Outbreak of Dysentery Associated with Ceftriaxone-Resistant Shigella sonnei: First Report of Plasmid-Mediated CMY-2-Type AmpC -Lactamase Res
http://www.100md.com
微生物临床杂志 2005年第6期
Department of Pediatrics, Veterans General Hospital—Kaohsiung, Kaohsiung, Taiwan
Department of Pediatrics, Chang Gung Children's Hospital, Taoyuan, Taiwan
National Yang-Ming University, Taipei, Taiwan
Department of Pediatrics, E-DA Hospital/I-Shou University, Kaohsiung County, Taiwan
ABSTRACT
We document the first report of plasmid-encoded CMY-2-type AmpC -lactamase identified among Shigella sonnei isolates resistant to ceftriaxone and obtained after an outbreak of bacillary dysentery in Taiwan. One hundred eighty-two children in two elementary schools in Yu-Li, Taiwan, where an outbreak occurred after a typhoon hit this area in 2001, were enrolled in this study. Clinical and epidemiologic data on the infected children were collected. Pulsed-field gel electrophoresis (PFGE) was performed on the isolates to determine the genetic relatedness of outbreak strains. Plasmid analysis and PCR were performed to identify -lactamase genes responsible for ceftriaxone resistance. Forty-seven children from the two elementary schools were culture positive for S. sonnei in this outbreak. Twenty-three children were asymptomatic. Of the total isolates 55.3% were resistant to ampicillin. One hundred percent of the isolates obtained from children in school A were initially susceptible to both ampicillin and ceftriaxone. Of isolates obtained from school B 96.2% were nonsusceptible to ceftriaxone. However, two isolates from school A developed resistance to ampicillin during the course of treatment. All 18 available isolates showed closely related PFGE patterns (4, 4a, 4b, and 4c). CMY-2-type AmpC -lactamase was responsible for ceftriaxone resistance in ceftriaxone-nonsusceptible isolates; Southern blot hybridization confirmed that such a resistance gene was located on the plasmid. This is the first report of plasmid-mediated CMY-2-type AmpC -lactamase in S. sonnei. Ampicillin-resistant isolates can develop during the course of antibiotic treatment.
INTRODUCTION
Shigellosis is primarily a disease of poor, crowded communities that do not have adequate sanitation or clean water. Among the four Shigella species, S. flexneri is the most commonly isolated species in the developing world. However, in industrialized countries, the predominant species is S. sonnei, which accounts for 90% of the infections caused by Shigella spp. (25). Despite the continual improvement of sanitary conditions and the socioeconomic level of the population in Taiwan, outbreaks of shigellosis still occur after natural disasters such as typhoons or earthquakes.
Resistance of Shigella spp. to various antibiotics is increasing (26); however, plasmid-encoded resistance of Shigella spp. to extended-spectrum cephalosporins is still rare. Genetic testing is required to identify the relationship and mechanism of antimicrobial resistance in outbreak situations. Pulsed-field gel electrophoresis (PFGE) is a useful epidemiological tool but provides limited information about the genetic (plasmid) profile of the microorganisms. The purpose of this study was to investigate the epidemiological, clinical, and microbiological features of an outbreak of Shigella dysentery caused by S. sonnei in Yu-Li, Hua-Lien, Taiwan. Of note, this is the first report documenting production of CMY-2-type AmpC -lactamase by S. sonnei.
MATERIALS AND METHODS
Patients and population. An outbreak of bacillary dysentery caused by S. sonnei occurred in two elementary schools in Yu-Li, Hua-Lien, Taiwan, in September to October 2001, after a typhoon hit Taiwan. The clinical and epidemiologic data for the infected children were collected retrospectively from the medical records. Children who were admitted to Yu-Li Veterans Hospital with shigellosis were enrolled in the study. Stool culture (or rectal swab) was performed for all the students in the two elementary schools after S. sonnei was isolated from the first student who had diarrhea.
Methods. (i) Bacterial isolates. Identification of Shigella organisms was performed according to accepted bacteriological procedures and completed by agglutination with specific antisera (Wellcome Labs, Poole, Dorset, England) (14).
(ii) Antimicrobial susceptibility. Antimicrobial susceptibility was examined by the disk diffusion method. The antimicrobial agents examined included ampicillin, chloramphenicol, ciprofloxacin, trimethoprim-sulfamethoxazole, and ceftriaxone. Susceptible and nonsusceptible isolates were defined according to the criteria suggested by the CLSI (formerly NCCLS) (19, 20).
(iii) Chromosomal DNA analysis by PFGE. PFGE was performed following the procedures described previously (32) with modifications. For PFGE, bacterial colonies were suspended in 10 mM Tris-0.1 mM EDTA and cast into a plug, which was treated with ESP buffer (0.5 M EDTA, 1% sodium lauroyl sarcosine, 0.5 mg of proteinase K per ml) for 24 h. The plug was washed and digested at 37°C with 40 U of XbaI in NEB2 (New England Biolabs 2) buffer overnight. The plug was then loaded onto a 1% agarose gel, and PFGE was carried out with a CHEF Mapper XA System (Bio-Rad Laboratories). The gel was run with a linear increase in switching times (from 4 to 70 s) over a period of 26 h, a 120-degree switch angle, and a gradient of 6.0 V per cm. The gel was stained with ethidium bromide and photographed with UV illumination. The DNA fingerprints generated by PFGE were analyzed according to the criteria proposed by Tenover et al. (33).
PCR and Southern blot hybridization. Genes for -lactamases responsible for ceftriaxone resistance were amplified by PCR using primer sets previously described for detecting blaSHV-5, blaCTX-M, and blaCMY-2 genes (4, 10, 15, 18, 24). PCR products were purified by using the Microcon PCR centrifugal filter device (Millipore) and were sequenced with an ABI 377 automatic sequencer (Perkin-Elmer Applied Biosystems). The nucleotide sequences obtained were compiled and analyzed using Pcgene software (Intelligenetics). The search for homologous sequences was done in the GenBank database using FASTA software through the Internet.
Plasmid profiles of the isolates were determined by the alkaline lysis method (13) and then transferred to a membrane for the following hybridization experiment. To locate the resistance gene, DNA-DNA hybridization with the PCR product as a probe was performed by the method of Southern (31).
RESULTS
Description of the outbreak. Yu-Li is located on the east side of Taiwan, where sanitary conditions are generally poorer than those of the west side. In September 2001, a typhoon hit the area and caused extensive flooding. Two elementary schools, located about 30 kilometers apart in Yu-Li, were affected in this outbreak. There were 98 students at school A and 84 students at school B. Twenty children in school A initially presented with fever, abdominal pain, and bloody stool and were admitted to Yu-Li Veterans Hospital in mid-September. S. sonnei was isolated from the stool cultures of all 20 students. These isolates were all susceptible to ampicillin. Surveillance cultures were performed on the other 78 students in school A. One asymptomatic child was also culture positive for S. sonnei. Subsequently, four children in school B had the same symptoms and were admitted during early October. Another 22 asymptomatic children from school B were found to be culture positive for S. sonnei. All the stool cultures obtained from school B yielded S. sonnei strains that were nonsusceptible to ampicillin.
Demographics and clinical features. Altogether 47 children from the two elementary schools were culture positive for S. sonnei in this outbreak (21 from school A and 26 from school B). The demographic data and symptoms at presentation are shown in Table 1. Fifty-one percent (24/47) of the culture-positive children exhibited signs and symptoms of shigellosis (20 from school A and 4 from school B). Twenty-three children were asymptomatic.
Antimicrobial susceptibility, course of treatment, and outcome. The antibiotic susceptibility data are shown in Table 2. All the isolates from the children of school A were susceptible to ampicillin initially. The 21 culture-positive children from school A received a 5-day course of ampicillin. Fifty-seven percent (12/21) had negative follow-up cultures, while S. sonnei was still isolated after 5 days of treatment in 42.8% (9/21). Twenty-two percent (two/nine) of these isolates were now nonsusceptible to ampicillin. These nine children received another 2-day treatment with ceftriaxone. The follow-up cultures were all negative after ceftriaxone treatment.
In contrast, all the isolates from the children of school B were nonsusceptible to ampicillin. Ampicillin was the initial treatment for the 26 children of school B before the susceptibility data were available. Ampicillin was used for 3 days, and then the antibiotic was switched to ceftriaxone in 20 children. The treatment of the 20 children was switched to a 5-day course of ciprofloxacin due to the continued stool positivity after 2 days of ceftriaxone treatment. The treatment of the other six children was directly shifted from ampicillin to the 5-day course of ciprofloxacin.
No patient developed bacteremia or other complications such as hemolytic-uremic syndrome, toxic megacolon, intestinal perforation, encephalopathy, or death. The follow-up stool cultures performed 24 and 48 h after discontinuance of the last course of antibiotics were all negative.
PFGE. Eighteen isolates were available for PFGE analysis (nine isolates from school A and nine isolates from school B). The results of PFGE are shown in Fig. 1. These 18 isolates appeared to originate from the same source, as their PFGE patterns were closely related according to the Tenover criteria (33). The isolates from school A showed the same PFGE pattern (pattern 4) except for one isolate (pattern 4a). The isolates from school B also showed an identical PFGE pattern (pattern 4b) except for two isolates (pattern 4c and pattern 4). Furthermore, the genotype of the nonsusceptible isolates from school A that were obtained after treatment was identical to that of the susceptible isolates from that school (Table 3).
PCR and Southern blot hybridization. Ten isolates (one from school A and nine from school B) were tested by PCR. AmpC -lactamase was responsible for ceftriaxone resistance in all ceftriaxone-resistant isolates, and Southern blot hybridization confirmed that the resistance gene was located on the plasmid (Table 3). DNA sequencing of the PCR products confirmed that all ceftriaxone-resistant isolates harbored CMY-2 and SHV-5 -lactamase genes. Two isolates contained the SHV-5 gene only and remained susceptible to ceftriaxone.
DISCUSSION
Shigella spp. have managed to survive the antibiotic era via an ingenious mechanism of resistance, the production of -lactamases. High rates of resistance to ampicillin among Shigella isolates are due to the production of -lactamases similar to TEM-1 or OXA-1 (21, 28, 29). In the past 2 decades both the isolation frequencies and the types of extended-spectrum -lactamase (ESBL) have gradually increased. ESBLs are detected most commonly in Klebsiella pneumoniae and Escherichia coli but have been noted in other members of the family Enterobacteriaceae as well. However, no ESBL-producing Shigella strains were reported until 1999, when an SHV-11 ESBL-producing S. dysenteriae strain was reported in India (2). Recently, four other ESBL-producing strains have been reported from four different countries: an S. sonnei strain with a CTX-M-14-type ESBL from Korea (22), an S. sonnei strain with a CTX-M-2-type ESBL from Argentina (23), an S. flexneri strain with an SHV-2-type ESBL from France (8), and an S. sonnei strain with a CTX-M-3-type ESBL from Turkey (1). To the authors' knowledge, this is the first report that CMY-2-type AmpC -lactamase was responsible for ceftriaxone resistance in resistant isolates of S. sonnei; Southern blot hybridization confirmed that the resistance gene was located on the plasmid.
The most common typing procedures currently used with Shigella spp. are plasmid analysis and PFGE (9, 11, 16, 17, 30). Shigella species usually harbor a heterogeneous population of plasmids, ranging in numbers from 2 to as many as 10 (12). Plasmid analysis has proven to be a useful typing technique (9, 11); it is inexpensive and quick to perform. However, many plasmids are unstable and may be easily gained and/or lost, thus limiting this method's capacity in delineating genetic relatedness. Conversely, PFGE has a high discriminatory power, yet the procedure is time-consuming and expensive. In this outbreak, PFGE analysis demonstrated that all the isolates expressed similar or identical DNA fingerprints, indicating that these isolates might originate from the same source. Remarkably, the antibiograms were distinctively different among the isolates from the two schools. Shigellosis is relatively infrequent in Taiwan, with the incidence being 0.006% in the year 2001. Although the direct association with a food/water source was not established, the outbreaks happened very close together in terms of timing and geographic distance. Thus, it is biologically plausible that the outbreaks at the two schools were closely related while a parental strain might have acquired the resistant plasmid, leading to the expression of different antibiograms and spread in one of the schools.
In community-based observational studies, Shigella species usually are isolated from children with diarrhea and are seldom isolated from control patients who do not have diarrhea (3, 5, 6). Therefore, it has been inferred that infection with Shigella is virtually synonymous with disease. However, in this study, nearly half of the patients (23/47) were asymptomatic. The symptoms/signs of infected children comprise a clinical spectrum ranging from asymptomatic infections and secretory diarrhea to bloody diarrhea. Notably, bloody stool was noted in only 16.7% of the symptomatic patients.
Of note, antibiotic resistance appears to develop during the course of treatment, as shown in the two isolates obtained from patients of school A who received 5-day ampicillin treatment. Emergence of the resistance gene because of selective pressure may have contributed to the newly developed resistance. In this study, nine children from school A received a 2-day course of treatment to eradicate the organism from the stool. A treatment course of 5 days has been recommended for shigellosis. However, a shorter treatment course has been proposed as well (7, 27) and has demonstrated clinical and bacteriological cure rates comparable to those of the longer course. This would limit the environmental pressure for selection and dissemination of resistance genes among bacterial populations. In those instances when antibiotics are mandatory, short but effective courses of treatment may be warranted. A double-blind randomized study is mandatory to determine the optimal regimen and duration of treatment for shigellosis.
In summary, this is the first report on the CMY-2-type AmpC -lactamase in S. sonnei. The gene was located on a plasmid and might be responsible for the spread of ceftriaxone resistance among isolates. Emergence of resistance could occur during the course of treatment; thus, close monitoring is mandatory.
ACKNOWLEDGMENTS
We thank Marilyn M. Wagener for her expertise and insight in reviewing the manuscript and for her helpful comments.
This work was supported by the Veterans General Hospital Kaohsiung grant number VGHKS 93-085.
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Department of Pediatrics, Chang Gung Children's Hospital, Taoyuan, Taiwan
National Yang-Ming University, Taipei, Taiwan
Department of Pediatrics, E-DA Hospital/I-Shou University, Kaohsiung County, Taiwan
ABSTRACT
We document the first report of plasmid-encoded CMY-2-type AmpC -lactamase identified among Shigella sonnei isolates resistant to ceftriaxone and obtained after an outbreak of bacillary dysentery in Taiwan. One hundred eighty-two children in two elementary schools in Yu-Li, Taiwan, where an outbreak occurred after a typhoon hit this area in 2001, were enrolled in this study. Clinical and epidemiologic data on the infected children were collected. Pulsed-field gel electrophoresis (PFGE) was performed on the isolates to determine the genetic relatedness of outbreak strains. Plasmid analysis and PCR were performed to identify -lactamase genes responsible for ceftriaxone resistance. Forty-seven children from the two elementary schools were culture positive for S. sonnei in this outbreak. Twenty-three children were asymptomatic. Of the total isolates 55.3% were resistant to ampicillin. One hundred percent of the isolates obtained from children in school A were initially susceptible to both ampicillin and ceftriaxone. Of isolates obtained from school B 96.2% were nonsusceptible to ceftriaxone. However, two isolates from school A developed resistance to ampicillin during the course of treatment. All 18 available isolates showed closely related PFGE patterns (4, 4a, 4b, and 4c). CMY-2-type AmpC -lactamase was responsible for ceftriaxone resistance in ceftriaxone-nonsusceptible isolates; Southern blot hybridization confirmed that such a resistance gene was located on the plasmid. This is the first report of plasmid-mediated CMY-2-type AmpC -lactamase in S. sonnei. Ampicillin-resistant isolates can develop during the course of antibiotic treatment.
INTRODUCTION
Shigellosis is primarily a disease of poor, crowded communities that do not have adequate sanitation or clean water. Among the four Shigella species, S. flexneri is the most commonly isolated species in the developing world. However, in industrialized countries, the predominant species is S. sonnei, which accounts for 90% of the infections caused by Shigella spp. (25). Despite the continual improvement of sanitary conditions and the socioeconomic level of the population in Taiwan, outbreaks of shigellosis still occur after natural disasters such as typhoons or earthquakes.
Resistance of Shigella spp. to various antibiotics is increasing (26); however, plasmid-encoded resistance of Shigella spp. to extended-spectrum cephalosporins is still rare. Genetic testing is required to identify the relationship and mechanism of antimicrobial resistance in outbreak situations. Pulsed-field gel electrophoresis (PFGE) is a useful epidemiological tool but provides limited information about the genetic (plasmid) profile of the microorganisms. The purpose of this study was to investigate the epidemiological, clinical, and microbiological features of an outbreak of Shigella dysentery caused by S. sonnei in Yu-Li, Hua-Lien, Taiwan. Of note, this is the first report documenting production of CMY-2-type AmpC -lactamase by S. sonnei.
MATERIALS AND METHODS
Patients and population. An outbreak of bacillary dysentery caused by S. sonnei occurred in two elementary schools in Yu-Li, Hua-Lien, Taiwan, in September to October 2001, after a typhoon hit Taiwan. The clinical and epidemiologic data for the infected children were collected retrospectively from the medical records. Children who were admitted to Yu-Li Veterans Hospital with shigellosis were enrolled in the study. Stool culture (or rectal swab) was performed for all the students in the two elementary schools after S. sonnei was isolated from the first student who had diarrhea.
Methods. (i) Bacterial isolates. Identification of Shigella organisms was performed according to accepted bacteriological procedures and completed by agglutination with specific antisera (Wellcome Labs, Poole, Dorset, England) (14).
(ii) Antimicrobial susceptibility. Antimicrobial susceptibility was examined by the disk diffusion method. The antimicrobial agents examined included ampicillin, chloramphenicol, ciprofloxacin, trimethoprim-sulfamethoxazole, and ceftriaxone. Susceptible and nonsusceptible isolates were defined according to the criteria suggested by the CLSI (formerly NCCLS) (19, 20).
(iii) Chromosomal DNA analysis by PFGE. PFGE was performed following the procedures described previously (32) with modifications. For PFGE, bacterial colonies were suspended in 10 mM Tris-0.1 mM EDTA and cast into a plug, which was treated with ESP buffer (0.5 M EDTA, 1% sodium lauroyl sarcosine, 0.5 mg of proteinase K per ml) for 24 h. The plug was washed and digested at 37°C with 40 U of XbaI in NEB2 (New England Biolabs 2) buffer overnight. The plug was then loaded onto a 1% agarose gel, and PFGE was carried out with a CHEF Mapper XA System (Bio-Rad Laboratories). The gel was run with a linear increase in switching times (from 4 to 70 s) over a period of 26 h, a 120-degree switch angle, and a gradient of 6.0 V per cm. The gel was stained with ethidium bromide and photographed with UV illumination. The DNA fingerprints generated by PFGE were analyzed according to the criteria proposed by Tenover et al. (33).
PCR and Southern blot hybridization. Genes for -lactamases responsible for ceftriaxone resistance were amplified by PCR using primer sets previously described for detecting blaSHV-5, blaCTX-M, and blaCMY-2 genes (4, 10, 15, 18, 24). PCR products were purified by using the Microcon PCR centrifugal filter device (Millipore) and were sequenced with an ABI 377 automatic sequencer (Perkin-Elmer Applied Biosystems). The nucleotide sequences obtained were compiled and analyzed using Pcgene software (Intelligenetics). The search for homologous sequences was done in the GenBank database using FASTA software through the Internet.
Plasmid profiles of the isolates were determined by the alkaline lysis method (13) and then transferred to a membrane for the following hybridization experiment. To locate the resistance gene, DNA-DNA hybridization with the PCR product as a probe was performed by the method of Southern (31).
RESULTS
Description of the outbreak. Yu-Li is located on the east side of Taiwan, where sanitary conditions are generally poorer than those of the west side. In September 2001, a typhoon hit the area and caused extensive flooding. Two elementary schools, located about 30 kilometers apart in Yu-Li, were affected in this outbreak. There were 98 students at school A and 84 students at school B. Twenty children in school A initially presented with fever, abdominal pain, and bloody stool and were admitted to Yu-Li Veterans Hospital in mid-September. S. sonnei was isolated from the stool cultures of all 20 students. These isolates were all susceptible to ampicillin. Surveillance cultures were performed on the other 78 students in school A. One asymptomatic child was also culture positive for S. sonnei. Subsequently, four children in school B had the same symptoms and were admitted during early October. Another 22 asymptomatic children from school B were found to be culture positive for S. sonnei. All the stool cultures obtained from school B yielded S. sonnei strains that were nonsusceptible to ampicillin.
Demographics and clinical features. Altogether 47 children from the two elementary schools were culture positive for S. sonnei in this outbreak (21 from school A and 26 from school B). The demographic data and symptoms at presentation are shown in Table 1. Fifty-one percent (24/47) of the culture-positive children exhibited signs and symptoms of shigellosis (20 from school A and 4 from school B). Twenty-three children were asymptomatic.
Antimicrobial susceptibility, course of treatment, and outcome. The antibiotic susceptibility data are shown in Table 2. All the isolates from the children of school A were susceptible to ampicillin initially. The 21 culture-positive children from school A received a 5-day course of ampicillin. Fifty-seven percent (12/21) had negative follow-up cultures, while S. sonnei was still isolated after 5 days of treatment in 42.8% (9/21). Twenty-two percent (two/nine) of these isolates were now nonsusceptible to ampicillin. These nine children received another 2-day treatment with ceftriaxone. The follow-up cultures were all negative after ceftriaxone treatment.
In contrast, all the isolates from the children of school B were nonsusceptible to ampicillin. Ampicillin was the initial treatment for the 26 children of school B before the susceptibility data were available. Ampicillin was used for 3 days, and then the antibiotic was switched to ceftriaxone in 20 children. The treatment of the 20 children was switched to a 5-day course of ciprofloxacin due to the continued stool positivity after 2 days of ceftriaxone treatment. The treatment of the other six children was directly shifted from ampicillin to the 5-day course of ciprofloxacin.
No patient developed bacteremia or other complications such as hemolytic-uremic syndrome, toxic megacolon, intestinal perforation, encephalopathy, or death. The follow-up stool cultures performed 24 and 48 h after discontinuance of the last course of antibiotics were all negative.
PFGE. Eighteen isolates were available for PFGE analysis (nine isolates from school A and nine isolates from school B). The results of PFGE are shown in Fig. 1. These 18 isolates appeared to originate from the same source, as their PFGE patterns were closely related according to the Tenover criteria (33). The isolates from school A showed the same PFGE pattern (pattern 4) except for one isolate (pattern 4a). The isolates from school B also showed an identical PFGE pattern (pattern 4b) except for two isolates (pattern 4c and pattern 4). Furthermore, the genotype of the nonsusceptible isolates from school A that were obtained after treatment was identical to that of the susceptible isolates from that school (Table 3).
PCR and Southern blot hybridization. Ten isolates (one from school A and nine from school B) were tested by PCR. AmpC -lactamase was responsible for ceftriaxone resistance in all ceftriaxone-resistant isolates, and Southern blot hybridization confirmed that the resistance gene was located on the plasmid (Table 3). DNA sequencing of the PCR products confirmed that all ceftriaxone-resistant isolates harbored CMY-2 and SHV-5 -lactamase genes. Two isolates contained the SHV-5 gene only and remained susceptible to ceftriaxone.
DISCUSSION
Shigella spp. have managed to survive the antibiotic era via an ingenious mechanism of resistance, the production of -lactamases. High rates of resistance to ampicillin among Shigella isolates are due to the production of -lactamases similar to TEM-1 or OXA-1 (21, 28, 29). In the past 2 decades both the isolation frequencies and the types of extended-spectrum -lactamase (ESBL) have gradually increased. ESBLs are detected most commonly in Klebsiella pneumoniae and Escherichia coli but have been noted in other members of the family Enterobacteriaceae as well. However, no ESBL-producing Shigella strains were reported until 1999, when an SHV-11 ESBL-producing S. dysenteriae strain was reported in India (2). Recently, four other ESBL-producing strains have been reported from four different countries: an S. sonnei strain with a CTX-M-14-type ESBL from Korea (22), an S. sonnei strain with a CTX-M-2-type ESBL from Argentina (23), an S. flexneri strain with an SHV-2-type ESBL from France (8), and an S. sonnei strain with a CTX-M-3-type ESBL from Turkey (1). To the authors' knowledge, this is the first report that CMY-2-type AmpC -lactamase was responsible for ceftriaxone resistance in resistant isolates of S. sonnei; Southern blot hybridization confirmed that the resistance gene was located on the plasmid.
The most common typing procedures currently used with Shigella spp. are plasmid analysis and PFGE (9, 11, 16, 17, 30). Shigella species usually harbor a heterogeneous population of plasmids, ranging in numbers from 2 to as many as 10 (12). Plasmid analysis has proven to be a useful typing technique (9, 11); it is inexpensive and quick to perform. However, many plasmids are unstable and may be easily gained and/or lost, thus limiting this method's capacity in delineating genetic relatedness. Conversely, PFGE has a high discriminatory power, yet the procedure is time-consuming and expensive. In this outbreak, PFGE analysis demonstrated that all the isolates expressed similar or identical DNA fingerprints, indicating that these isolates might originate from the same source. Remarkably, the antibiograms were distinctively different among the isolates from the two schools. Shigellosis is relatively infrequent in Taiwan, with the incidence being 0.006% in the year 2001. Although the direct association with a food/water source was not established, the outbreaks happened very close together in terms of timing and geographic distance. Thus, it is biologically plausible that the outbreaks at the two schools were closely related while a parental strain might have acquired the resistant plasmid, leading to the expression of different antibiograms and spread in one of the schools.
In community-based observational studies, Shigella species usually are isolated from children with diarrhea and are seldom isolated from control patients who do not have diarrhea (3, 5, 6). Therefore, it has been inferred that infection with Shigella is virtually synonymous with disease. However, in this study, nearly half of the patients (23/47) were asymptomatic. The symptoms/signs of infected children comprise a clinical spectrum ranging from asymptomatic infections and secretory diarrhea to bloody diarrhea. Notably, bloody stool was noted in only 16.7% of the symptomatic patients.
Of note, antibiotic resistance appears to develop during the course of treatment, as shown in the two isolates obtained from patients of school A who received 5-day ampicillin treatment. Emergence of the resistance gene because of selective pressure may have contributed to the newly developed resistance. In this study, nine children from school A received a 2-day course of treatment to eradicate the organism from the stool. A treatment course of 5 days has been recommended for shigellosis. However, a shorter treatment course has been proposed as well (7, 27) and has demonstrated clinical and bacteriological cure rates comparable to those of the longer course. This would limit the environmental pressure for selection and dissemination of resistance genes among bacterial populations. In those instances when antibiotics are mandatory, short but effective courses of treatment may be warranted. A double-blind randomized study is mandatory to determine the optimal regimen and duration of treatment for shigellosis.
In summary, this is the first report on the CMY-2-type AmpC -lactamase in S. sonnei. The gene was located on a plasmid and might be responsible for the spread of ceftriaxone resistance among isolates. Emergence of resistance could occur during the course of treatment; thus, close monitoring is mandatory.
ACKNOWLEDGMENTS
We thank Marilyn M. Wagener for her expertise and insight in reviewing the manuscript and for her helpful comments.
This work was supported by the Veterans General Hospital Kaohsiung grant number VGHKS 93-085.
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