Global population structure and genotyping framework for genomic surveillance of the major dysentery pathogen, Shigella sonnei.

Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring the spread of resistant clones, including those that have recently emerged, at local and global scales.

Antibodies Elicited by the Shigella sonnei GMMA Vaccine in Adults Trigger Complement-Mediated Serum Bactericidal Activity: Results From a Phase 1 Dose Escalation Trial Followed by a Booster Extension.

Shigella is the second most deadly diarrheal disease among children under five years of age, after rotavirus, with high morbidity and mortality in developing countries. Currently, no vaccine is widely available, and the increasing levels of multidrug resistance make Shigella a high priority for vaccine development. The single-component candidate vaccine against Shigella sonnei (1790GAHB), developed using the GMMA technology, contains the O antigen (OAg) portion of lipopolysaccharide (LPS) as active moiety. The vaccine was well tolerated and immunogenic in early-phase clinical trials. In a phase 1 placebo-controlled dose escalation trial in France (NCT02017899), three doses of five different vaccine formulations (0.06/1, 0.3/5, 1.5/25, 3/50, 6/100 µg of OAg/protein) were administered to healthy adults. In the phase 1 extension trial (NCT03089879), conducted 2-3 years following the parent study, primed individuals who had undetectable antibody levels before the primary series received a 1790GAHB booster dose (1.5/25 µg OAg/protein). Controls were unprimed participants immunized with one 1790GAHB dose. The current analysis assessed the functionality of sera collected from both studies using a high-throughput luminescence-based serum bactericidal activity (SBA) assay optimized for testing human sera. Antibodies with complement-mediated bactericidal activity were detected in vaccinees but not in placebo recipients. SBA titers increased with OAg dose, with a persistent response up to six months after the primary vaccination with at least 1.5/25 µg of OAg/protein. The booster dose induced a strong increase of SBA titers in most primed participants. Correlation between SBA titers and anti-S. sonnei LPS serum immunoglobulin G levels was observed. Results suggest that GMMA is a promising OAg delivery system for the generation of functional antibody responses and persistent immunological memory.

Screening of the Dichloromethane: Methanolic Extract of Centella asiatica for Antibacterial Activities against Salmonella typhi, Escherichia coli, Shigella sonnei, Bacillus subtilis, and Staphylococcus aureus.

Bacterial infections are responsible for a large number of deaths every year worldwide. On average, 80% of the African population cannot afford conventional drugs. Moreover, many synthetic antibiotics are associated with side effects and progressive increase in antimicrobial resistance. Currently, there is growing interest in discovering new antibacterial agents from ethnomedicinal plants. About 60% of the population living in developing countries depends on herbal drugs for healthcare needs. This study involved the screening of Centella asiatica commonly used by herbal medicine practitioners in Kisii County to treat symptoms related to bacterial infections. Standard bioassay methods were applied throughout the study. They included preliminary screening of dichloromethane: methanolic extract of Centella asiatica against human pathogenic bacteria including Salmonella typhi ATCC 19430, Escherichia coli ATCC 25922, Shigella sonnei ATCC 25931, Bacillus subtilis ATCC 21332, and Staphylococcus aureus ATCC 25923 using agar disc diffusion, broth microdilution method, and time-kill kinetics with tetracycline as a positive control. Phytochemical screening was carried out to determine the different classes of compounds in the crude extracts. Data were analyzed using one way ANOVA and means separated by Tukey's test. Dichloromethane: methanolic extract of Centella asiatica was screened against the selected bacterial strains. Time-kill kinetic studies of the extracts showed dose- and time-dependent kinetics of antibacterial properties. Phytochemical screening of the DCM-MeOH extract revealed the presence of alkaloids, flavonoids, phenolics, terpenoids, cardiac glycosides, saponins, steroids, and tannins. The present study indicates that the tested plant can be an important source of antibacterial agents and recommends that the active phytoconstituents be isolated, identified, and screened individually for activities and also subjected further for in vivo and toxicological studies.

Isolation of ß-glucan from Eleusine coracana and its antibiofilm, antidiabetic, antioxidant, and biocompatible activities.

Eleusine coracana (Finger millet) has high nutritional value with numerous health benefits and is of low cost. Isolation of beta-glucan (ßG) from E. coracana (Ec-ßG) has gained increasing research attention. UV-vis spectroscopy used to measure the surface plasmon resonance at 361 nm to confirm the presence of polysaccharides (glucan molecules) in Ec-ßG. X-ray diffraction analysis of Ec-ßG displayed a crystalline nature and confirmed the presence of the ßG molecule. Further, the bioactive compounds of Ec-ßG were screened using gas chromatography-mass spectrometry. The antibacterial activity of Ec-ßG against both Gram-positive (Lysinibacillus fusiformis, Enterococcus faecalis) and Gram-negative (Proteus vulgaris, Shigella sonnei) bacteria were assessed through minimum inhibitory concentrations <70 µg/ml of Ec-ßG. In addition, the antibiofilm activity and bacterial viability of Ec-ßG at 100 µg/ml was confirmed by light and confocal laser scanning microscopy. Furthermore, Ec-ßG inhibits α-amylase and α-glucosidase at an IC50 -value of 1.23 and 1.42 µg/ml, respectively. Superoxide anion scavenging activity at IC50-1.4 µg/ml and DPPH radical scavenging activity at IC50-1.2 µg/ml showed that Ec-ßG had potential antioxidant property. The in vitro hemolysis assay for biocompatibility of Ec-ßG at 200 µg/ml showed 0.06 ± 0.09%. Therefore, Ec-ßG has the potential to act as a suggestive agent for antibacterial, antidiabetic, and antioxidant activity.

Shigella sonnei O-Antigen Inhibits Internalization, Vacuole Escape, and Inflammasome Activation.

Two Shigella species, Shigella flexneri and Shigella sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle- and high-income countries. S. flexneri is a prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species.IMPORTANCE Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.

R18C is a new viable P2-like bacteriophage of rabbit origin infecting Citrobacter rodentium and Shigella sonnei strains.

Here, we report a novel virulent P2-like bacteriophage, R18C, isolated from rabbit faeces, which, in addition to Escherichia coli K-12 strains, was able to be propagated on Citrobacter rodentium strain ICC169 and a range of Shigella sonnei strains with high efficiency of plating (EOP). It represents the first lytic bacteriophage originating from rabbit and the first infectious P2-like phage of animal origin. In the three characteristic moron-containing regions of P2-like phages, R18C contains genes with unknown function that have so far only been found in cryptic P2-like prophages.

Dissecting the molecular evolution of fluoroquinolone-resistant Shigella sonnei.

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance.

Development of FAcE (Formulated Alhydrogel competitive ELISA) method for direct quantification of OAg present in Shigella sonnei GMMA-based vaccine and its optimization using Design of Experiments approach.

Many formulated vaccines, including 1790GAHB Shigella sonnei GMMA-based vaccine, contain Alhydrogel (aluminum hydroxide), consequently the antigen content must be determined in the formulated final vaccine product, as required by regulatory authorities. The direct quantification of antigens adsorbed on aluminum salts is difficult, and antigens may need to be extracted using laborious and often ineffective desorption procedures. To directly quantify the sugar vaccine target in the LPS of 1790GAHB, we have developed a new FAcE (Formulated Alhydrogel competitive ELISA) method. FAcE is an immunoassay based on the competition between S. sonnei LPS, coated on the ELISA plate, and the LPS in formulated S. sonnei GMMA, in binding a specific monoclonal antibody. To optimize the method, which is as easy to perform as a standard ELISA, we have applied a Design of Experiments (DOE) approach. A model was found to define the significant assay variables and to predict their impact on the output responses. Results obtained using the DOE optimized FAcE assay showed that the method is sensitive (0.02 µg/mL lower detection limit), precise, reproducible and can accurately quantify independently formulated drug products, making it a useful tool in routine tests of Alhydrogel-based vaccines. We are currently using this method to determine S. sonnei vaccine potency, stability and lot-to-lot variations, and are broadening its applicability to quantify active ingredients of other Alhydrogel GMMA-vaccines and in multivalent vaccines formulations.

Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput Shigella Serum Bactericidal Assay.

Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with Shigella vaccines containing LPS can elicit antibodies capable of killing Shigella in a serotype-specific manner. Thus, to facilitate Shigella vaccine development, we have developed a serum bactericidal assay (SBA) specific for three Shigella serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against S. flexneri 2a, S. flexneri 3a, and S. sonnei Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The Shigella SBA, combined with a reference serum, should facilitate the development of Shigella vaccines across the field.IMPORTANCEShigella is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective Shigella vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring Shigella-specific functional antibodies in vitro We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate Shigella vaccine development.

Shigella sonnei Does Not Use Amoebae as Protective Hosts.

Shigella flexneri and Shigella sonnei bacteria cause the majority of all shigellosis cases worldwide. However, their distributions differ, with S. sonnei predominating in middle- and high-income countries and S. flexneri predominating in low-income countries. One proposed explanation for the continued range expansion of S. sonnei is that it can survive in amoebae, which could provide a protective environment for the bacteria. In this study, we demonstrate that while both S. sonnei and S. flexneri can survive coculture with the free-living amoebae Acanthamoebae castellanii, bacterial growth is predominantly extracellular. All isolates of Shigella were degraded following phagocytosis by A. castellanii, unlike those of Legionella pneumophila, which can replicate intracellularly. Our data suggest that S. sonnei is not able to use amoebae as a protective host to enhance environmental survival. Therefore, alternative explanations for S. sonnei emergence need to be considered.IMPORTANCE The distribution of Shigella species closely mirrors a country's socioeconomic conditions. With the transition of many populous nations from low- to middle-income countries, S. sonnei infections have emerged as a major public health issue. Understanding why S. sonnei infections are resistant to improvements in living conditions is key to developing methods to reduce exposure to this pathogen. We show that free-living amoebae are not likely to be environmental hosts of S. sonnei, as all Shigella strains tested were phagocytosed and degraded by amoebae. Therefore, alternative scenarios are required to explain the emergence and persistence of S. sonnei infections.

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.

The Shigella ProU system is required for osmotic tolerance and virulence.

To cope with hyperosmotic stress encountered in the environments and in the host, the pathogenic as well as non-pathogenic microbes use diverse transport systems to obtain osmoprotectants. To study the role of Shigella sonnei ProU system in response to hyperosmotic stress and virulence, we constructed deletion and complementation strains of proV and used an RNAi approach to silence the whole ProU operon. We compared the response between wild type and the mutants to the hyperosmotic pressure in vitro, and assessed virulence properties of the mutants using gentamicin protection assay as well as Galleria mellonella moth larvae model. In response to osmotic stress by either NaCl or KCl, S. sonnei highly up-regulates transcription of proVWX genes. Supplementation of betaine greatly elevates the growth of the wild type S. sonnei but not the proV mutants in M9 medium containing 0.2 M NaCl or 0.2 M KCl. The proV mutants are also defective in intracellular growth compared with the wild type. The moth larvae model of G. mellonella shows that either deletion of proV gene or knockdown of proVWX transcripts by RNAi significantly attenuates virulence. ProU system in S. sonnei is required to cope with osmotic stress for survival and multiplication in vitro, and for infection.

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

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

An innate immune system-mimicking, real-time biosensing of infectious bacteria.

An animal cell-based biosensor was investigated to monitor bacterial contamination in an unattended manner by mimicking the innate immune response. The cells (RAW 264.7 cell line) were first attached onto the solid surfaces of a 96-well microtiter plate and co-incubated in the culture medium with a sample that might contain bacterial contaminants. As Toll-like receptors were present on the cell membrane surfaces, they acted as a sentinel by binding to pathogen-associated molecular patterns (PAMPs) of any contaminant. Such biological recognition initiates signal transmission along various pathways to produce different proinflammatory mediators, one of which, tumor necrosis factor-α (TNF-α) was measured using an immunosensor. To demonstrate automated bacterium monitoring, a capture antibody specific for TNF-α was immobilized on an optical fiber sensor tip and then used to measure complex formation in a label-free sensor system (e.g., Octet Red). The sensor response time depended significantly on the degree of agitation of the culture medium, controlling the biological recognition and further autocrine/paracrine signaling by cytokines. The response, particularly under non-agitated conditions, was also influenced by the medium volume, revealing a local gradient change of the cytokine concentration and also acidity, caused by bacterial growth near the bottom surfaces. A biosensor system retaining 50 µL medium and not employing agitation could be used for the early detection of bacterial contamination. This novel biosensing model was applied to the real-time monitoring of different bacteria, Shigella sonnei, Staphylococcus aureus, and Listeria monocytogenes. They (<100 CFU mL(-1)) could be detected automatically within the working time. Such analysis was carried out without any manual handling regardless of the bacterial species, suggesting the concept of non-targeted bacterial real-time monitoring. This technique was further applied to real sample testing (e.g., with milk) to exemplify, for example, the food quality control process without using any additional sample pretreatment such as magnetic concentration.

The Rising Dominance of Shigella sonnei: An Intercontinental Shift in the Etiology of Bacillary Dysentery.

Shigellosis is the major global cause of dysentery. Shigella sonnei, which has historically been more commonly isolated in developed countries, is undergoing an unprecedented expansion across industrializing regions in Asia, Latin America, and the Middle East. The precise reasons underpinning the epidemiological distribution of the various Shigella species and this global surge in S. sonnei are unclear but may be due to three major environmental pressures. First, natural passive immunization with the bacterium Plesiomonas shigelloides is hypothesized to protect populations with poor water supplies against S. sonnei. Improving the quality of drinking water supplies would, therefore, result in a reduction in P. shigelloides exposure and a subsequent reduction in environmental immunization against S. sonnei. Secondly, the ubiquitous amoeba species Acanthamoeba castellanii has been shown to phagocytize S. sonnei efficiently and symbiotically, thus allowing the bacteria access to a protected niche in which to withstand chlorination and other harsh environmental conditions in temperate countries. Finally, S. sonnei has emerged from Europe and begun to spread globally only relatively recently. A strong selective pressure from localized antimicrobial use additionally appears to have had a dramatic impact on the evolution of the S. sonnei population. We hypothesize that S. sonnei, which exhibits an exceptional ability to acquire antimicrobial resistance genes from commensal and pathogenic bacteria, has a competitive advantage over S. flexneri, particularly in areas with poorly regulated antimicrobial use. Continuing improvement in the quality of global drinking water supplies alongside the rapid development of antimicrobial resistance predicts the burden and international distribution of S. sonnei will only continue to grow. An effective vaccine against S. sonnei is overdue and may become one of our only weapons against this increasingly dominant and problematic gastrointestinal pathogen.

Molecular characterization and PCR-based replicon typing of multidrug resistant Shigella sonnei isolates from an outbreak in Thimphu, Bhutan.

BACKGROUND: Shigella species are an important cause of diarrhea in developing countries. These bacteria normally acquire their antibiotic resistance via several different mobile genetic elements including plasmids, transposons, and integrons involving gene cassettes. During a diarrhea surveillance study in Thimphu, Bhutan in June and July, 2011, Shigella sonnei were isolated more frequently than expected. This study describes the antibiotic resistance of these S. sonnei isolates. METHODS: A total of 29 S. sonnei isolates from Thimphu, Bhutan was characterized for antimicrobial susceptibility by disc diffusion assay and minimum inhibitory concentration (MIC) assay. All isolates were tested by pulsed-field gel electrophoresis (PFGE) with restriction enzyme XbaI and were tested for plasmid. The plasmid patterns and the PFGE patterns were analyzed by Bionumerics software. DNA sequencing was performed on amplified products for gyraseA gene and class 1 and class 2 integrons. S. sonnei isolates were classified for incompatibility of plasmids by PCR-based replicon typing (PBRT). RESULTS: These S. sonnei were resistant to multiple drugs like ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, streptomycin, and tetracycline but susceptible to azithromycin. All isolates had class 2 integrons dfrA1, sat1 and aadA1 genes. Two point mutations in Gyrase A subunit at position Ser83Leu and Asp87Gly were detected in these quinolone resistant isolates. The plasmid and PFGE patterns of S. sonnei isolates suggested a clonal relationship of the isolates. All isolates carried common ColE plasmid. ColE plasmid co-resided with B/O plasmid (nine isolates) or I1 plasmid (one isolate). CONCLUSIONS: The characteristics of 29 S. sonnei isolates from Thimphu, Bhutan in June and July, 2011 are identical in PFGE, plasmid and resistance pattern. This study suggests that these recent S. sonnei isolates are clonally related and multidrug-resistant.

Tracking the establishment of local endemic populations of an emergent enteric pathogen.

Shigella sonnei is a human-adapted pathogen that is emerging globally as the dominant agent of bacterial dysentery. To investigate local establishment, we sequenced the genomes of 263 Vietnamese S. sonnei isolated over 15 y. Our data show that S. sonnei was introduced into Vietnam in the 1980s and has undergone localized clonal expansion, punctuated by genomic fixation events through periodic selective sweeps. We uncover geographical spread, spatially restricted frontier populations, and convergent evolution through local gene pool sampling. This work provides a unique, high-resolution insight into the microevolution of a pioneering human pathogen during its establishment in a new host population.

Study of probiotic potential of four wild Lactobacillus rhamnosus strains.

The four wild Lactobacillus rhamnosus strains were examined in vitro for resistance to simulated gastro and intestinal juices, adhesion to HT-29 cells, antagonistic activity against enteric pathogens and immunomodulating activity. The strains L. rhamnosus SB5L, J5L and IN1L were able to survive in simulated gastro juice while the strain L. rhamnosus SB31L lost viability exposed to simulated gastro juice for 3 h. The four strains had high viability in simulated small intestinal juice with little loss (<1.0 cycle reduction). The strains SB5L, J5L and IN1L antagonized against Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium ATCC 14028, Shigella sonnei ATCC 25931. The strain L. rhamnosus IN1L had the highest adhesive capability to HT-29 cells in vitro (251 bacteria cells per 100 HT-29 cells) compared to the other three L. rhamnosus strains. The live bacteria, cell wall and DNA of the four L. rhamnosus induced the secretion of pro-inflammatory cytokines IL-12 (p70), IFN-γ and TNF-α by human peripheral blood mononuclear cells (PBMCs). The levels of IL-12 (p70), IFN-γ and TNF-α produced by stimulated PBMCs were significantly higher (P < 0.05) than those of the control. Those data indicated that the four L. rhamnosus strains have the potential as the probiotic for human being use, although further studies are still needed.