RESUMO
Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype-based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid pro-filing showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.
Assuntos
Escherichia coli/classificação , Microbiologia da Água , Bangladesh , Resistência Microbiana a Medicamentos , Eletroforese em Gel de Campo Pulsado/métodos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Fenótipo , Plasmídeos , Sorotipagem/métodos , Fatores de Virulência/genéticaAssuntos
DNA Bacteriano/análise , Infecções por Salmonella/epidemiologia , Infecções por Salmonella/microbiologia , Salmonella enteritidis , Meios de Cultura , Surtos de Doenças , Fezes/microbiologia , Humanos , Japão/epidemiologia , Filogenia , Salmonella enteritidis/classificação , Salmonella enteritidis/genética , Salmonella enteritidis/isolamento & purificação , Salmonella enteritidis/metabolismoRESUMO
We analyzed the resistance to cefotaxime of a Salmonella enterica serovar Enteritidis isolate from a stool culture of a 4-year-old boy. It produced a beta-lactamase CTX-M-14, encoded by two related R plasmids. The region surrounding the blaCTX-M-14 gene had an original mosaic structure containing insertion sequences (IS26 and IS903D).
Assuntos
Antibacterianos/farmacologia , Cefotaxima/farmacologia , Resistência às Cefalosporinas/genética , Salmonella enteritidis/enzimologia , beta-Lactamases/metabolismo , Pré-Escolar , Fezes/microbiologia , Humanos , Japão , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fatores R/genética , Infecções por Salmonella , Salmonella enteritidis/efeitos dos fármacos , Análise de Sequência de DNA , beta-Lactamases/genéticaRESUMO
Escherichia coli strain TUM2139 was isolated from a stool sample from a 9-year-old girl on 16 June 2004. This strain was categorized as Shiga toxin-producing Escherichia coli (STEC) because the Shiga-like toxin gene stx(1) was detected by immunochromatography and PCR assay. The strain was highly resistant to cefotaxime (256 microg/ml) and was also resistant to cefepime, cefpodoxime, ceftriaxone, and aztreonam. In the presence of 4 microg of clavulanic acid per ml, the MIC of cefotaxime decreased to < or =0.12 microg/ml, indicating that this strain was an extended-spectrum beta-lactamase (ESBL) producer. Cefotaxime resistance was transferred to E. coli C600 by conjugation at a frequency of 3.0 x 10(-6). A PCR assay was performed with primer sets specific for TEM-type and SHV-type ESBLs and for the CTX-M-2 (Toho-1), CTX-M-3, and CTX-M-9 groups of ESBLs. A specific signal was observed with the primer set specific for the CTX-M-9 group of beta-lactamases. This beta-lactamase was confirmed to be the ESBL CTX-M-18 by DNA sequencing. This is the first report of an ESBL-producing STEC isolate.
Assuntos
Escherichia coli/enzimologia , Toxina Shiga/genética , beta-Lactamases/biossíntese , Criança , Conjugação Genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/patogenicidade , Feminino , Humanos , Testes de Sensibilidade MicrobianaRESUMO
We performed susceptibility testing with Shigella sonnei isolates from imported and domestic cases of infection in Japan during 2001 and 2002. Some S. sonnei isolates were resistant to nalidixic acid, tetracycline, and trimethoprim-sulfamethoxazole. Most of the nalidixic acid-resistant strains showed reduced susceptibility to fluoroquinolones but did not show fluoroquinolone resistance.
Assuntos
Fluoroquinolonas/farmacologia , Shigella sonnei/efeitos dos fármacos , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Ácido Nalidíxico/farmacologia , Shigella sonnei/genéticaRESUMO
Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T. Sato, T. Shimizu, M. Watarai, M. Kobayashi, S. Kano, T. Hamabata, Y. Takeda, and S. Yamasaki, Gene 309:35-48, 2003). Region V, which is ca. 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli (STEC) on the basis of the diversity of region V. When region V was amplified by long and accurate-PCR (LA-PCR) with five control E. coli strains carrying six different Stx phages such as E. coli strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained. Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains. When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained. Two STEC O157 strains, however, did not produce any bands. Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups. The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area. To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay. In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern. Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks.
Assuntos
Infecções por Escherichia coli/epidemiologia , Escherichia coli/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Toxinas Shiga/biossíntese , Surtos de Doenças , Eletroforese em Gel de Campo Pulsado , Escherichia coli/classificação , Infecções por Escherichia coli/microbiologia , Humanos , Epidemiologia MolecularRESUMO
Among roughly one thousand incidents of shigellosis annually in Japan, approximately 70% of the cases are estimated to be associated with overseas travel. However, at the end of 2001, reports of domestically acquired Shigella sonnei infections suddenly increased. We report here the first multi-prefectural outbreak of Shigella sonnei infections linked to the consumption of imported oysters in Japan at the end of 2001. Isolates of S. sonnei from patients epidemiologically linked to eating contaminated oysters and from the imported oysters themselves showed an indistinguishable pulsed-field gel electrophoresis pattern and drug resistance pattern.
Assuntos
Surtos de Doenças , Disenteria Bacilar/epidemiologia , Disenteria Bacilar/microbiologia , Doenças Transmitidas por Alimentos/microbiologia , Ostreidae/microbiologia , Shigella sonnei/classificação , Shigella sonnei/isolamento & purificação , Animais , Técnicas de Tipagem Bacteriana , Impressões Digitais de DNA , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Doenças Transmitidas por Alimentos/epidemiologia , Humanos , Japão/epidemiologia , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Polimorfismo de Fragmento de Restrição , Shigella sonnei/efeitos dos fármacosRESUMO
A multiplex polymerase chain reaction (PCR) assay was developed for the identification of Salmonella enterica serovar Typhimurium. Three sets of primers were designed for detecting O4, H:i, and H:1,2 antigen genes from the antigen-specific genes rfbJ, fliC, and fljB, respectively. These were evaluated in a multiplex PCR assay by using DNAs from S. enterica serovar Typhimurium, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens. Multiplex PCR proved to be capable of identifying S. enterica serovar Typhimurium specifically and differentiating it from other Salmonella serovars in addition to non-Salmonella enteric pathogens. Thus, this multiplex PCR assay can be practically applied to the identification of S. enterica serovar Typhimurium.
Assuntos
Reação em Cadeia da Polimerase/métodos , Salmonella typhimurium/isolamento & purificação , Salmonella typhimurium/classificação , Salmonella typhimurium/genéticaAssuntos
Citrobacter , Enterobacter , Infecções por Enterobacteriaceae , Infecções por Proteus , Proteus , Antibacterianos/uso terapêutico , Citrobacter/patogenicidade , Enterobacter/patogenicidade , Infecções por Enterobacteriaceae/complicações , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/microbiologia , Humanos , Hospedeiro Imunocomprometido , Infecções Oportunistas/complicações , Infecções Oportunistas/microbiologia , Proteus/patogenicidade , Infecções por Proteus/microbiologiaRESUMO
Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.
Assuntos
Anti-Infecciosos/farmacologia , Resistência Microbiana a Medicamentos/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Salmonella paratyphi A/efeitos dos fármacos , Salmonella typhi/efeitos dos fármacos , DNA Girase/genética , DNA Girase/metabolismo , Fluoroquinolonas , Humanos , Testes de Sensibilidade Microbiana , Mutação , Febre Paratifoide/microbiologia , Salmonella paratyphi A/enzimologia , Salmonella paratyphi A/genética , Salmonella typhi/enzimologia , Salmonella typhi/genética , Febre Tifoide/microbiologiaAssuntos
Microbiologia de Alimentos , Intoxicação Alimentar por Salmonella/microbiologia , Infecções por Salmonella/microbiologia , Salmonella enteritidis/genética , Tipagem de Bacteriófagos , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Japão/epidemiologia , Dados de Sequência Molecular , Plasmídeos , Intoxicação Alimentar por Salmonella/epidemiologia , Infecções por Salmonella/epidemiologia , Salmonella enteritidis/classificação , Salmonella enteritidis/patogenicidade , Análise de Sequência de DNARESUMO
Enterohemorrhagic Escherichia coli O157 is one of the leading worldwide public health concerns, causing large outbreaks of hemorrhagic colitis as well as numerous small outbreaks and sporadic cases. The variability of restriction enzyme-digestion patterns of O157 genomes, which is widely used to distinguish strains in the molecular epidemiology of O157 infections, suggests the presence of some genomic diversity among the strains. Based on the complete genome sequence of O157 Sakai, we analyzed the whole genome structures of eight O157 strains displaying diverse XbaI-digestion patterns by a systematic PCR analysis that we have named whole genome PCR scanning. This analysis identified not only the O157-specific sequences that are highly conserved among the strains, but also revealed an unexpectedly high degree of genomic diversity. In particular, prophages, including Shiga toxin-transducing phages, exhibited extensive structural and positional diversity, implying that variation of bacteriophages is a major factor in generating genomic diversity among the O157 lineage.
Assuntos
Escherichia coli O157/genética , Genoma Bacteriano , Reação em Cadeia da Polimerase , Cromossomos Bacterianos , Colífagos/genética , Variação Genética , Toxina Shiga/genéticaRESUMO
The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.
Assuntos
Anti-Infecciosos/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Salmonella paratyphi A/efeitos dos fármacos , Salmonella typhi/efeitos dos fármacos , Fluoroquinolonas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Salmonella paratyphi A/enzimologia , Salmonella paratyphi A/genética , Salmonella typhi/enzimologia , Salmonella typhi/genética , Análise de Sequência de DNAAssuntos
Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Bacillus subtilis/genética , Primers do DNA/genética , Genes Bacterianos/genética , Reação em Cadeia da Polimerase/métodos , Antraz/microbiologia , Artefatos , Bacillus subtilis/isolamento & purificação , Bioterrorismo , Especificidade por SubstratoRESUMO
Genotyping of 1,102 enterohemorrhagic Escherichia coli isolates by the use of pulsed-field gel electrophoresis (PFGE) carried out from January to November 2000 has revealed the high genomic diversity of these isolates in Japan. By combining the results of genotyping of the isolates with the information from other epidemiological investigations of the cases, we identified a diffuse outbreak in Japan in the year 2000 that seemed to be sporadic but was actually linked. Isolates with only the Shiga toxin 2 gene derived from patient specimens and the contaminated food involved in this diffuse outbreak showed an indistinguishable PFGE profile and the same phage type. Based on the diversity of genotypes among the isolates of enterohemorrhagic E. coli O157:H7/- in Japan, we suggest the presence of a few other possible diffuse outbreaks due to the organisms, showing indistinguishable genotypes.
Assuntos
Surtos de Doenças , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Variação Genética/genética , Genoma Bacteriano , Eletroforese em Gel de Campo Pulsado , Escherichia coli O157/classificação , Escherichia coli O157/patogenicidade , Microbiologia de Alimentos , Genes Bacterianos/genética , Genótipo , Humanos , Japão/epidemiologia , Filogenia , Toxina Shiga II/genéticaRESUMO
The PCR primers for O, H, and Vi antigen genes, tyv (rfbE), prt (rfbS), fliC-d, fliC-a, and viaB, were designed and used for the rapid identification of Salmonella enterica serovars Typhi and Paratyphi A with multiplex PCR. The results showed that all the clinical isolates examined of Salmonella serovars Typhi and Paratyphi A were accurately identified by this assay.