Determinants of immune responses predictive of protection against shigellosis in an endemic zone: a systems analysis of antibody profiles and function.

BACKGROUND: Shigella is the third leading global cause of moderate or severe diarrhoea among children younger than 5 years globally, and is the leading cause in children aged 24-59 months. The mechanism of protection against Shigella infection and disease in endemic areas is uncertain. We aimed to compare the Shigella-specific antibody responses in individuals living in Shigella-endemic and non-endemic areas, and to identify correlates of protection in a Shigella-endemic location. METHODS: We applied a systems approach to retrospectively analyse serological responses to Shigella across endemic and non-endemic populations. We profiled serum samples collected from 44 individuals from the USA without previous exposure to Shigella and who were experimentally challenged with Shigella sonnei (non-endemic setting), and serum samples collected from 55 Peruvian army recruits (endemic setting). In the endemic setting, a subset of 37 samples collected from individuals infected with culture-confirmed Shigella flexneri 2a were divided into two groups: susceptible, which included individuals infected within 90 days of entering the camp (n=29); or resistant, which included individuals infected later than 90 days after entering the camp (n=8). We analysed Shigella-specific antibody isotype, subclass, and Fc receptor binding profiles across IpaB, IpaC, IpaD, and lipopolysaccharide from S flexneri 2a, 3a, and 6, and S sonnei, and O-specific polysaccharide (OSP) from S flexneri 2a and 3a and S sonnei. We also evaluated antibody-mediated complement deposition and innate immune cell activation. The main outcome of interest was the detection of antibody markers and functionality associated with protection against shigellosis in a high-burden endemic setting. FINDINGS: Adults with endemic exposure to Shigella possessed broad and functional antibody responses across polysaccharide, glycolipid, and protein antigens compared with individuals from non-endemic regions. In a setting with high Shigella burden, elevated levels of OSP-specific Fcα receptor (FcαR) binding antibodies were associated with resistance to shigellosis, whereas total OSP-specific IgA was not, suggesting a potentially unique functionality. OSP-specific FcαR binding IgA found in resistant individuals activated bactericidal neutrophil functions including phagocytosis, degranulation, and production of reactive oxygen species. Moreover, IgA depletion from resistant serum significantly reduced binding of OSP-specific antibodies to FcαR and antibody-mediated activation of neutrophils and monocytes. INTERPRETATION: Our findings suggest that OSP-specific functional IgA responses contribute to protective immunity against Shigella infection in a high-burden setting. These findings will assist in the development and evaluation of Shigella vaccines. FUNDING: US National Institutes of Health.

Emergence of lineage III of Shigella sonnei ST152 belonging to a high-risk clone harboring the blaCTX-M-15 gene in Peru.

Multidrug-resistant Shigella sonnei ST152, global lineage III, is a high-risk clone, whose dissemination has limited therapeutic options for shigellosis. This study aimed to characterize two isolates of S. sonnei, which were recovered in Lima, Peru, during November 2019, exhibiting resistance to extended-spectrum cephalosporins and quinolones, and concurrently harboring blaCTX-M-15 and qnrS1 genes, in addition to mutations in gyrA-S83L. These isolates were resistant to ceftriaxone, ciprofloxacin and trimethoprim/sulfamethoxazole. The molecular analysis showed that both isolates belonged to lineage III, sublineages IIIa and IIIb. The blaCTX-M-15 gene was located in the same genetic platform as qnrS1, flanked upstream by ISKpn19, on a conjugative plasmid belonging to the IncI-γ group. To the best of our knowledge, this would be the first report on S. sonnei isolates carrying the blaCTX-M-15 gene in Peru. The global dissemination of S. sonnei ST152, co-resistant to ß-lactams and quinolones, could lead to a worrisome scenario in the event of potential acquisition of genetic resistance mechanisms to azithromycin.

Identification of Shiga-Toxin-Producing Shigella Infections in Travel and Non-Travel Related Cases in Alberta, Canada.

It has long been accepted that Shiga toxin (Stx) only exists in Shigella dysenteriae serotype 1. However, in recent decades, the presence of Shiga toxin genes (stx) in other Shigella spp. have been reported. We screened 366 Shigella flexneri strains from Alberta, Canada (2003 to 2016) for stx and 26 positive strains were identified. These isolates are highly related with the majority originating from the Dominican Republic and three isolates with Haiti origin. Both phylogenetic and spanning tree analysis of the 26 Alberta and 29 stx positive S. flexneri originating from the U.S., France, Canada (Quebec) and Haiti suggests that there are geographic specific distribution patterns (Haiti and Dominican Republic clades). This study provides the first comprehensive whole genome based phylogenetic analysis of stx positive S. flexneri strains as well as their global transmission, which signify the public health risks of global spreading of these strains.

Historical, current, and emerging tools for identification and serotyping of Shigella.

The Shigella genus includes serious foodborne disease etiologic agents, with 4 species and 54 serotypes. Identification at species and serotype levels is a crucial task in microbiological laboratories. Nevertheless, the genetic similarity between Shigella spp. and Escherichia coli challenges the correct identification and serotyping of Shigella spp., with subsequent negative repercussions on surveillance, epidemiological investigations, and selection of appropriate treatments. For this purpose, multiple techniques have been developed historically ranging from phenotype-based methods and single or multilocus molecular techniques to whole-genome sequencing (WGS). To facilitate the selection of the most relevant method, we herein provide a global overview of historical and emerging identification and serotyping techniques with a particular focus on the WGS-based approaches. This review highlights the excellent discriminatory power of WGS to more accurately elucidate the epidemiology of Shigella spp., disclose novel promising genomic targets for surveillance methods, and validate previous well-established methods.

How microbiological tests reflect bacterial pathogenesis and host adaptation.

Historically, clinical microbiological laboratories have often relied on isolation of pure cultures and phenotypic testing to identify microorganisms. These clinical tests are often based on specific biochemical reactions, growth characteristics, colony morphology, and other physiological aspects. The features used for identification in clinical laboratories are highly conserved and specific for a given group of microbes. We speculate that these features might be the result of evolutionary selection and thus may reflect aspects of the life cycle of the organism and pathogenesis. Indeed, several of the metabolic pathways targeted by diagnostic tests in some cases may represent mechanisms for host colonization or pathogenesis. Examples include, but are not restricted to, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella enterica, Shigella spp., and enteroinvasive Escherichia coli (EIEC). Here, we provide an overview of how some common tests reflect molecular mechanisms of bacterial pathogenesis.

Outbreak of Central American born Shigella sonnei in two youth camps in Belgium in the summer of 2019.

In 2019, an outbreak of Shigella sonnei occurred during two youth camps in Belgium. The clustering of isolates from both camps was confirmed by next-generation sequencing, as well as a secondary infection of a technician. The outbreak strain clustered with internationally isolated strains from patients with recent travel history to Central America. This report exemplifies enhanced surveillance and international collaboration between public health institutes by enabling to link local outbreaks to region-specific sublineages circulating abroad.

Epidemiology of Shigella infections and diarrhea in the first two years of life using culture-independent diagnostics in 8 low-resource settings.

Culture-independent diagnostics have revealed a larger burden of Shigella among children in low-resource settings than previously recognized. We further characterized the epidemiology of Shigella in the first two years of life in a multisite birth cohort. We tested 41,405 diarrheal and monthly non-diarrheal stools from 1,715 children for Shigella by quantitative PCR. To assess risk factors, clinical factors related to age and culture positivity, and associations with inflammatory biomarkers, we used log-binomial regression with generalized estimating equations. The prevalence of Shigella varied from 4.9%-17.8% in non-diarrheal stools across sites, and the incidence of Shigella-attributable diarrhea was 31.8 cases (95% CI: 29.6, 34.2) per 100 child-years. The sensitivity of culture compared to qPCR was 6.6% and increased to 27.8% in Shigella-attributable dysentery. Shigella diarrhea episodes were more likely to be severe and less likely to be culture positive in younger children. Older age (RR: 1.75, 95% CI: 1.70, 1.81 per 6-month increase in age), unimproved sanitation (RR: 1.15, 95% CI: 1.03, 1.29), low maternal education (<10 years, RR: 1.14, 95% CI: 1.03, 1.26), initiating complementary foods before 3 months (RR: 1.10, 95% CI: 1.01, 1.20), and malnutrition (RR: 0.91, 95% CI: 0.88, 0.95 per unit increase in weight-for-age z-score) were risk factors for Shigella. There was a linear dose-response between Shigella quantity and myeloperoxidase concentrations. The burden of Shigella varied widely across sites, but uniformly increased through the second year of life and was associated with intestinal inflammation. Culture missed most clinically relevant cases of severe diarrhea and dysentery.

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.

In silico design of a vaccine candidate based on autotransporters and HSP against the causal agent of shigellosis, Shigella flexneri.

Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.

Virulence-related genes are associated with clinical and nutritional outcomes of Shigella/Enteroinvasive Escherichia coli pathotype infection in children from Brazilian semiarid region: A community case-control study.

Shigella/Enteroinvasive Escherichia coli (EIEC) pathotype is a major enteropathogen associated with diarrhea and malnutrition in children from developing countries. This study aimed to correlate Shigella/EIEC virulence-related genes (VRGs) with clinical symptoms, nutritional status and coenteropathogens in children from the Brazilian semiarid region. We designed a case-control study of community diarrhea in six cities of the Brazil semiarid region with 1200 children aging 2-36 months. Standardized questionnaire was applied for collecting sociodemographic, nutritional status and clinical information of the children. DNA samples were extracted from stools and diagnosed for Shigella/EIEC using PCR-based approaches. Positive samples were tested for 28 VRGs using four multiplex PCRs. Intestinal inflammation was determined by measuring fecal myeloperoxidase (MPO). Shigella/EIEC pathotype was detected in 5% of the children and was significantly associated with diarrhea. The genes sen (encoding Shigella enterotoxin 2), ipgB2, ipgB1 (both encoding type 3 secretion system-T3SS effectors that modulate actin filament), and ospF (encoding a T3SS effector involved in suppression of host responses) were further associated with diarrhea in Shigella/EIEC positive children. Among children presenting diarrhea, virA gene (encoding a T3SS effector that promotes microtubule destabilization) was associated with fever, while virB (encoding a major transcriptional activator) was associated with low height-for-age z-score. In addition, these VRGs were associated with increased fecal MPO, and coinfection with Salmonella spp. was associated with increased abdominal pain. These data reinforce the impact of Shigella/EIEC on diarrhea in children from Brazilian semiarid region and highlighted the contributions of specific virulence genes for its pathobiology.

Burden of laboratory-confirmed shigellosis infections in Guatemala 2007-2012: results from a population-based surveillance system.

BACKGROUND: We describe the epidemiology and antimicrobial susceptibility patterns of culture-confirmed Shigella infections in facility-based surveillance sites in Guatemala. Current studies using quantitative molecular diagnostics suggest Shigella may contribute most to the global diarrheal disease burden. Since identification of Shigella requires culturing techniques using stool specimens and few laboratories in Guatemala routinely culture for this pathogen, little is known about the true burden of Shigella in Guatemala or, importantly, the antimicrobial resistance patterns. METHODS: Clinical, epidemiological, and laboratory data were collected on 5399 patients with acute diarrhea (≥3 loose stools in 24 h) from June 2007-August 2012. Multidrug resistance (MDR) was defined as resistance to ampicillin and trimethoprim/sulfamethoxazole. RESULTS: Five percent (261) of stool specimens yielded Shigella spp. The annual incidence of laboratory-confirmed infections ranged from 5.0 to 24.1 per 100,000 persons in Santa Rosa and 0.3 to 6.2 per 100,000 in Quetzaltenango; 58% of cases occurred in children < 5 years of age. Thirty patients were hospitalized; one patient died. Oral rehydration or intravenous solution was used to treat 72% of hospitalized and 15% of ambulatory cases. Fifty-nine percent of cases were S. flexneri and 51% of cases were MDR. CONCLUSIONS: Shigella is an important cause of bacterial diarrhea in children and prevalence of MDR highlights the importance of appropriate treatment regimens. This study demonstrates that strengthening laboratory capacity in Guatemala can help determine causes which can lead to prevention of diarrheal diseases, particularly in children. Such capacity building is also critical for rapid detection and control of public health threats at their source and therefore for global health security.

[Shigella flexneri bacteremia in children: two case report and review of literature]. / Bacteriemia por Shigella flexneri en dos lactantes.

Shigella spp. is the most frequent micro-biological isolation in bacterial diarrhea in Argentina. It causes a watery diarrhea or dysenteric disease. It rarely causes extraintestinal problems. It has an incidence of bacteremia of 0,4-7,3%, and its appearance compels us to look for associated risk factors, as children under one year of age and immunodeficiency, among others. We describe two children with Shigella flexneri bacteremia. They presented with fever and diarrhea. One of them had primary immune deficiency.

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.

Distribution and characterization of Shiga toxin converting temperate phages carried by Shigella flexneri in Hispaniola.

Shigella infections account for a considerable burden of acute diarrheal diseases worldwide and remain a major cause of childhood mortality in developing countries. Although, all four species of Shigella (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) cause bacillary dysentery, historically only S. dysenteriae type 1 has been recognized as carrying the genes for Shiga toxin (stx). Recent epidemiological data, however, have suggested that the emergence of stx carrying S. flexneri strains may have originated from bacteriophage-mediated inter-species horizontal gene transfer in one specific geographical area, Hispaniola. To test this hypothesis, we analyzed whole genome sequences of stx-encoding phages carried by S. flexneri strains isolated in Haiti and S. flexneri S. boydii and S. dysenteriae strains isolated from international travelers who likely acquired the infection in Haiti or the Dominican Republic. Phylogenetic analysis showed that phage sequences encoded in the Shigella strains from Hispaniola were bacteriophage φPOC-J13 and they were all closely related to a phage isolated from a USA isolate, E. coli 2009C-3133 serotype O119:H4. In addition, despite the low genetic heterogeneity of phages from different Shigella spp. circulating in the Caribbean island between 2001 and 2014, two distinct clusters emerged in Haiti and the Dominican Republic. Each cluster possibly originated from phages isolated from S. flexneri 2a, and within each cluster several instances of horizontal phage transfer from S. flexneri 2a to other species were detected. The implications of the emergence of stx-producing non-S. dysenteriae type 1 Shigella species, such as S. flexneri, spans not only the basic science behind horizontal phage spread, but also extends to medical treatment of patients infected with this pathogen.

Shigella infection in children under 5 years old in western French Guiana.

French Guiana, a tropical country, is characterised by a young and multi-ethnic population. Difficulties in accessing safe water sources lead to outbreaks of gastroenteritis. The objectives of this study were (1) to describe the microbiological profile of shigella strains isolated in western French Guiana, including antimicrobial susceptibility and the distribution of strains in terms of species and serotypes and (2) to estimate the incidence of shigellosis in children under 5 years old. A retrospective observational study was conducted of 213 cases of shigellosis diagnosed in the biology department of the hospital centre for western French Guiana between 2000 and 2012 in children under 5 years old. The serogroups (formerly known as species) that predominates in French Guiana was Shigella flexneri. No resistance was observed to fluoroquinolones or to third-generation cephalosporins. The average incidence of shigellosis in children under 5 years old in western French Guiana was estimated at 189.6 cases per 100 000 inhabitants per year. Shigellosis is a public health problem in western French Guiana. These infections suggest the difficulties in accessing safe water sources and the lack of public sanitation. A quadrivalent vaccine containing Shigella sonnei and three serotypes of S. flexneri (S. flexneri 2a, 3a and 6) could provide broad coverage against shigella infections.

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

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.

Differences in tetracycline resistance determinant carriage among Shigella flexneri and Shigella sonnei are not related to different plasmid Inc-type carriage.

OBJECTIVES: The aim of this study was to establish the prevalence of the most common molecular mechanisms involved in tetracycline resistance as well as their relationship with plasmid incompatibility (Inc) groups in a collection of Shigella spp. causing traveller's diarrhoea. METHODS: Tetracycline susceptibility was established in 187 Shigella spp. (74 Shigella flexneri and 113 Shigella sonnei), of which 153 isolates were recovered as a confirmed cause of traveller's diarrhoea. The prevalence of the tet(A), tet(B) and tet(G) genes was analysed by PCR. Eighteen plasmid Inc groups was determined in a subset of 59 isolates. RESULTS: Among 154 tetracycline-resistant isolates, 122 (79.2%) harboured at least tet(A) or tet(B). The tet(B) gene was the most frequently detected, being present in 70 isolates (45.5%), whilst tet(A) was detected in 57 isolates (37.0%). The tet(G) gene was present in only 11 (7.2%) isolates. Moreover, the tet(A) gene was more frequent in S. sonnei (P=0.0007), whilst the tet(B) gene was more frequent in S. flexneri (P<0.0001). Plasmids belonging to Inc group B (P<0.05) were significantly more frequent among S. flexneri, whilst those belonging to groups K, FIC and FIIA (P<0.05) were preferentially detected among S. sonnei. CONCLUSION: The prevalence of the tet(A) and tet(B) genes differed between S. sonnei and S. flexneri. Moreover, the prevalence of plasmid Inc groups in S. flexneri and S. sonnei differed. However, no relationship was found between the two phenomena.

Molecular characterization of virulence and antimicrobial resistance profile of Shigella species isolated from children with moderate to severe diarrhea in northeastern Brazil.

Molecular characterization of virulence and antimicrobial resistance profiles were determined for Shigella species isolated from children with diarrhea in Fortaleza, Brazil. Fecal specimens were collected along with socioeconomic and clinical data from children with moderate to severe diarrhea requiring emergency care. Shigella spp. were isolated by standard microbiological techniques, and we developed 4 multiplex polymerase chain reaction assays to detect 16 virulence-related genes (VRGs). Antimicrobial susceptibility tests were performed using disk diffusion assays. S. flexneri and S. sonnei were the predominant serogroups. S. flexneri was associated with low monthly incomes; more severe disease; higher number of VRGs; and presence of pic, set, and sepA genes. The SepA gene was associated with more intense abdominal pain. S. flexneri was correlated with resistance to ampicillin and chloramphenicol, whereas S. sonnei was associated with resistance to azithromycin. Strains harboring higher numbers of VRGs were associated with resistance to more antimicrobials. We highlight the correlation between presence of S. flexneri and sepA, and increased virulence and suggest a link to socioeconomic change in northeastern Brazil. Additionally, antimicrobial resistance was associated with serogroup specificity in Shigella spp. and increased bacterial VRGs.