A high number of multidrug-resistant and predominant genetically related cluster of Shigella flexneri strains isolated over 34 years in Brazil.

Shigella flexneri has been a major public health problem in developing countries. This work analyzed the frequency of 16 virulence genes, the genotypic diversity, and the antimicrobial resistance profiles of 130 S. flexneri strains isolated in Brazil. The ipaH gene was found in all the 130 strains. The frequencies of the other genes were variable ial (88.5%), sigA (82.3%), iuc (74.6%), virA (73%), pic (72.3%), virF (57.7%), sat (48.5%), ipaBCD (37%), sen (36%), set1A (35.4%), sepA (30%), set1B (30%), virB (14%), icsA (10%), and ipgD (5.4%). A total of 57 (43.8%) strains were multidrug-resistant. ERIC-PCR grouped 96 of the strains into a single cluster with ≥ 70.4% of similarity, 75 of these strains presented a similarity ≥ 80.9%. PFGE grouped 120 of the strains into a single cluster with 57.4% of similarity and 82 of these strains presented a similarity ≥ 70.6%. In conclusion, the high frequency of some virulence genes reinforces the pathogenic potential of the strains studied. The high rates of MDR strains are alarming once it may lead to failure when antimicrobial treatment is necessary. Genotype techniques reveled a major cluster with high genetic similarity including S. flexneri strains from the different Brazilian states and distinct years of isolation, showing that they probably emerged from a common ancestor.

PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation.

Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.

Design of Two Multiplex PCR Assays for Serotyping Shigella flexneri.

Shigella flexneri is a major health problem in developing countries. There are 19 serotypes recognized based on O-antigen structure and its typing is important for epidemiological purposes. However, the diversity of serotypes and the difficulties presented by phenotypic serotyping, for example, unavailable antisera for less common antigens, require the implementation of molecular techniques. In this study, we developed two multiplex PCR assays targeting the O-antigen synthesis genes and the O-antigen modification genes, for the rapid identification of S. flexneri serotypes 1/7, 2, 4, 5, and 6 (PCR A) and serotype 7 and group antigenic factors (3,4; 6; 7,8; E1037) (PCR B). A total of 73 S. flexneri strains representing 18 serotypes, except serotype 1d, were used in the study. Specific amplification patterns were obtained for each of the different serotypes. All strains tested had concordant results with phenotypic and genotypic serotyping; therefore, its implementation in the microbiology clinical laboratory will significantly improve S. flexneri serotyping.

Prevalence and Virulence Genes of Shigella spp. Isolated from Patients with Diarrhea in Rosario, Argentina.

The aim of this study was to determine the prevalence and virulence factors of Shigella species isolated from patients with diarrhea. Shigella species were isolated from 1,022 stool samples collected from different hospitals in Rosario, Argentina. The isolates were characterized using phenotypic tests, serotyping, and detection of virulence genes by PCR. One hundred strains (9.8% of samples collected) of Shigella were isolated. Shigella flexneri was the most frequently identified species (74%), followed by S. sonnei (26%). S. flexneri was also the predominant species isolated from children aged 6-14 years. These clinical strains of Shigella were then tested for the presence of ipaH, virA, ial, sen, and set using specific primers. virA was present in all strains, whereas ipaH was detected in 98% of strains and ial in 83%. sen was found in 71.6% of S. flexneri and 42.3% of S. sonnei isolates, and 41.9% of S. flexneri isolates were positive for set. Furthermore, 32.4% of S. flexneri isolates were positive for both set and sen. This study provides data on the prevalence and distribution of diverse Shigella strains.

AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri.

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100% correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological data.

Clinical isolates of Shiga toxin 1a-producing Shigella flexneri with an epidemiological link to recent travel to Hispañiola.

Shiga toxins (Stx) are cytotoxins involved in severe human intestinal disease. These toxins are commonly found in Shigella dysenteriae serotype 1 and Shiga-toxin-producing Escherichia coli; however, the toxin genes have been found in other Shigella species. We identified 26 Shigella flexneri serotype 2 strains isolated by public health laboratories in the United States during 2001-2013, which encode the Shiga toxin 1a gene (stx1a). These strains produced and released Stx1a as measured by cytotoxicity and neutralization assays using anti-Stx/Stx1a antiserum. The release of Stx1a into culture supernatants increased ≈100-fold after treatment with mitomycin C, suggesting that stx1a is carried by a bacteriophage. Infectious phage were found in culture supernatants and increased ≈1,000-fold with mitomycin C. Whole-genome sequencing of several isolates and PCR analyses of all strains confirmed that stx1a was carried by a lambdoid bacteriophage. Furthermore, all patients who reported foreign travel had recently been to Hispañiola, suggesting that emergence of these novel strains is associated with that region.

Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates.

Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm.

A new multiplex PCR for differential identification of Shigella flexneri and Shigella sonnei and detection of Shigella virulence determinants.

Most of the multiplex PCR (mPCR) used to identify Shigella do not discriminate between Shigella species or serotypes. We designed a mPCR to differentiate between S. flexneri and S. sonnei strains based on the detection of markers associated with the she pathogenicity island described in Shigella. In addition, specific primers were included to detect the Shigella virulence determinants ShET-1 and ShET-2 enterotoxin genes. The analysis of 304 Shigella strains from Chile and 79 Shigella strains from other geographic locations indicated that the mPCR described here detected all Shigella species and specifically differentiated S. flexneri and S. sonnei. The technique was sensitive, reproducible, specific and simple to perform, providing a new tool with the potential to be employed for epidemiological and diagnostic purposes.

Molecular epidemiology of Shigella flexneri in a diarrhoea-endemic area of Lima, Peru.

A year-long community-based study of diarrhoeal diseases was conducted in Canto Grande, a periurban community in Lima, Peru. In 109 (34%) houses out of 323 that were visited, at least one individual was detected with shigellosis. The frequency of the 161 shigella isolates obtained was as follows: 117 S. flexneri (73%), 21 S. boydii (13%), 15 S. dysenteriae (9%), and 8 S. sonnei (5%). Using a non-radioactive ipaH gene probe as a molecular epidemiological tool, a total of 41 S. flexneri strains were shown to be distributed in 25 intra-family comparisons by pairs (icp). Further subdivision, based on a comparison of the serotype, plasmid profile, antibiotic resistances and ipaH hybridization patterns indicated that Group I, with 11 icp (44%), had strains that were identical. Group II with 8 icp (32%), had strains that were different and Group III with 6 icp (24%), had strains with the same serotype and identical ipaH profiles but with differences in other markers. This data indicates that a diversity of shigella clones circulated in this community resulting from both clonal spread and horizontal transfer of genetic elements. Furthermore, ipaH profiling of isolates can be used not only to differentiate between closely related shigella strains but also with other parameters, help to understand the dynamics of the generation of new clones of pathogenic bacteria.

Shigella flexneri strains produce bacteriocins active against members of the human microbial intestinal flora.

The principal aim of this work was to detect the bacteriocinogenic capacity of S. flexneri strains on members of the human intestinal flora. A total of 49 bacteriocinogenic S. flexneri strains were isolated from individuals of both sexes and different ages. The bacteriocins were detected by means of the drop method using E. coli and B. fragilis as target strains. The serotypes of the S. flexneri were determined. The producer capacity of bacteriocins was analysed in 10 different colonies of the same cellular clone and also the arbitrary units were determined. The highest number of bacteriocinogenic S. flexneri strains were obtained from diarrhoeal individuals from 0-10 years old and the S. flexneri serotype 2a was the most abundant. It was demonstrated that E. coli and B. fragilis isolated from the normal intestinal flora of healthy individuals were susceptible to the bacteriocinogenic S. flexneri strains. By means of the determination of arbitrary units per ml of the bacteriocin, it was demonstrated that colonies from a single colony isolate of a same clone of bacteriocinogenic S. flexneri produce different quantities of bacteriocin.

[Acute diarrhea outbreak caused by Shigella flexneri at a school in Madrid, Cundinamarca: phenotypic and genotypic characterization of the isolates]. / Brote de enfermedad diarreica aguda causado por Shigella flexneri en una escuela de Madrid, Cundinamarca: caracterización fenotípica y genotípica de los aislamientos.

Shigellosis is an acute diarrhoeal disease that is the main cause of morbidity and mortality in developing countries. In 1997, the Colombian Instituto Nacional de Salud Microbiology Group organized a network surveillance program with the country's Public Health Laboratories (PHLs) to monitor the principal etiological agents responsible for acute diarrhoeal disease. In May, 2001, the PHL of the state of Cundinamarca reported a food poisoning outbreak involving an elementary school community. The main goal of the Microbiology Group involvement was to establish the molecular relationships among the isolates from the outbreak by phenotypic and genotypic methods of characterization. Stool cultures were obtained from 22 of 195 affected individuals. The Microbiology Group confirmed the identification of the isolates by biochemical and serological probes. The antimicrobial susceptibilities were tested against the following battery of antibiotics: chloramphenicol, trimehoprim-sulfamethozazole, cefotaxime, gentamicin, ampicillin and ciprofloxacin. The isolates were subjected to pulsed field gel electrophoresis (PFGE) using the following CDC (U.S. Centers for Disease Control) protocols: Xbal restriction enzyme, Shigella sonnei CDC F2353 as the reference standard, and lambda phage as a molecular weight marker. In 15 of 22 (68%) stool cultures Shigella was recovered, all isolates were identified as Shigella flexneri serotype 6 biotype Newcastle with the same antimicrobial susceptibility profile. PFGE showed that 3 (20%) isolates were identical (100% genetic similarity) and the other 12 (80%) were very closely related (genetic similarity between 86-98%). The network system permitted the INS ready access to the isolates and the implementation of the PFGE permitted a quantitative characterization of the clonal relationship among the isolates from the outbreak.

Use of AFLP, plasmid typing and phenotyping in a comparative study to assess genetic diversity of Shigella flexneri strains.

Shigella flexneri infections are one of the main causes of acute diarrhoea in Cuba. Twenty strains isolated from sporadic cases in nine different Cuban provinces were characterized. Serotyping, antibiotic-resistance typing, plasmid-typing and AFLP-typing were used to determine their suitability for use in epidemiological studies of S. flexneri. The predominant serotypes were serotype 6 (35%) and serotype 2 (35%). Eleven different plasmid profiles were detected (Diversity Index = 0.92). AFLP-typing discriminated 12 different patterns (DI = 0.95), these patterns were not coincident with plasmid-typing patterns. Both techniques combined distinguished 14 patterns among the 20 studied strains (DI = 0.99). There was no consistent relationship between plasmid-typing and AFLP-typing patterns or antibiotic-resistance typing patterns. Ninety-five percent of S. flexneri strains were multiresistant.

An arginine residue at the active site of beta-lactamase from Shigella flexneri UCSF-129.

The role of a residue of arginine at the active site of beta-lactamase from Shigella flexneri UCSF-129 was studied. It is a local pathogenic strain which produces intestinal problems, especially in children. Purified enzymes were obtained by affinity chromatography on phenylboronic acid-agarose gels. The enzyme was serine dependent with a molecular weight of 23.6 kD. It was specifically modified with phenylglyoxal (1/830 molar ratio) and incubated for 20 min in the presence of 50 mM sodium phosphate buffer at pH 8.3. The chemical change was established by isoelectric focusing, since a loss of one positive charge was detected. Protection by cephradine, a substrate, indicated the presence of a residue of arginine at its active site. Controls were conducted by differential spectroscopy. Similar results were obtained with 2,3,butanedione. This vital arginine stabilizes the negative charge of the carboxylic group of C-3 or C-4 from the substrates.