Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence.

Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.

Shigella sonnei: virulence and antibiotic resistance.

Shigella sonnei is the emerging pathogen globally, as it is the second common infectious species of shigellosis (bloody diarrhoea) in low- and middle-income countries (LMICs) and the leading one in developed world. The multifactorial processes and novel mechanisms have been identified in S. sonnei, that are collectively playing apart a substantial role in increasing its prevalence, while replacing the S. flexneri and other Gram-negative gut pathogens niche occupancy. Recently, studies suggest that due to improvement in sanitation S. sonnei has reduced cross-immunization from Plesiomonas shigelliodes (having same O-antigen as S. sonnei) and also found to outcompete the two major species of Enterobacteriaceae family (Shigella flexneri and Escherichia coli), due to encoding of type VI secretion system (T6SS). This review aimed to highlight S. sonnei as an emerging pathogen in the light of recent research with pondering aspects on its epidemiology, transmission, and pathogenic mechanisms. Additionally, this paper aimed to review S. sonnei disease pattern and related complications, symptoms, and laboratory diagnostic techniques. Furthermore, the available treatment reigns and antibiotic-resistance patterns of S. sonnei are also discussed, as the ciprofloxacin and fluoroquinolone-resistant S. sonnei has already intensified the global spread and burden of antimicrobial resistance. In last, prevention and controlling strategies are briefed to limit and tackle S. sonnei and possible future areas are also explored that needed more research to unravel the hidden mysteries surrounding S. sonnei.

Comparative genome analysis of 12 Shigella sonnei strains: virulence, resistance, and their interactions.

Shigellosis is a highly infectious disease that is mainly transmitted via fecal-oral contact of the bacteria Shigella. Four species have been identified in Shigella genus, among which Shigella flexneri is used to be the most prevalent species globally and commonly isolated from developing countries. However, it is being replaced by Shigella sonnei that is currently the main causative agent for dysentery pandemic in many emerging industrialized countries such as Asia and the Middle East. For a better understanding of S. sonnei virulence and antibiotic resistance, we sequenced 12 clinical S. sonnei strains with varied antibiotic-resistance profiles collected from four cities in Jiangsu Province, China. Phylogenomic analysis clustered antibiotic-sensitive and resistant S. sonnei into two distinct groups while pan-genome analysis reveals the presence and absence of unique genes in each group. Screening of 31 classes of virulence factors found out that type 2 secretion system is doubled in resistant strains. Further principle component analysis based on the interactions between virulence and resistance indicated that abundant virulence factors are associated with higher levels of antibiotic resistance. The result present here is based on statistical analysis of a small sample size and serves basically as a guidance for further experimental and theoretical studies.

Genetic Mechanisms behind the Spread of Reduced Susceptibility to Azithromycin in Shigella Strains Isolated from Men Who Have Sex with Men in Québec, Canada.

We analyzed 254 Shigella species isolates collected in Québec, Canada, during 2013 and 2014. Overall, 23.6% of isolates showed reduced susceptibility to azithromycin (RSA) encoded by mphA (11.6%), ermB (1.7%), or both genes (86.7%). Shigella strains with RSA were mostly isolated from men who have sex with men (68.8% or higher) from the Montreal region. A complete sequence analysis of six selected plasmids from Shigella sonnei and different serotypes of Shigella flexneri emphasized the role of IS26 in the dissemination of RSA.

Anti-adhesion of probiotic Enterococcus faecium WEFA23 against five pathogens and the beneficial effect of its S-layer proteins against Listeria monocytogenes.

Enterococcus faecium WEFA23 is a potential probiotic strain isolated from Chinese infant feces. In this study, the antagonistic activity of E. faecium WEFA23 on adhesion to pathogens was investigated. Enterococcus faecium WEFA23 was able to compete, exclude, and displace the adhesion of Escherichia coli O157:H7, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes CMCC54007, Staphylococcus aureus CMCC26003, and Shigella sonnei ATCC 25931 to Caco-2 cells. Among them, L. monocytogenes achieved the strongest inhibition rate in both competition and displacement assays. Those anti-adhesion capacities were related to the bacterial physicochemical properties (hydrophobicity, auto-aggregation, and co-aggregation) of the bacterial surface. For L. monocytogenes, the anti-adhesion capacity was affected by the heat treatment, cell density, and growth phase of E. faecium WEFA23; 108 colony-forming units of viable cells per millilitre at the stationary phase exhibited the strongest anti-adhesion activity. In addition, removal of S-layer proteins of E. faecium WEFA23 by treatment with 5 mol/L LiCl significantly decreased its adhesion capacity, and those S-layer proteins were able to compete, displace, and exclude L. monocytogenes at different levels. Both cells and S-layer proteins of E. faecium WEFA23 significantly reduced the apoptosis of Caco-2 cells induced by L. monocytogenes, which was mediated by caspase-3 activation. This study might be helpful in understanding the anti-adhesion mechanism of probiotics against pathogens.

Azithromycin Resistance in Shigella spp. in Southeast Asia.

Infection by Shigella spp. is a common cause of dysentery in Southeast Asia. Antimicrobials are thought to be beneficial for treatment; however, antimicrobial resistance in Shigella spp. is becoming widespread. We aimed to assess the frequency and mechanisms associated with decreased susceptibility to azithromycin in Southeast Asian Shigella isolates and use these data to assess appropriate susceptibility breakpoints. Shigella isolates recovered in Vietnam and Laos were screened for susceptibility to azithromycin (15 µg) by disc diffusion and MIC. Phenotypic resistance was confirmed by PCR amplification of macrolide resistance loci. We compared the genetic relationships and plasmid contents of azithromycin-resistant Shigella sonnei isolates using whole-genome sequences. From 475 available Shigella spp. isolated in Vietnam and Laos between 1994 and 2012, 6/181 S. flexneri isolates (3.3%, MIC ≥ 16 g/liter) and 16/294 S. sonnei isolates (5.4%, MIC ≥ 32 g/liter) were phenotypically resistant to azithromycin. PCR amplification confirmed a resistance mechanism in 22/475 (4.6%) isolates (mphA in 19 isolates and ermB in 3 isolates). The susceptibility data demonstrated the acceptability of the S. flexneri (MIC ≥ 16 g/liter, zone diameter ≤ 15 mm) and S. sonnei (MIC ≥ 32 g/liter, zone diameter ≤ 11 mm) breakpoints with a <3% discrepancy. Phylogenetic analysis demonstrated that decreased susceptibility has arisen sporadically in Vietnamese S. sonnei isolates on at least seven occasions between 2000 and 2009 but failed to become established. While the proposed susceptibility breakpoints may allow better recognition of resistant isolates, additional studies are required to assess the impact on the clinical outcome. The potential emergence of azithromycin resistance highlights the need for alternative options for management of Shigella infections in countries where Shigella is endemic.

Aptamer-Immobilized Surface Plasmon Resonance Biosensor for Rapid and Sensitive Determination of Virulence Determinant.

Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.

Inhibition of Shigella sonnei-induced epithelial barrier disruption by surface-layer associated proteins of lactobacilli from Chinese fermented food.

Surface-layer associated proteins (SLAP) of Lactobacillus paracasei ssp. paracasei M5-L and Lactobacillus casei Q8-L were examined to identify the functional basis for their protection within intestinal epithelial cells. The results showed that SLAP of M5-L and Q8-L remained active in a trypsin solution and retained a 45-kDa protein band, similar to that observed in controls. In contrast, under conditions of simulated gastric juice, the SLAP were partially degraded. Inhibitory effects of SLAP on adherence of Shigella sonnei to HT-29 cells were assessed with use of exclusion, competition, and replacement assays. In response to M5-L at 50 µg/mL SLAP, an inhibition ratio of 33% was obtained, while for Q8-L at 400 µg/mL SLAP, the inhibition ratio was 48%. Hoechst 33258 test results showed that cells infected with S. sonnei and co-incubated with SLAP of M5-L and Q8-L were only partially apoptotic, with apoptosis rates of 37.67 and 43.67%, respectively. These levels of apoptosis were substantially lower than that observed with cells infected with S. sonnei alone. In addition, the SLAP of Q8-L and M5-L reduced downstream caspase-1 activity and further modified apoptotic cell damage. Finally, SLAP of M5-L and Q8-L were also able to prevent S. sonnei-induced membrane damage by inhibiting delocalization of zonula occludens (ZO)-1 and reducing the amount of occludin produced by S. sonnei.

The Multivalent Adhesion Molecule SSO1327 plays a key role in Shigella sonnei pathogenesis.

Shigella sonnei is a bacterial pathogen and causative agent of bacillary dysentery. It deploys a type III secretion system to inject effector proteins into host epithelial cells and macrophages, an essential step for tissue invasion and immune evasion. Although the arsenal of bacterial effectors and their cellular targets have been studied extensively, little is known about the prerequisites for deployment of type III secreted proteins during infection. Here, we describe a novel S. sonnei adhesin, SSO1327 which is a multivalent adhesion molecule (MAM) required for invasion of epithelial cells and macrophages and for infection in vivo. The S. sonnei MAM mediates intimate attachment to host cells, which is required for efficient translocation of type III effectors into host cells. SSO1327 is non-redundant to IcsA; its activity is independent of type III secretion. In contrast to the up-regulation of IcsA-dependent and independent attachment and invasion by deoxycholate in Shigella flexneri, deoxycholate negatively regulates IcsA and MAM in S. sonnei resulting in reduction in attachment and invasion and virulence attenuation in vivo. A strain deficient for SSO1327 is avirulent in vivo, but still elicits a host immune response.

An O antigen capsule modulates bacterial pathogenesis in Shigella sonnei.

Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.

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.

Expression of different bacterial cytotoxins is controlled by two global transcription factors, CRP and Fis, that co-operate in a shared-recruitment mechanism.

Pet is a cytotoxic autotransporter protein secreted by the pathogenic enteroaggregative Escherichia coli strain 042. Expression of Pet is co-dependent on two global transcription regulators: CRP (cyclic AMP receptor protein) and Fis (factor for inversion stimulation). At the pet promoter CRP binds to a single site centred at position -40.5 upstream of the start site for transcription. Due to the suboptimal positioning of this site, CRP alone activates transcription poorly and requires Fis to bind upstream to promote full activation. Here, we show that CRP and Fis control the expression of other important autotransporter toxins, namely Sat from uropathogenic E. coli (UPEC) and SigA from Shigella sonnei, and that this regulation has been conserved in different pathogens. Furthermore, we investigate the mechanism of Fis-mediated co-activation, exploiting a series of semi-synthetic promoters, with similar architecture to the pet promoter. We show that, when bound at position -40.5, CRP recruits RNA polymerase inefficiently and that Fis compensates by aiding polymerase recruitment through a direct protein-protein interaction. We demonstrate that other suitably positioned upstream transcription factors, which directly recruit RNA polymerase, can also compensate for the inappropriate positioning of CRP. We propose that this is a simple 'shared-recruitment' mechanism, by which co-dependence of promoters on two transcription factors could evolve.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Escherichia coli categories.

The mass profiles of cell-free extracts of 180 commensal and pathogenic strains of Escherichia coli were determined by MALDI-TOF mass spectrometry (MS). While some peaks were highly conserved in all E. coli, several peaks occurred only in some strains, showing heterogeneity among them. We did not detect strain-specific peaks for any of the E. coli categories tested. However, review of the fully conserved and the variable peaks suggested that MALDI-TOF MS has the potential to distinguish commensal and uropathogenic E. coli strains. Additionally, eight Shigella sonnei isolates were tested and found to be indistinguishable from E. coli by MALDI-TOF MS under the test conditions.

Competitive inhibition of three novel bacteria isolated from faeces of breast milk-fed infants against selected enteropathogens.

Numerous in vitro and in vivo studies conducted using different probiotic micro-organisms have demonstrated their ability to interfere with the growth and virulence of a variety of enteropathogens. The reported beneficial effects of the use of probiotics to complement antibiotic therapy or prevent diarrhoea or gastrointestinal infection in infants have increased in recent years. In the present study, we demonstrated the capacity of supernatants obtained from three novel probiotics (Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036) isolated from the faeces of breastfed infants to inhibit the growth of enterotoxigenic and enteropathogenic (EPEC) bacteria, such as Escherichia coli, Salmonella and Shigella. To assess their potential antimicrobial activity, the 17 and 24 h cell-free supernatants broth concentrates (10×) having 1, 2 or 4 % of the three probiotics were incubated with EPEC bacteria strains. After 17 h of co-culture, the supernatants were able to inhibit the growth of E. coli, Salmonella and Shigella up to 40, 55 and 81 %, respectively. However, the inhibitory capacity of some supernatants was maintained or completely lost when the supernatants (pH 3·0) were neutralised (pH 6·5). Overall, these results demonstrated that L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036 produce compounds that exhibited strain-specific inhibition of enterobacteria and have the potential to be used as probiotics in functional foods.

A Shigella sonnei outbreak traced to imported basil--the importance of good typing tools and produce traceability systems, Norway, 2011.

On 9 October 2011, the University Hospital of North Norway alerted the Norwegian Institute of Public Health (NIPH) about an increase in Shigella sonnei infections in Tromsø. The isolates had an identical 'multilocus variable-number tandem repeat analysis' (MLVA) profile. Most cases had consumed food provided by delicatessen X. On 14 October, new S. sonnei cases with the same MLVA-profile were reported from Sarpsborg, south-eastern Norway. An outbreak investigation was started to identify the source and prevent further cases. All laboratory-confirmed cases from both clusters were attempted to be interviewed. In addition, a cohort study was performed among the attendees of a banquet in Tromsø where food from delicatessen X had been served and where some people had reported being ill. A trace-back investigation was initiated. In total, 46 cases were confirmed (Tromsø= 42; Sarpsborg= 4). Having eaten basil pesto sauce or fish soup at the banquet in Tromsø were independent risk factors for disease. Basil pesto was the only common food item that had been consumed by confirmed cases occurring in Tromsø and Sarpsborg. The basil had been imported and delivered to both municipalities by the same supplier. No basil from the specific batch was left on the Norwegian market when it was identified as the likely source. As a result of the multidisciplinary investigation, which helped to identify the source, the Norwegian Food Safety Authority, together with NIPH, planned to develop recommendations for food providers on how to handle fresh plant produce prior to consumption.

Simultaneous infection with Shigella sonnei and Vibrio cholerae in a young child.

A 20-month-old infant presented to the emergency department with diarrhea and vomiting of six days duration. Blood, stool and urine specimens were collected for both bacteriological culture and parasitic workup. Concurrent infections with both Shigella sonnei and Vibrio cholerae were determined to be the cause of the infectious diarrhea. This case illustrates the importance of the microbiology laboratory in detecting infrequently seen enteric pathogens. We will also review the pathogenesis and laboratory diagnosis of Vibrio cholerae and Shigella sonnei.

Broadly protective Shigella vaccine based on type III secretion apparatus proteins.

Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine.

Pathogenomic analyses of Shigella isolates inform factors limiting shigellosis prevention and control across LMICs.

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMICs), are increasingly antimicrobial resistant and have no widely available licenced vaccine. We performed genomic analyses of 1,246 systematically collected shigellae sampled from seven countries in sub-Saharan Africa and South Asia as part of the Global Enteric Multicenter Study (GEMS) between 2007 and 2011, to inform control and identify factors that could limit the effectiveness of current approaches. Through contemporaneous comparison among major subgroups, we found that S. sonnei contributes ≥6-fold more disease than other Shigella species relative to its genomic diversity, and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against ciprofloxacin, the current WHO-recommended antimicrobial for the treatment of shigellosis, among Shigella isolates. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, providing a framework for the focused application of comparative genomics to guide vaccine development, and the optimization of control and prevention strategies for other pathogens relevant to public health policy considerations.

Outbreak of Shigella sonnei infection in Norway linked to consumption of fresh basil, October 2011.

We report a Shigella sonnei outbreak of 46 cases that occurred in Norway during October 2011. Two municipalities were involved. A large cluster (42 cases)was concentrated in north Norway, while a smallcluster (4 cases) occurred in the south-east region.Epidemiological evidence and trace back investigations have linked the outbreak to the consumption of imported fresh basil. The product has been withdrawn from the market. No further cases have been reported since 25 October.

Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA).

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.