374; SH3006 and SH2955; SH11430 and SH11443; SH17656 and SH17669), isolates of the same group demonstrated the same PFGE pattern. For two groups (SH9154 and SH9155; SH17279 and SH17289), isolates of the same group demonstrated PFGE patterns different in five bands, indicating two genetic events between the isolates (21). For the remaining six groups (SH2308, SH2557, and SH2590; SH8923, SH8924, and SH8957; SH9658, SH9672, and SH9677; SH11119 and SH11143; SH11662 and SH11646; SH19698, SH19699, and SH19723), however, isolates of the same group demonstrated PFGE patterns only different in one or two bands, indicating a single genetic event between them (21).

Classification of the 51 subtypes by presence or absence of three NotI bands.

Detailed examination of the PFGE patterns of the 51 subtypes and type A indicates that three specific bands in the type A pattern were deleted at high frequency (Table 3). These are bands 19, 31, and 32. Based on the presence or absence of these three bands, the 51 subtypes can be divided into six classes (classes I to VI; Table 3). Subtypes that have bands 19 and 32 but not band 31 belong to class I. Subtypes that have bands 19 and 31 but not band 32 belong to class II. Subtypes that have bands 31 and 32 but not band 19 belong to class III. Subtypes that have only one of the three bands belong to class IV. Subtypes that do not have any of the three bands belong to class V. Subtypes that have all of the three bands belong to class VI. By using this classification, 15, 7, 7, 8, 6, and 8 subtypes belong to classes I, II, III, IV, V, and VI, respectively. Fifty-three percent (10 out of 19) of subtypes involving a deletion in band 19 also had an addition of band 20. For classes I, II, and III, subtypes of the same class were mostly placed in the same genetic cluster with similarity of 0.90 or greater (Fig. 2B).

PFGE of other Shigella isolates in Renai and the neighboring townships.

Isolates of Shigella spp. other than S. flexneri serotype 2a also seemed to be recovered at a relatively high frequency in Renai and neighboring townships after the outbreak in 1996 (Table 2). In order to identify possible relationships between the outbreak strain and the non-S. flexneri serotype 2a isolates, PFGE was also performed with the non-S. flexneri serotype 2a isolates recovered in Renai and neighboring townships from 1997 to 2000. The results indicate that all of them had differences of more than 10 bands in their NotI-PFGE patterns compared with that of the outbreak pattern. They are not related to either type A or any of the 51 subtypes, and no major pattern can be found among them. A representative photograph showing the NotI-PFGE patterns of 8 non-S. flexneri serotype 2a isolates is shown in Fig. 2A.

DISCUSSION

Nantou County is located in central Taiwan and consists of 12 townships and 1 city, of which Renai and Hsini townships are the two largest and are the only two mountainous townships in Nantou County. Shigellosis was rare in Nantou County before August 1996 but was frequently found in Renai and neighboring townships since an outbreak of S. flexneri serotype 2a in Renai Township in August 1996 (Table 2). An S. flexneri serotype 2a outbreak strain with the type A pattern was the main strain observed during and after the outbreak. Seven and 45 closely related strains with subtypes A2 to A8 patterns and A3, A4, and A9 to A51 patterns, respectively, appeared during and after the outbreak. They were recovered far less frequently than the outbreak strain, indicating that transmission of these closely related strains occurred less frequently than transmission of the outbreak strain. Compared to the outbreak strain, 84% (42 out of 50) of the closely related strains had deletions of at least one of three specific NotI bands in their PFGE patterns, suggesting deletion hot spots in the genome of the outbreak strain.

Of the 180 S. flexneri serotype 2a isolates recovered from 1996 to 2000, 12 epidemiologically related groups were identified (Table 1). For four groups, isolates of the same group showed identical PFGE patterns, indicating transmission of the same strain among members of the group. For the other two groups (SH9154 and SH9155; SH17279 and SH17289), isolates of the same group showed a difference of five bands in their PFGE patterns. While SH17279 (subtype 45) andSH17289 (subtype 23) were recovered from two neighbors during the outbreak in 1999 (Table 1) and were probably not truly epidemiologically related, the relationship between SH9154 and SH9155 was somewhat complicated. SH9154 (subtype A24) and SH9155 (subtype A25) were recovered from two family members. One month earlier, three separate strains (type A and subtypes A21 and A11) had been recovered from the same two persons and another family member, respectively. Although the possibility of independent origins can not be excluded, the two isolates were likely to have evolved from complex genetic rearrangements among isolates of type A and subtypes A21 and A11 during the 1-month period in the family. Furthermore, for the remaining 6 groups, isolates of the same group showed differences of only one or two bands in their PFGE patterns, indicating a single genetic event among them (21). It appears that for these six groups one isolate had evolved directly from the other isolate of the same group. Based on these epidemiological observations and the presence of deletion hot spots in the genome of the outbreak strain, we hypothesized that many, if not all, of the 50 closely related strains recovered during and after the 1996 outbreak were derived from the outbreak strain or other closely related strains by various genetic events, such as tandem duplication, deletion, translocation, and inversion (see below).

Several lines of evidence further support our hypothesis. First, during the two outbreaks in 1996 and 1999 isolates that had no apparent epidemiological relationships showed a single genetic event among them. For example, isolates of subtypes  A3 to A8 were recovered during the outbreak in 1996. Subtypes A3, A5, A6, A7, and A8 differed from subtype A4 in two, two, one, two, and one band, respectively. Isolates of subtype A3, A5, A6, A7, and A8 can thus be explained by separate single events from isolates of subtype A4 (Table 1). Similarly, isolates of type A and subtypes A4, A23, A30, A45, A46, and A47 were recovered during the outbreak in October 1999. Subtypes A4 and A47 differed from type A in one and two bands, respectively, subtype A46 differed from subtype A23 in one band, and subtype A45 differed from subtype A30 in one band. Isolates of subtype A4, A47, A46, and A45 can thus be explained by single events from isolates of type A and subtypes A23 and A30 (Table 1). Second, the distribution of isolates of the subtypes roughly correlates with the distribution of the outbreak strain (Fig. 1). Third, numbers of S. flexneri serotype 2a isolates recovered decreased yearly from 1997 to 2000. The decrease is related to yearly decrease in the actual numbers of the outbreak strain isolates and also to the relative numbers of the outbreak strain isolates to all S. flexneri serotype 2a isolates recovered in that year. As the outbreak strain transmitted much better than the closely related strains, one possible explanation for the correlation would be de novo generation of closely related strains in human colons from the outbreak strain. Inside human colons the newly generated, closely related strains might be preferentially selected over the outbreak strain, as adaptive immunity would initially be elicited by infection with the outbreak strain.

Rearrangements of large DNA pieces are usually caused by homologous recombination between long repeat sequences, such as rRNA operons, transposons, and IS (insertion sequence) elements. Depending upon whether the two repeat sequences are the same or are in reversed orientation, tandem duplication, deletion, translocation, and inversion of the intervening sequence could occur. Mobility of IS elements would increase the variety of rearrangements (10). Shigella species carry multiple copies of insertion sequences, such as IS1, IS600, and IS629 in their genomes (14). IS-mediated deletions and tandem amplifications of DNA of 10 to 100 kb have been reported for S. dysenteriae and Yersinia pestis (5, 15). Futhermore, S. flexneri serotype 2a carry at least four pathogenicity islands (1, 13, 16, 19, 20, 22). Pathogenicity islands are large (often more than 30 kb), unstable DNA fragments that carry many virulence-associated genes and mobility genes (including insertion sequences, integrases, transposases, and origin of plasmid replication) (6). It has been demonstrated that pathogenicity islands are involved in spontaneous deletions of 51 and 99 kb of DNA in S. flexneri serotype 2a (1, 20). Both insertion sequences and pathogenicity islands could account for the frequent generation of closely related strains and deletion hot spots observed in this study. We propose that some important factors present in the outbreak strain were lost or inactivated during the course of generating the closely related strains, leading to poor transmission of the strains. These factors might be associated with the prolonged survival of the bacteria in the environment, as recently reported for poly P kinase of Shigella and Salmonella spp. (11), or for intracellular persistence of the bacteria in human enteric cells.

Shigella species, like Salmonella species, are facultative intracellular pathogens. Both bacteria not only cause gastrointestinal diseases but also survive in enteric epithelium cells. Studies with Salmonella spp. in a mouse model indicated that gastroenteritis symptoms could be separated from intracellular accumulation and systemic spreading of the bacteria and that genes for gastroenteritis symptoms were mainly located on pathogenicity island SPI1 and that genes for intracellular accumulation and systemic spreading of the bacteria were located on pathogenicity island SPI2 (8). Clements et al. (3) demonstrated that a single mutation in a global regulator gene, the polynucleotide phosphorylase gene, in Salmonella enterica could switch the acute gastroenteritis symptom into a systemic infection in the mouse model. It is likely that a similar regulation system might also exist in S. flexneri. Recovery from gastroenteritis symptoms caused by S. flexneri usually takes about a week, but survival of the bacteria in human enteric cells of up to 17 months has been reported (12). These asymptomatic carriers are suspected as the source of transmission of shigellosis (7, 9). Although we did not screen the asymptomatic contacts as was done in a previous study, the five S. flexneri serotype 2a isolates recovered from asymptomatic contacts in 1996, indeed, were the outbreak strain (2). This and the fact that only the outbreak strain could be recovered twice from the same persons within a 2-week period (SH10492 and SH10592 and SH11119 and SH11201; Table 1) indicated that the outbreak strain had a better surviving capability than the closely related strains had, though they might have similar capabilities in eliciting the gastroenteritis symptom in patients.

In summary, our PFGE results show that a single S. flexneri serotype 2a strain responsible for the outbreak in 1996 continued to be the main strain causing shigellosis in the area from 1997 to 2000. Forty-five closely related but poorly transmissible strains emerged from the outbreak strain during the 4-year period. Compared to the outbreak strain, 87% (39 out of 45) of them had deletions of at least one of three specific NotI bands in their PFGE patterns. Characterization of these three NotI bands is in progress.

Acknowledgments

This work was supported by research grant DOH-90-DC-2018 from the Department of Health, Taiwan, Republic of China.

We thank the staff from the Renai Township Health Station for assistance in the epidemiological investigation and from the Puli Christian Hospital and the Puli Veteran Hospital for providing the Shigella isolates.

REFERENCES

  • 1.Al-Hasani, K., K. Kajakumar, B. Dieter, R. Robin-Browne, B. Adler, and H. Sakellaris. 2001. Genetic organization of the she pathogenicity island in Shigella flexneri 2a. Microb. Pathog. 30:1-8. [DOI] [PubMed] [Google Scholar]
  • 2.Chiou, C.-S., W.-B. Hsu, H.-L. Wei, and J.-H. Chen. 2001. Molecular epidemiology of a Shigella flexneri outbreak in a mountainous township in Taiwan, Republic of China. J. Clin. Microbiol. 39:1048-1056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Clements, M. O., S. Eriksson, A. Thompson, S. Lucchini, J. C. D. Hinton, S. Normark, and M. Rhen. 2002. Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica. Proc. Natl. Acad. Sci. USA 99:8784-8789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Department of Health, Taiwan, R. O. C. 2002. Statistically important characteristics in public health in Taiwan, Republic of China. Department of Health, Taipei, Taiwan, Republic of China.
  • 5.Fetherston, J. D., P. Schuetze, and R. D. Perry. 1992. Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element. Mol. Microbiol. 6:2693-2704. [DOI] [PubMed] [Google Scholar]
  • 6.Hacker, J., G. Blum-Oehler, I. Muhldorfer, and H. Tschape. 1997. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol. Microbiol. 23:1089-1097. [DOI] [PubMed] [Google Scholar]
  • 7.Haider, K., M. I. Huq, A. R. Samadi, and K. Ahmad. 1985. Plasmid characterization of Shigella spp. isolated from children with shigellosis and asymptomatic excretors. J. Antimicrob. Chemother. 16:691-698. [DOI] [PubMed] [Google Scholar]
  • 8.Hanson-Wester, I., and M. Hensel. 2001. Salmonella pathogenicity islands encoding type III secretion systems. Microb. Infect. 3:549-559. [DOI] [PubMed] [Google Scholar]
  • 9.Hossain, M. A., K. Z. Hasan, and M. J. Albert. 1994. Shigella carriers among non-diarrhoeal children in an endemic area of shigellosis in Bangladesh. Trop. Geograph. Med. 46:40-48. [PubMed] [Google Scholar]
  • 10.Hughes, D. 2000. Evaluating genome dynamics: the constraints on rearrangements with bacterial genomes. Genome Biol. 1:0006.1-0006.8. [DOI] [PMC free article] [PubMed]
  • 11.Kim, K. S., N. N. Rao, C. D. Fraley, and A. Kornberg. 2002. Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp. Proc. Natl. Acad. Sci. USA 99:7675-7680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Levine, M. M., H. L. Dupont, M. Khodabandelou, and R. B. Hornick. 1973. Long-term Shigella-carrier state. N. Engl. J. Med. 288:1169-1171. [DOI] [PubMed] [Google Scholar]
  • 13.Luke, S. N., S. A. Turner, K. Kajakumar, H. Sakellaris, and B. Adler. 2001. Ferric dicitrate transport system (Fec) of Shigella flexneri 2a YSH6000 is encoded on a novel pathogenicity island carrying multiple antibiotic resistance genes. Infect. Immun. 69:6012-6021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Matsutani, S., and E. Ohtsubo. 1993. Distribution of the Shigella sonnei insertion elements in Enterobacteriaceae. Gene 127:111-115. [DOI] [PubMed] [Google Scholar]
  • 15.McDonough, M. A., and J. R. Butterton. 1999. Spontaneous tandem amplification and deletion of the Shiga toxin operon in Shigella dysenteriae 1. Mol. Microbiol. 34:1058-1069. [DOI] [PubMed] [Google Scholar]
  • 16.Moss, J. E., T. J. Cardozo, A. Zychlinsky, and E. A. Croisman. 1999. The selC-associated SHI-2 pathogenicity island of Shigella flexneri. Mol. Microbiol. 33:74-83. [DOI] [PubMed] [Google Scholar]
  • 17.Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and F. C. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed. American Society for Microbiology Press, Washington, D.C.
  • 18.Nei, M., and W. H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76:5269-5273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Rajakumar, K., C. Sasakawa, and B. Adler. 1996. A spontaneous 99-kb chromosomal deletion results in multi-antibiotic susceptibility and an attenuation of contact haemolysis in Shigella flexneri 2a. J. Med. Microbiol. 45:64-75. [DOI] [PubMed] [Google Scholar]
  • 20.Rajakumar, K., C. Sasakawa, and B. Adler. 1997. Use of novel approach, termed island probing, identifies the Shigella flexneri she pathogenicity island which encodes a homolog of the immunoglobulin A protease-like family of proteins. Infect. Immun. 65:4606-4614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Vokes, S. A., S. A. Reeves, A. G. Torres, and S. M. Pane. 1999. The aerobactin iron transport system genes in Shigella flexneri. Mol. Microbiol. 33:63-73. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES