Multiple- locus variable-number tandem-repeat analysis (MLVA) of Shigella sonnei isolates of 2012 outbreak I. R. Iran.

Shigella sonnei is a major cause of diarrhea especially in children. Molecular study can help to determine the outbreak of this bacterium. Multiple-Locus Variable number tandem repeat Analysis (MLVA) will largely influence the public health field by introducing newer, faster, safer, and effective procedure for typing of microorganisms. A total of fifty shigella isolates were collected between November 2012 to October 2013 in Tehran, Iran. The strains were identified base on biochemical and molecular tests. Subsequently, all shigella species were confirmed by species-specific polymerase chain reaction (PCR). Virulence factors were detected using PCR for ial, set1A, and set1B genes. The strains were genotyped by MLVA typing method. All of the isolates were identified as S. sonnei by biochemical and molecular (PCR) methods. Virulence genes identified among all isolates included ial, and set1A genes in 20% and 5% of all isolates, respectively. On the other hand, none of isolates were positive for set1B gene. Using MLVA method 22 MLVA types were identified. MLVA type 11 accounted for 32% of isolates. Moreover, all virulence factors were only detected in MLVA type 11, 9, 5, 4. The results of this study indicate that the Iranian 2012-2013 S. sonnei outbreak isolates were virulent and clonaly related. Furthermore, this study showed that MLVA can be used as useful method for S. sonnei genotyping in epidemiological investigations.

Towards a peptide-based vaccine against Shigella sonnei: A subtractive reverse vaccinology based approach.

Shigella sonnei is one of the major causes of shigellosis in technically advanced countries and reports of its unprecedented increase are published from the Middle East, Latin America, and Asia. The pathogen exhibits resistance against first and second line antibiotics which highlights the need for the development of an effective broad-spectrum vaccine. A computational based approach comprising subtractive reverse vaccinology was used for the identification of potential peptide-based vaccine candidates in the proteome of S. sonnei reference strain (53G). The protocol revealed three essential, host non-homologous, highly virulent, antigenic, conserved and adhesive vaccine proteins: TolC, PhoE, and outer membrane porin protein. The cellular interactome of these proteins supports their direct and indirect involvement in biologically significant pathways, essential for pathogen survival. Epitope mapping of these candidates reveals the presence of surface exposed 9-mer B-cell-derived T-cell epitopes of an antigenic, virulent, non-allergen nature and have broad-spectrum potency. In addition, molecular docking studies demonstrated the deep binding of the epitopes in the binding groove and the stability of the complex with the most common binding allele in the human population, DRB1*0101. Future characterization of the screened epitopes in order to further investigate the immune protection efficacy in animal models is highly desirable.

Hybrid genome assembly of Shigella sonnei reveals the novel finding of chromosomal integration of an IncFII plasmid carrying a mphA gene.

Azithromycin is increasingly being used for the treatment of shigellosis despite a lack of interpretative guidelines and with limited clinical evidence. The present study determined azithromycin susceptibility and correlated this with macrolide-resistance genes in Shigella spp. isolated from stool specimens in Vellore, India. The susceptibility of 332 Shigella isolates to azithromycin was determined using the disc diffusion method. Of these, 31 isolates were found to be azithromycin resistant. The azithromycin minimum inhibitory concentration (MIC) was determined using the broth microdilution method. In addition, isolates were screened for mphA and ermB genes using conventional PCR. Furthermore, an isolate that was positive for resistance genes was subjected to complete genome analysis, and was analysed for mobile genetic elements. The azithromycin MIC for the 31 resistant Shigella isolates ranged between 2 and 16 mg l-1. PCR results showed that a single isolate of Shigella sonnei carried a mphA gene. Complete genome analysis revealed integration of an IncFII plasmid into the chromosome of S. sonnei , which was also found to carry the following resistance genes: sul1, bla DHA1, qnrB4, mphA, tetR. Mutations in the quinolone-resistance-determining region (QRDR) were also observed. Additionally, prophages, insertion sequences and integrons were identified. The novel finding of IncFII plasmid integration into the chromosome of S. sonnei highlights the potential risk of Shigella spp. becoming resistance to azithromycin in the future. These suggests that it is imperative to monitor Shigella susceptibility and to study the resistance mechanism of Shigella to azithromycin considering the limited treatment choices for shigellosis.

The Prevalence of Shiga Toxin-1 in Non-Shigella Dysenteriae Isolates Collected from Diarrhea Samples in Patients, Ahvaz, Iran.

BACKGROUND: Acute diarrhea is a major public health problem, particularly in developing countries. Shigellosis is one of the substantial causative agents of microbial dysentery and still has a remarkable prevalence, particularly in areas with poor hygienic infrastructures. The probable existence of the deadly Shiga toxin (Stx) protein in some Shigella strains would manifest life-threatening clinical symptoms of the infection. METHODS: The aim of this study was to determine the presence of Shigella toxin 1 (Stx1) in isolated from patients with diarrhea. Totally, 227 Shigella species, including 60 S. flexneri, 157 S. sonnei, and 10 S. boydii were collected from diarrheal patients in the tropical infectious diseases research center of Ahvaz, Iran, during 2013-2015. The isolates were collected mostly from the intensive care unit, infectious disease, and surgery settings. The isolates were identified, and the polymerase chain reaction (PCR) was performed to detect the stx gene. RESULTS: The results indicated that none of them encode the stx1 gene. CONCLUSION: Isolates of this study were not capable of stx1 encoding. Future investigations should consider the relations between other Shigella species and Shigella toxin in Iran.

Surface-enhanced Raman spectroscopic-based aptasensor for Shigella sonnei using a dual-functional metal complex-ligated gold nanoparticles dimer.

Herein, we constructed an aptamer-based sensor for the sensitive and highly specific detection of Shigella sonnei via surface enhanced Raman spectroscopy (SERS) analysis. A composite material integrated of the Raman active 4-MBA ligand of the Eu-complex and citrate-stabilized Au nanoparticles (cit-Au NPs) was synthesized and served as both active substrate and Raman reporter. Aptamers targeted to S. Sonnei was then modified onto the surface of this dual-functional material. With the introduction of S. Sonnei, aptamer bound with target with high affinity and specificity, leaving the dual-functional material onto the bacteria. The SERS intensity response showed a strong positive linear correlation (R = 0.9956) with increasing concentrations of S. sonnei (ranging from 10 to 106 cfu/mL). High specificity was achieved at Shigella species (S. dysenteriae, S. flexneri, S. boydii) and other common bacteria (Salmonella typhimurium, Staphylococcus aureus and Escherichia coli). When applied in real samples, the approach showed recoveries from 92.6 to 103.8 %. The designed approach holds great potential for the construction of various aptasensors for the effective and convenient detection of different food hazards.

Increasing clinical resistance rate of Shigella sonnei to cefotaxime in Jiangsu Province, China, between 2012 and 2015.

BACKGROUND: The objective of this study is to evaluate the prevalence of Shigella sonnei (S. sonnei) and characterize the mechanism of its increasing resistance to cefotaxime, a third-generation cephalosporin agent between 2012 and 2015. METHODS: We investigated the drug resistance in 95 isolates of S. sonnei by K-B dilution method and isolates with the extended-spectrum beta-lactamases (ESBLs)-producing genes were detected by polymerase chain reaction (PCR) and sequencing. RESULTS: Over a 4-year period, the resistance rate of S. sonnei to cefotaxime increased from 31.6% to 64.3%, between 2012 and 2015. Molecular characterization of the ESBL genes, comprising 28 strains of CTX-M-1 group: blaCTX-M-55 (n=22), blaCTX-M-3 (n=3) and blaCTX-M-15 (n=3); 11 strains of CTX-M-9 group: blaCTX-M-14 (n=9) and blaCTX-M-65 (n=2), and 36 strains with blaTEM-1 gene. None of S. sonnei isolates carried blaCTX-M-2 group and SHV-type. CONCLUSIONS: The antimicrobial resistance rate of S. sonnei to cefotaxime significantly increased. Accordingly, regular surveillance of the cephalosporin-resistant S. sonnei should be emphasized. Moreover, exploration of the mechanism underlying the resistance of S. sonnei to cefotaxime contributes to the prophylaxis of further emergence of drug resistance.

[Point-of-care tests for the rapid diagnosis of shigellosis]. / Tests de diagnostic rapide des shigelloses.

Worldwide, it is estimated that 140 million people suffer from shigellosis annually. The traditional identification of Shigella spp. by culture lacks sensitivity. Rapid diagnosis of shigellosis is important because it allows to engage appropriate antimicrobial treatment that shortens the duration and severity of the illness and reduces microbial carriage, thus the spread of infection in the community. Onestep immunochromatographic dipstick tests have been successfully developed at Institut Pasteur for Shigella spp., Shigella flexneri 2a, Shigella sonnei, and Shigella dysenteriae 1. The present work describes the evaluation of these four rapid diagnostic tests (RDT) that addressed the issue of rapid diagnosis of Shigella diarrhea and dysentery testing from bacterial cultures, stools, and rectal swabs which is usually how the specimen is often collected or received from the field or from remote settings. The evaluations have been performed in Chile, Democratic Republic of Congo, Senegal, Djibouti, Vietnam, India, and France, in dispensaries, in emergency room, on the field, in public health laboratories, and by the French Army. The dipstick method used requires minimal technical skill, and the test can be read between 5 and 15 minutes. Stool cultures and the immunochromatographic test showed concordant results in the comparative studies when RDT for S. sonnei was tested in Chile, Vietnam, India, and France; specificity (Sp) was 96% and sensitivity (Se) was 100%. When RDT for S. flexneri 2a was tested in Vietnam, Se was 91.5% and Sp was 99.2%. In Chile, Se was 83.3% and Sp was 100%. When RDT for S. dysenteriae 1 was tested in India, Vietnam, Senegal, and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the Sp was 98.7% and the Se was 91.7%. In Chile, the initial finding for a simple RDT to diagnose Shigella spp. demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys. Additionally, the dipsticks can be stored at room temperature in a humidity-proof plastic bag, making them easily transportable. Considering the potential impact these RDT have for the clinical management of the disease and for epidemiological studies, industrialization of these tests is in progress.

Comparative Genomic and Phylogenetic Analysis of a Shiga Toxin Producing Shigella sonnei (STSS) Strain.

Shigella strains are important agents of bacillary dysentery, and in recent years Shigella sonnei has emerged as the leading cause of shigellosis in industrialized and rapidly developing countries. More recently, several S. sonnei and Shigella flexneri strains producing Shiga toxin (Stx) have been reported from sporadic cases and from an outbreak in America. In the present study we aimed to shed light on the evolution of a recently identified Shiga toxin producing S. sonnei (STSS) isolated in Europe. Here we report the first completely assembled whole genome sequence of a multidrug resistant (MDR) Stx-producing S. sonnei (STSS) clinical strain and reveal its phylogenetic relations. STSS 75/02 proved to be resistant to ampicillin, streptomycin, tetracycline, chloramphenicol, thrimetoprim, and sulfomethoxazol. The genome of STSS 75/02 contains a 4,891,717 nt chromosome and seven plasmids including the 214 kb invasion plasmid (pInv) harboring type III secretion system genes and associated effectors. The chromosome harbors 23 prophage regions including the Stx1 converting prophage. The genome carries all virulence determinants necessary for an enteroinvasive lifestyle, as well as the Stx1 encoding gene cluster within an earlier described inducible converting prophage. In silico SNP genotyping of the assembled genome as well as 438 complete or draft S. sonnei genomes downloaded from NCBI GenBank revealed that S. sonnei 75/02 belongs to the more recently diverged global MDR lineage (IIIc). Targeted screening of 1131 next-generation sequencing projects taken from NCBI Short Read Archive of confirms that only a few S. sonnei isolates are Stx positive. Our results suggest that the acquisition of Stx phages could have occurred in different environments as independent events and that multiple horizontal transfers are responsible for the appearance of Stx phages in S. sonnei strains.

Shigella sonnei Encodes a Functional T6SS Used for Interbacterial Competition and Niche Occupancy.

Shigella is a leading cause of dysentery worldwide, with the majority of infections caused by two subgroups, S. flexneri and S. sonnei. Although S. flexneri has been highly prevalent in low-income countries, global development has brought an increase in S. sonnei at the expense of S. flexneri. However, the mechanisms behind this shift are not understood. Here we report that S. sonnei, but not S. flexneri, encodes a type VI secretion system (T6SS) that provides a competitive advantage in the gut. S. sonnei competes against E. coli and S. flexneri in mixed cultures, but this advantage is reduced in T6SS mutant strains. In addition, S. sonnei can persist as well as outcompete E. coli and S. flexneri in mice in a T6SS-dependent manner. These findings suggest that S. sonnei has a competitive advantage over S. flexneri and potentially explain the increasing global prevalence of S. sonnei.

A study of different buffers to maximize viability of an oral Shigella vaccine.

Live, whole cell killed and subunit vaccines are being developed for diarrheal diseases caused by V. cholerae, Shigella species, ETEC, and Campylobacter. Some of these vaccines can be administered orally since this route best mimics natural infection. Live vaccines administered orally have to be protected from the harsh acidic gastric environment. Milk and bicarbonate solutions have been administered to neutralize the stomach acid. For many Shigella vaccine trials, 100-120 ml of a bicarbonate solution is ingested followed by the live vaccine candidate, which is delivered in 30 ml of bicarbonate, water or saline. It is not clear if maximum bacterial viability is achieved under these conditions. Also, volumes of neutralizing buffer that are optimal for adults may be unsuitable for children and infants. To address these questions, we performed studies to determine the viability and stability of a Shigella sonnei vaccine candidate, WRSS1, in a mixture of different volumes of five different buffer solutions added to hydrochloric acid to simulate gastric acidity. Among the buffers tested, bicarbonate solution, rotavirus buffer and CeraVacx were better at neutralizing acid and maintaining the viability of WRSS1. Also, a much smaller volume of the neutralizing buffer was sufficient to counteract stomach acid while maintaining bacterial viability.