Characterization of Shigella flexneri Serotype 6 Strains Isolated from Bangladesh and Identification of a New Phylogenetic Cluster.

A significant cause of shigellosis in Bangladesh and other developing countries is Shigella flexneri serotype 6. This serotype has been subtyped, on the basis of the absence or presence of a group-specific antigen, E1037, into S. flexneri 6a and 6b, respectively. Here, we provided rationales for the subclassification, using several phenotypic and molecular tools. A set of S. flexneri 6a and 6b strains isolated between 1997 and 2015 were characterized by analyzing their biochemical properties, plasmid profiles, virulence markers, pulsed-field gel electrophoresis (PFGE) results, and ribotype. Additionally, the genomic relatedness of these subserotypes was investigated with global isolates of serotype 6 using publicly available genomes. Both subserotypes of S. flexneri 6 agglutinated with monoclonal antiserum against S. flexneri (MASF) B and type VI-specific antiserum (MASF VI) and were PCR positive for O-antigen flippase-specific genes and virulence markers (ipaH, ial, sen, and sigA). Unlike S. flexneri 6a strains, S. flexneri 6b strains seroagglutinated with anti-E1037 antibodies, MASF IV-I. Notably, these two antigenically distinct subserotypes were clonally diverse, showing two distinct PFGE patterns following the digestion of chromosomal DNA with either XbaI or IceuI. In addition, hybridization of a 16S rRNA gene probe with HindIII-digested genomic DNA yielded two distinguishing ribotypes. Genomic comparison of S. flexneri subserotype 6a and 6b strains from Bangladesh indicated that, although these strains were in genomic synteny, the majority of them formed a unique phylogroup (PG-4) that was missing for the global isolates. This study supports the subserotyping and emphasizes the need for global monitoring of the S. flexneri subserotypes 6a and 6b. IMPORTANCE Shigella flexneri serotype 6 is one of the predominant serotypes among shigellosis cases in Bangladesh. Characterization of a novel subserotype of S. flexneri 6 (VI:E1037), agglutinated with type 6-specific antibody and anti-E1037, indicates a unique evolutionary ancestry. PFGE genotyping supports the finding that these two antigenically distinct subserotypes are clonally diverse. A phylogenetic study based on single-nucleotide polymorphism (SNP) data revealed that these two subserotypes were in genomic synteny, although their genomes were reduced. Interestingly, a majority of the S. flexneri 6 strains isolated from Bangladesh form a novel phylogenetic cluster. Therefore, this report underpins the global monitoring and tracking of the novel subserotype.

Virulence factors and molecular characteristics of Shigella flexneri isolated from calves with diarrhea.

BACKGROUND: The natural hosts of Shigella are typically humans and other primates, but it has been shown that the host range of Shigella has expanded to many animals. Although Shigella is becoming a major threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. RESULTS: Fifty-four S. flexneri isolates from Gansun, Shanxi, Qinghai, Xinjiang and Tibet obtained during 2014 to 2016 possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100 %, 100 %, 77.78 %, 79.63 %, 48.15 %, 48.15 and 0 %, respectively. Multilocus variable number tandem repeat analysis (MLVA) based on 8 variable number of tandem repeat (VNTR) loci discriminated the isolates into 39 different MLVA types (MTs), pulsed field gel electrophoresis (PFGE) based on NotI digestion divided the 54 isolates into 31 PFGE types (PTs), and multilocus sequence typing (MLST) based on 15 housekeeping genes differentiated the isolates into 7 MLST sequence types (STs). CONCLUSIONS: The findings from this study enrich our knowledge of the molecular characteristics of S. flexneri collected from calves with diarrhea, which will be important for addressing clinical and epidemiological issues regarding shigellosis.

Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose.

Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids.

Comparative analysis of Bacterial Vaginosis microbiota among pregnant and non-pregnant females and isolation of phages against Enterococcus faecalis, Enterococcus faecium, and Shigella flexneri strains.

BACKGROUND AND OBJECTIVES: Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age. It shifts the paradigms of the vagina from healthy, beneficial microbiota to facultative and strict anaerobes. BV remains one of the most arduous and controversial challenges in modern-day clinical microbiology because of its high prevalence and relapse rates. A lot of research has been carried out on it. Still, its etiology is unknown, which gave this infection global importance. The current study was designed to investigate and compare the microbiota of pregnant and non-pregnant females suffering from BV, and phages were isolated against BV microbiota. MATERIAL AND METHODS: The samples were collected from the vagina by using a speculum, and swabs were streaked on different media to isolate bacteria. The microbiological analysis was performed by microscopy, biochemical testing, and antibiotic susceptibility was determined by using Metronidazole and Clindamycin. Furthermore, the phages were isolated and characterized against BV strains. RESULTS AND CONCLUSION: The Gram staining showed high prevalence of Staphylococcus (36% vs. 33%), followed by Streptococcus (31% vs. 14%) and Enterococcus (7% vs. 14%) in non-pregnant and pregnant females' respectively. However, the exception was observed in non-pregnant BV positive females, who had Shigella flexneri in their samples. The antibiotic sensitivity showed Metronidazole was resistant against all BV microbiota, and Clindamycin showed susceptibility against 3 strains. Phages were isolated against three bacterial strains, i.e. E. faecalis, E. faecium, and S. flexneri. Bacterial reduction assay showed bacterial growth decreases in the presence of phage suspension, pH stability showed phages' maximum lytic activity at pH 7 for E. faecalis and E. faecium and pH 9 for S. flexneri. However, the thermal stability showed phages' highest lytic activity at 55 °C for E. faecalis, 70 °C for E. faecium, and 40 °C for S. flexneri. Phage genome isolation showed that all phages nucleic acid was DNA in nature and between 15 and 20kbp. SEM analysis showed they were circular in shape and might belong to the Podoviridae family. This study provides an understanding of pathogens involved in BV and helps the doctors to treat the patients accordingly. Furthermore, this study showed that Bacterial Vaginosis and BV secondary bacteria have associations. BV secondary microbiota is also involved in the pathogenesis of this infection, whereas bacteriophage therapy has the potential to be used as an alternative treatment to antibiotics.

Molecular epidemiology of Shigella flexneri isolated from pediatrics in a diarrhea-endemic area of Khyber Pakhtunkhwa, Pakistan.

Shigella flexneri is considered as an important causative agent of Shigellosis causing diarrhea in the countries with a low socioeconomic status. No study has been carried out on the molecular prevalence of S. flexneri in Khyber Pakhtunkhwa, Pakistan. So this study was designed to evaluate the molecular prevalence of S. flexneri and their associated risk factors. A total of 2014 diarrheal stool samples were collected from January 2016 to May 2017 from pediatrics patients of Khyber Pakhtunkhwa followed by identification of S. flexneri through biochemical, serological, and molecular methods. The overall prevalence of Shigella species was found to be 7.9% (n = 160). The predominant Shigella specie was S. flexneri (n = 155, 96.8%) followed by S. boydii (n = 5, 3.1%). Interestingly, no sample was found positive for S. sonnei and S. dysenteriae. The majority of Shigellosis cases occurred from June to September. Potential risk factors related with Shigellosis were unhygienic latrine usage, bad hand washing, and consumption of unhygienic food and water, and pipe leakage in the sewage system. In this study, we have observed a high number of Shigellosis cases especially those caused by S. flexneri. It is suggested that effective health awareness programs should be organized by the regional health authorities to minimize the magnitude of pediatrics Shigellosis.

Evaluating Shigella flexneri Pathogenesis in the Human Enteroid Model.

The enteric pathogen Shigella is one of the leading causes of moderate-to-severe diarrhea and death in young children in developing countries. Transformed cell lines and animal models have been widely used to study Shigella pathogenesis. In addition to altered physiology, transformed cell lines are composed of a single cell type that does not sufficiently represent the complex multicellular environment of the human colon. Most available animal models do not accurately mimic human disease. The human intestinal enteroid model, derived from LGR5+ stem cell-containing intestinal crypts from healthy subjects, represents a technological leap in human gastrointestinal system modeling and provides a more physiologically relevant system that includes multiple cell types and features of the human intestine. We established the utility of this model for studying basic aspects of Shigella pathogenesis and host responses. In this study, we show that Shigellaflexneri is capable of infecting and replicating intracellularly in human enteroids derived from different segments of the intestine. Apical invasion by S. flexneri is very limited but increases ∼10-fold when enteroids are differentiated to include M cells. Invasion via the basolateral surface was at least 2-log10 units more efficient than apical infection. Increased secretion of interleukin-8 and higher expression levels of the mucin glycoprotein Muc2 were observed in the enteroids following S. flexneri infection. The human enteroid model promises to bridge some of the gaps between traditional cell culture, animal models, and human infection.

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.

A mouse model of shigellosis by intraperitoneal infection.

In human and nonhuman primates, Shigella spp. cause bacillary dysentery by invading colon epithelium and promoting a strong inflammatory response; however, adult mice are resistant to oral Shigella infection. In this study, intraperitoneal challenge with virulent S. flexneri 2a (YSH6000) resulted in diarrhea and severe body weight loss in adult B6 mice. Of note, virulent S. flexneri 2a could invade and colonize not only systemic tissues but also the serosa and lamina propria region of the large intestine. In addition, epithelial shedding, barrier integrity, and goblet cell hyperplasia were found in the large intestine by 24 hours post-intraperitoneal Shigella infection. Of note, predominant expression of proinflammatory cytokines and chemokines were found in the large intestine after intraperitoneal challenge. Monocytes played a critical role in attenuating diarrhea and in providing protective efficacy against intraperitoneal Shigella infection. Most importantly, mice prevaccinated with attenuated S. flexneri 2a (SC602) strain were protected against intraperitoneal challenge with YSH6000. When taken together, these findings show that intraperitoneal challenge with virulent S. flexneri 2a can provoke bacillary dysentery and severe pathogenesis in adult mice. This model may be helpful for understanding the induction mechanism of bacillary dysentery and for evaluating Shigella vaccine candidates.

Development of a Shigella conjugate vaccine targeting Shigella flexneri 6 that is immunogenic and provides protection against virulent challenge.

Immunity protective against shigella infection targets the bacterial O-specific polysaccharide (OSP) component of lipopolysaccharide. A multivalent shigella vaccine would ideally target the most common global Shigella species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. We previously reported development of shigella conjugate vaccines (SCVs) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using a platform squaric acid chemistry conjugation approach and carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. Here we report development of a SCV targeting S. flexneri 6 (SCV-Sf6) using the same platform approach. We demonstrated that SCV-Sf6 was recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG and IgM responses, as well as rTTHc-specific IgG responses. Immune responses were increased when administered with aluminum phosphate adjuvant. Vaccination induced bactericidal antibody responses against S. flexneri 6, and vaccinated animals were protected against lethal challenge with virulent S. flexneri 6. Our results assist in the development of a multivalent vaccine protective against shigellosis.

The relationship between integrons, antibiotic resistance genes and SXT resistance in Shigella flexneri strains.

OBJECTIVE: To investigate the correlation between sulfamethoxazole-trimethoprim (SXT) resistance in Shigella flexneri (S.flexneri) and the presence of integrons and relevant antibiotic resistance genes. METHODS: We collected 115 strains of Shigella flexneri isolated from feces of children with diarrhea in Jinan from 2012 to 2020 and determined the minimum inhibitory concentration (MIC) of SXT by Etest method. The presence of class 1, class 2, and class 3 integron genes, variable region antibiotic resistance gene cassettes, and sul1, sul2, sul3, and SXT elements were detected using polymerase chain reaction (PCR). Positive results were further analyzed by DNA sequencing and BLAST comparison. RESULTS: In total, the resistance rate to SXT was 60.9% among the 115 S.flexneri strains. The prevalence of class 1 and class 2 integrons were 88.7% and 87.0%, respectively, with no class 3 integrons detected. Among the strains, 13.0% carried typical class 1 integrons with variable region antibiotic resistance gene cassettes dfrA17-aadA5 and dfrV, while 85.2% carried atypical class 1 integrons with variable region antibiotic resistance gene cassette blaoxa-30-aadA1. The variable region antibiotic resistance gene cassettes of class 2 integrons were all dfrA1+sat1+aadA1. There was a statistical difference between the presence of class 1 integrons and class 2 integrons between the SXT-sensitive and resistant S.flexneri strains (χ2=22.800, χ2=16.365, P<0.01, P<0.01). Integrons carrying dfrV and dfrA1 by integrons also showed a statistical difference in SXT resistance (χ2=9.422, χ2=16.365, P<0.01, P<0.01). PCR revealed the presence of sul1 and sul2 in 13.0% and 47.0% of strains, respectively, with neither sul3 nor SXT elements detected. There was a significant difference between the presence of sul1, sul2 between the SXT-sensitive and resistant S.flexneri strains (χ2=9.588, χ2=65.445, P<0.01, P<0.01). CONCLUSION: In summary, integrons are involved in SXT resistance of S.flexneri, and dfrV, dfrA1, sul1, sul2 are closely related to SXT resistance of S.flexneri.

Development of Shigella conjugate vaccines targeting Shigella flexneri 2a and S. flexneri 3a using a simple platform-approach conjugation by squaric acid chemistry.

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.

Azithromycin alters Colony Stimulating Factor-1R (CSF-1R) expression and functional output of murine bone marrow-derived macrophages: A novel report.

Antibiotic treatment may lead to side effects that require mechanistic explanation. We investigated the effect of azithromycin (AZM) treatment on bone marrow-derived macrophage (Mφ) generation, their functional output, and the subsequent effect on bacterial clearance in a mouse model of S. flexneri infection. To our fascination, AZM increased PU.1, C/EBPß, CSF-1R/pCSF-1R expressions leading to M2-skewed in vitro BMDM generation. Altered Mφ-functions like- phagocytosis, oxidative stress generation, inflammasome-activation, cytokine release, and phenotype (pro-inflammatory-M1, anti-inflammatory-M2) even in the presence of infection were observed with AZM treatment. AZM increased CD206, egr2, arg1 (M2-marker) expression and activity while reducing CD68, inducible nitric oxide (iNOS) expression, and activity (M1-marker) in Mφs during infection. Pro-inflammatory cytokines (TNF-α, IL-12, IL-1ß) were reduced and anti-inflammatory IL-10 release was augmented by AZM-treated-iMφs (aiMφs) along with decreased asc, nlrp3, aim2, nlrp1a, caspase1 expressions, and caspase3 activity signifying that aMφs/aiMφs were primed towards an anti-inflammatory phenotype. Interestingly, CSF-1R blockade increased NO, IL-12, TNF-α, IL-1ß, decreased TGF-ß release, and CD206 expression in aiMφs. T-cell co-stimulatory molecule cd40, cd86, and cd80 expressions were decreased in ai/aM1-Mφs and co-cultured CD8+, CD4+ T-cells had decreased proliferation, t-bet, IFN-γ, IL-17, IL-2 but increased foxp3, TGF-ß, IL-4 which were rescued with CSF-1R blockade. Thus AZM affected Mφ-functions and subsequent T-cell responses independent of its antibacterial actions. This was validated in the balb/c model of S. flexneri infection. We conclude that AZM skewed BMDM generation to anti-inflammatory M2-like via increased CSF-1R expression. This warrants further investigation of AZM-induced altered-Mφ-generation during intracellular infections.

Combined effects of microplastics and antibiotic-resistant bacteria on Daphnia magna growth and expression of functional genes.

Microplastics could act as vectors for the transport of harmful bacteria, such as pathogens and antibiotic resistance bacteria (ARB), but their combined effects have not been reported yet. Here, ARB Shigella flexneri with sulfonamides resistance and micro-polystyrene (micro-PS) were used to investigate their possible combined effects on the growth and expression of functional genes in Daphnia magna. Results showed that micro-PS colonized with S. flexneri were ingested by D. magna and blocked in their intestine after 24 h exposure. Changes were observed in the life history and morphology of D. magna, as well as the expression of functional genes in all treatments, but with no difference in the survival rate. We also determined the expression of six functional genes involved in energy and metabolism (arginine kinase, AK) and oxidative stress response (thioredoxin reductase, TRxR, catalase, CAT, and glutathione S-transferases, GSTs), as well as in growth, development and reproduction (vitellogenin, Vtg1 and ecdysone receptor, EcR). AK and Vtg1 did not show significant differences, however, EcR was down-regulated and the other three genes (TRxR, CAT, GSTs) were up-regulated in the combined-treated group. Antibiotic resistance gene (ARGs) sul1 was detected when exposed to micro-PS colonized with S. flexneri., suggesting that D. magna could acquire resistance genes through microplastic biofilms. These results indicated that MPs could act as a carrier of ARB to transfer ARGs into D. magna, and affect the life history, morphology, and the expression of related functional genes of D. magna, to adapt to the stress caused by MPs and ARB.

In silico and in vivo approaches to recombinant multi-epitope immunogen of GroEL provides efficient cross protection against S. Typhimurium, S. flexneri, and S. dysenteriae.

OBJECTIVES: Stress or Heat Shock Proteins (HSPs) have been included in various operations like protein folding, autophagy, and apoptosis. HSP families recognize as protective antigens in a wide range of bacteria because they have been conserved through evolution. Due to their homology as well as antigenicity they are competent for applying in cross-protection against bacterial diseases. METHODS: In the present study, bioinformatics approaches utilized to design epitope-based construction of Hsp60 (or GroEL) protein. In this regard, potential B-cell and T-cell epitopes except for allergenic sequences were selected by immunoinformatic tools. The structural and functional aspects of the DNA, RNA, and protein levels were assessed by bioinformatics software. Following in silico investigations, recombinant GroEL multi-epitope of Salmonella typhi was expressed, purified, and validated. Mouse groups were immunized with recombinant protein and humoral immune response was measured by enzyme linked immunosorbent assay (ELISA). Animal challenge against Salmonella Typhimurium, Shigella flexneri, and Shigella dysenteriae was evaluated. RESULTS: recombinant protein expression and purification with 14.3 kilodaltons (kDa) was confirmed by SDS-PAGE and western blotting. After animal administration, the immunoglobulins evaluated increase after each immunization. Immunized antisera exhibited 80%, 40%, and 40% protection against the lethal dose infection by S. Typhimurium, S. flexneri, and S. dysenteriae respectively. Passive immunization conferred 50%, 30%, and 30% protection in mice against S. Typhimurium, S. flexneri and S. dysentery respectively. In addition, bacterial organ load had exhibited a significant decrease in colony forming unit (CFU) in the liver and spleen of the immunized mice compared to the control. CONCLUSION: Our study demonstrates the efficacy of S. Typhi recombinant GroEL multi-epitope to consider as a universal immunogen candidate versus multiple bacterial pathogens.

Diarrhea in an infant due to Shigella flexneri 1 carrying multiple cephalosporinase-encoding genes.

BACKGROUND: Infections caused by multidrug-resistant shigellae resistant to broad-spectrum cephalosporins are becoming more prevalent in the Middle East. We report a case of severe diarrhea due to a multiresistant Shigella flexneri 1 strain carrying four different ß-lactamase genes. CASE PRESENTATION: A one-year-old Syrian infant presented with severe acute diarrhea, vomiting and dehydration. She did not respond to empirical treatment with amoxicillin-clavulanic acid followed by cefotaxime. Later, stool culture revealed S. flexneri 1 resistant to both these drugs. The patient was successfully treated with meropenem to which S. flexneri 1 was susceptible. The isolate was resistant to eight classes of antibiotics, and the whole genome sequence (WGS) identified four ß-lactamase genes (blaCTX-M-15, blaEC-8, blaOXA-1, and blaTEM-1) along with genes mediating resistance to seven other antibiotic classes. The WGS also identified several virulence genes including senA that encodes ShET-2 which induces watery diarrhea. Phylogenetically, the isolate was closely related to isolates from South Asia. CONCLUSIONS: This report highlights the emergence of extremely resistant Shigella that has acquired multiple resistance genes to cephalosporins rendering these drugs ineffective.

Secondary Structural Transformation of Bovine Lactoferricin Affects Its Antibacterial Activity.

Lactoferricin (Lfcin) is a potent antibacterial peptide derived from lactoferrin by pepsin hydrolysis. It was hypothesized that structural transformation of Lfcin could affect its antibacterial function through forming and breaking of intramolecular disulfide bond. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with a disulfide bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism (CD) spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. Then, the secondary structure contents of the peptides were calculated on the basis of the CD spectra. The antibacterial activity of the peptides against Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Shigella flexneri ATCC 12022, and Staphylococcus aureus ATCC 25923 was evaluated. The results showed that bLfcin and bLfcin C36G had similar percentages of secondary structure in water, while bLfcin and bLfcin DB had similar ratios of secondary structure under less hydrophobic conditions. The synthetic peptides exhibited antibacterial activity against all the tested bacteria, except for S. aureus ATCC 25923. bLfcin demonstrated higher antibacterial activity compared with its derivatives. The results suggested that bLfcin could transform its structure under alterative ionic strengths and hydrophobic conditions, and the transformation of structures was beneficial to enhancing the antibacterial function.

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.

Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases.

In this study, we describe a multiplex PCR method for the detection of five food-relevant virulence pathogenicity genes of intestinal pathogens. Five pairs of primers were designed based on nuc gene for Staphylococcus aureus, hlyA gene of Listeria monocytogenes, ipaH gene of Shigella flexneri, lysP gene of Yersinia enterocolitica and tpi gene of Clostridium difficile. Conditions were optimized to amplify fragments of those genes simultaneously in one PCR amplification. After developing and optimizing the multiplex PCR reaction system, the specificity and sensitivity of the multiple PCR assays were evaluated. The optimized program is also applied to retail meat for testing. The result indicated that when the annealing temperature was 54 °C and the primer concentrations of S. aureus, L. monocytogenes, S. flexneri, Y. enterocolitica and C. difficile are 10, 10, 5, 3 and 2 µM, the five strains could expand 484, 345, 204, 156, 88 bp of clear fragments, respectively. So was the multiple PCR in artificially contaminated beef produce. All cultures were cultured and separated by traditional methods. The multiplex PCR method offers a rapid, simple, and accurate identification of pathogens and could be used in food safety investigations, clinical diagnosis as well as for the surveillance of the spreading determinants of pathogens in epidemiological studies.

Phylogenetic Characterization Reveals Prevalent Shigella flexneri ST100 Clone in Beijing, China, 2005 to 2018.

Shigella flexneri is a major cause of bacillary dysentery in Beijing, China. The genetic features and population structure of locally circulating clones remained unclear. In this study, we sequenced the genomes of 93 S. flexneri isolates from patients in Beijing from 2005 to 2018. Phylogenetic analysis revealed a predominant lineage comprised of ST100 isolates that had acquired an extensive repertoire of antimicrobial resistance determinants. A rapid local expansion of the largest clade of this lineage began in 2008 and gradually resulted in the dominance of serotype 2a. Other clades showed substantial evidence of interregional spread from other areas of China. Another lineage consisting of ST18 isolates was also identified and appeared to have persisted locally for nearly 6 decades. These findings suggest that S. flexneri epidemics in Beijing were caused by both local expansion and interregional transmission.IMPORTANCE Beijing is the largest transportation hub in China, with a highly mobile population. Shigella flexneri is a major cause of bacillary dysentery in Beijing. However, little is known about the genetic features and population structure of locally circulating S. flexneri clones. Whole-genome sequencing of 93 S. flexneri isolates revealed that S. flexneri epidemics in Beijing were predominantly caused by an ST100 clone. Interregional spread, rapid local expansion, and acquirement of antimicrobial resistance determinants have cocontributed to the epidemics of this clone. Another ST18 clone was also identified and showed long-term colonization in Beijing. Our study provides comprehensive insights into the population structure and evolutionary history of S. flexneri in Beijing.

Heterologous expression of Intimin and IpaB fusion protein in Lactococcus lactis and its mucosal delivery elicit protection against pathogenicity of Escherichia coli O157 and Shigella flexneri in a murine model.

Escherichia coli O157:H7 and Shigella flexneri are the predominant diarrhoeal pathogens and those strains producing Shiga toxins cause life-threatening sequelae including hemolytic uremic syndrome (HUS) upon their entry into the host. Intimate adherence of E. coli O157 and invasion of S. flexneri in the host intestinal epithelial cells is mainly mediated by Intimin and IpaB proteins, respectively. In this study, we have synthesized chimera of immunodominant regions of Intimin (eae) and IpaB (ipaB) designated as EI and expressed it in Lactococcus lactis (LL-EI) to develop a combinatorial oral vaccine candidate. Immune parameters and protective efficacy of orally administered LL-EI were assessed in the murine model. Significant EI-specific serum IgG, IgA, and fecal IgA antibody titer were observed in the LL-EI group. Considerable increase in EI-specific splenocyte proliferation and a concurrent upregulation of both Th1 and Th2 cytokines was observed in LL-EI immunized mice. Flow cytometry analysis also revealed a significant increase in CD4 and CD8 cell counts in LL-EI immunized group compared to PBS, LL control group.In vitro studies using LL-EI immunized mice sera showed substantial protection against bacterial adhesion and invasion caused by E. coli O157 and Shigella flexneri¸ respectively. LL-EI immunized group challenged with E. coli O157 ceased fecal shedding within 6 days, and mice challenged with S. flexneri showed 93% survival with minimal bacterial load in the lungs. Our results indicate that LL-EI immunization elicits systemic, mucosal and cell-mediated immune responses, and can be a promising candidate for oral vaccine development against these pathogens.