Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis as a reliable evidence for suspected Shigella spp. outbreaks

Abstract Background Shigellosis remains a serious public health problem and an important cause of morbidity and mortality worldwide. The aim of this study was to characterize fliC and the genetic relatedness of Shigella spp. isolated during a one-year period from children in a suspected outbreak in Tehran, Iran. Methods and results Fifty Shigella spp. were isolated from 3779 stool samples of children with diarrhea (prevalence rate: 1.32%). Among the isolates, 92% were characterized as Shigella sonnei, while 6% and 2% were identified as S. flexneri and S. boydii, respectively. S. dysenteriae was not recovered from the patients. All isolates were negative for fliC except for Shigella standard strains. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) profiles allowed differentiating the 50 isolates into 5 ERIC types, which were grouped into five clusters (ET1-ET5). Computer-assisted clustering of the strains showed a high degree of similarity among the isolates. Conclusion In conclusion, given the clonal correlation of the Shigella strains isolated in this study and the lack of fliC among them, we propose that probably a single or limited fliC-defected Shigella clone spread and caused the outbreak.

The importance of integrons for development and propagation of resistance in Shigella: the case of Latin America

Abstract In Latin America, the disease burden of shigellosis is found to coexist with the rapid and rampant spread of resistance to commonly used antibiotics. The molecular basis of antibiotic resistance lies within genetic elements such as plasmids, transposons, integrons, genomic islands, etc., which are found in the bacterial genome. Integrons are known to acquire, exchange, and express genes within gene cassettes and it is hypothesized that they play a significant role in the transmission of multidrug resistance genes in several Gram-negative bacteria including Shigella. A few studies have described antibiotic resistance genes and integrons among multidrug resistant Shigella isolates found in Latin America. For example, in Brazil, Bolivia, Chile, Costa Rica and Peru, class 1 and class 2 integrons have been detected among multidrug resistant strains of Shigella; this phenomenon is more frequently observed in S. flexneri isolates that are resistant to trimethoprim, sulfamethoxazole, streptomycin, ampicillin, chloramphenicol, and tetracycline. The gene cassette sul2, which is frequently detected in Shigella strains resistant to the sulfonamides, suggests that the sulfonamide-resistant phenotype can be explained by the presence of the sul2 genes independent of the integron class detected. It is to be noted that sul3 was negative in all isolates analyzed in these studies.The high frequency of sulfonamide (as encoded by sul2) and trimethoprim resistance is likely to be a result of the recurrent use of trimethoprim sulfamethoxazole as a popular regimen for the treatment of shigellosis. The observed resistance profiles of Shigella strains confirm that ampicillin and trimethoprim-sulfamethoxazole are ineffective as therapeutic options. In-depth information regarding antibiotic resistance mechanism in this pathogen is needed in order to develop suitable intervention strategies. There is a pressing need for regional and local antimicrobial resistance profiling of Shigella to be included as a part of the public health strategy.

Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region.

Background & objectives: There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. Methods: Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥0.25 μg/ml), SFM2 (≥4 μg/ml) and SFM3 (≥32 μg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥0.25 μg/ml) and SDM2 (≥4 μg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. Results: Mutations were observed in gyrA Ser83→Leu, Asp87→Asn/Gly, Val196→Ala and in parC Phe93→Val, Ser80→Ile, Asp101→Glu and Asp110→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance (p<0.05); while tolC and acrR expression levels did not. Interpretation & conclusions: Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val196→Ala in gyrA in clinical isolates and Phe93→Val, Asp101→Glu, Asp110→Glu and in parC in majority of laboratory-grown mutants.

Relationship among Shigella spp. and enteroinvasive Escherichia coli (EIEC) and their differentiation

Shigellosis produces inflammatory reactions and ulceration on the intestinal epithelium followed by bloody or mucoid diarrhea. It is caused by enteroinvasive E. coli (EIEC) as well as any species of the genus Shigella, namely, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. This current species designation of Shigella does not specify genetic similarity. Shigella spp. could be easily differentiated from E. coli, but difficulties observed for the EIEC-Shigella differentiation as both show similar biochemical traits and can cause dysentery using the same mode of invasion. Sequencing of multiple housekeeping genes indicates that Shigella has derived on several different occasions via acquisition of the transferable forms of ancestral virulence plasmids within commensal E. coli and form a Shigella-EIEC pathovar. EIEC showed lower expression of virulence genes compared to Shigella, hence EIEC produce less severe disease than Shigella spp. Conventional microbiological techniques often lead to confusing results concerning the discrimination between EIEC and Shigella spp. The lactose permease gene (lacY) is present in all E. coli strains but absent in Shigella spp., whereas β-glucuronidase gene (uidA) is present in both E. coli and Shigella spp. Thus uidA gene and lacY gene based duplex real-time PCR assay could be used for easy identification and differentiation of Shigella spp. from E. coli and in particular EIEC.

Plasmid-Encoded Multidrug Resistance: A Case study of Salmonella and Shigella from enteric diarrhea sources among humans

Salmonellosis and shigellosis are signifcant and persistent causes of diarrheal diseases among humans in developing countries. With that in mind, the current study investigates the occurrence of plasmid-encoded multidrug resistances in Salmonella and Shigella from diarrheal cases among humans. The isolates were characterized by serotyping, antimicrobial-susceptibility testing, transfer experiments and curing. The extended spectrum β-lactamase (ESBL) was detected by the double disc diffusion synergy test (DDST). A signifcant number of the plasmid-encoded multidrug resistant (PEMDR) Salmonella and Shigella isolates were found to harbour transferable plasmid genes resistant to antibiotics like ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, ceftriaxone, cefuroxime and to a lesser extent to ciprofoxacin and ofoxacin. The conjugative R-plasmids-encoded extended-spectrum β-lactamase also showed resistances to cephalosporins (ceftriaxone and cefuroxime) and ampicillin. Curing experiments showed chromosomal resistances to varied antibiotics. The fndings confrmed the presence of PEMDR in Salmonella and Shigella strains as a suitable adaptation to a changing antibiotic environment. The results therefore suggest the limited use of the commonly prescribed/or third generation cephalosporins as an empirical treatment of multidrug resistant Salmonella and Shigella because this may affect therapeutic outcomes.

Characterization of Shigella spp. by antimicrobial resistance and PCR detection of ipa genes in an infantile population from Porto Velho (Western Amazon region), Brazil

The incidence of Shigella spp. was assessed in 877 infants from the public hospital in Rondônia (Western Amazon region, Brazil) where Shigella represents the fourth cause of diarrhea. Twenty-five isolates were identified: 18 were Shigella flexneri, three Shigella sonnei, three Shigella boydii and one Shigella dysenteriae. With the exception of S. dysenteriae, all Shigella spp. isolated from children with diarrhea acquired multiple antibiotic resistances. PCR detection of ipa virulence genes and invasion assays of bloody diarrhea and fever (colitis) were compared among 25 patients testing positive for Shigella. The ipaH and ipaBCD genes were detected in almost all isolates and, unsurprisingly, all Shigella isolates associated with colitis were able to invade HeLa cells. This work alerts for multiple antibiotic resistant Shigella in the region and characterizes presence of ipa virulence genes and invasion phenotypesin dysenteric shigellosis.

Epidemiología clínica y molecular de las infecciones por Shigella spp en niños de la Región Metropolitana durante el verano 2004-2005 / Shigella spp Infections In children living In the Metropolitan RegionChile, during summer of 2004-2005

Background: Shigella spp is a frequent cause of diarrhea in children. Antimicrobials decrease the duration of diarrhea and pathogen excretion. However, the increasing resistance limits their therapeutic value. Aim: To study Shigella serotype distribution in the Metropolitan Region in Chile, and its relationship with severity of disease, antimicrobial resistance pattern and clonality. Material and methods: During summer 2004-2005, stool samples from children with diarrhea were collected in Cary Blair transpon medium and cultured. Shigella isolates were serotyped using monoclonal and polyclonal commercial antibodies. In vitro activity of ampicillin, amoxicillin/clavulanic acid, chloramphenicol, cotrimoxazol, nalidixic acid, ciprofloxacin, ceftriaxone and azythromycin was determined by minimal inhibitory concentration (MIC). Clonality was studied by pulsed-field gel electrophoresis (PFGE) using Xbal as restriction enzyme. Results: One hundred thirty nine Shigella strains were isolated (77 S sonnei and 62 S flexneri). S sonnei and S flexneri 2a serotypes were responsible for 95 percent of episodes. Children aged 2-4 years, showed a greater incidence of Shigella infections and 77 percent of episodes were treated on an ambulatory basis. High resistance levels were observed for ampicillin, cotrimoxazole, amoxicillin-clavulanic acid and chloramphenicol (67 percent, 60 percent, 56 percent and 45 percent, respectively). We found 11 resistance patterns and 61,2 percent of strains were multiresistant. There were multiple clones without a strict relationship with resistance patterns. Conclusions: Shigella infections in Metropolitan Region in Chile are associated to a restricted number of serotypes, representing a clonal expansion associated to different antimicrobial resistant patterns.

Shigellosis : challenges & management issues.

Infectious diseases kill about 11 million children each year while acute diarrhoeal diseases account for 3.1 million deaths in children under 5 yr of age, of which 6,00,000 deaths annually are contributed by shigellosis alone. Shigellosis, also known as acute bacillary dysentery, is characterized by the passage of loose stools mixed with blood and mucus and accompanied by fever, abdominal cramps and tenesmus. It may be associated with a number of complications of which haemolytic uraemic syndrome is the most serious. Shigellosis is caused by Shigella spp. which can be subdivided into four serogroups namely S.sonnei, S.boydii, S.flexneri and S.dysenteriae. Organisms as low as 10-100 in number can cause the disease. Shigellosis can occur in sporadic, epidemic and pandemic forms. Epidemics have been reported from Central American countries, Bangladesh, Sri Lanka, Maldives, Nepal, Bhutan, Myanmar and from the Indian subcontinent, Vellore, eastern India and Andaman and Nicobar islands. Plasmid profile of shigellae in Kolkata has shown a correlation between presence of smaller plasmids and shigellae serotypes- indicating epidemiological changes of the species. Diagnosis of shigellosis is essentially clinical. Laboratory diagnosis includes stool culture and polymerase chain reaction (PCR). Treatment includes use of an effective antibiotic, rehydration therapy (if there is dehydration) and appropriate feeding during and after an episode of shigellosis. Hand-washing is the single most important strategy for prevention of transmission of shigellosis from person to person. A safe and effective vaccine should be developed against the more important circulating strains i.e., S. dysenteriae type 1 and S. flexneri 2a.

Resistencia a antibióticos y epidemiología molecular de Shigella spp. en el nordeste argentino / Antibiotic resistance and molecular epidemiology of Shigella spp. in northeastern Argentina

OBJETIVOS: Evaluar la resistencia a antibióticos de cepas de Shigella spp. aisladas de muestras de heces en el nordeste argentino y caracterizarlas desde el punto de vista de su epidemiología molecular. MÉTODOS: Se estudiaron 132 aislamientos de Shigella spp. obtenidos de las heces de igual número de pacientes con diarrea que asistieron a diferentes laboratorios privados y estatales de las provincias del Chaco y Corrientes, Argentina, durante el período de 1998 a 2002. Cada cepa se caracterizó según su serotipo, su resistencia a 13 antibióticos individuales o combinados y su sensibilidad a las piocinas. A 52 cepas seleccionadas en función de sus perfiles de susceptibilidad antimicrobiana se les determinaron la dotación plasmídica mediante lisis alcalina y las secuencias repetitivas palindrómicas extragénicas mediante la amplificación de segmentos repetitivos de ADN con la reacción en cadena de la polimerasa (REP-RCP). Se aplicó la prueba de ji al cuadrado para comparar proporciones. El nivel de significación estadística fue de 0,05. RESULTADOS: Shigella flexneri fue la especie más frecuente (78 por ciento), seguida de S. sonnei (22 por ciento). En general, la resistencia de S. flexneri a los antibióticos estudiados fue mayor que la de S. sonnei y esta diferencia fue estadísticamente significativa (P <0,001) frente a ampicilina, tetraciclina, cloramfenicol y la combinación de ampicilina con sulbactama. Las cepas de S. flexneri también mostraron mayor multirresistencia que las de S. sonnei (84,5 por ciento frente a 31,0 por ciento; P <0,001). Las cepas aisladas de S. flexneri pudieron agruparse según 5 piocinotipos, 3 perfiles plasmídicos y 5 patrones de secuencias repetitivas palindrómicas. Por su parte, las cepas de S. sonnei conformaron 3 piocinotipos, 2 perfiles plasmídicos y 3 patrones de secuencias repetitivas palindrómicas. CONCLUSIONES: Las especies de Shigella estudiadas mostraron una elevada resistencia a los antibióticos de uso más frecuente, por lo que se deben poner en marcha actividades de vigilancia a fin de detectar y controlar la aparición de nuevas cepas resistentes. La aplicación de técnicas de tipificación epidemiológica puede ayudar a conocer con mayor precisión la distribución y evolución de cepas de microorganismos resistentes circulantes.