Pathogen elimination by probiotic Bacillus via signalling interference.

Probiotic nutrition is frequently claimed to improve human health. In particular, live probiotic bacteria obtained with food are thought to reduce intestinal colonization by pathogens, and thus to reduce susceptibility to infection. However, the mechanisms that underlie these effects remain poorly understood. Here we report that the consumption of probiotic Bacillus bacteria comprehensively abolished colonization by the dangerous pathogen Staphylococcus aureus in a rural Thai population. We show that a widespread class of Bacillus lipopeptides, the fengycins, eliminates S. aureus by inhibiting S. aureus quorum sensing-a process through which bacteria respond to their population density by altering gene regulation. Our study presents a detailed molecular mechanism that underlines the importance of probiotic nutrition in reducing infectious disease. We also provide evidence that supports the biological significance of probiotic bacterial interference in humans, and show that such interference can be achieved by blocking a pathogen's signalling system. Furthermore, our findings suggest a probiotic-based method for S. aureus decolonization and new ways to fight S. aureus infections.

Intestinal microbiota: The hidden gems in the gut?

Within the last decade, our understanding of the role of the intestinal microbiota in health and disease has rapidly increased due to significant advances in next-generation sequencing technologies. Scientists have discovered more and more gut microbes with supposedly "beneficial" roles for human health and are starting to identify the underlying mechanisms. In this review, we summarize the latest knowledge about the human intestinal microbiota, including the intestinal bacteriome, virome and mycobiome. We discuss the function that recent studies attribute to the intestinal microbiota in preventing or controlling selected diseases and present recent research on biotherapeutic approaches to control these diseases.

Staphylococcus aureus colonisation and strategies for decolonisation.

Staphylococcus aureus is a leading cause of death by infectious diseases worldwide. Treatment of S aureus infections is difficult due to widespread antibiotic resistance, necessitating alternative approaches and measures for prevention of infection. Because S aureus infections commonly arise from asymptomatic colonisation, decolonisation is considered a key approach for their prevention. Current decolonisation procedures include antibiotic-based and antiseptic-based eradication of S aureus from the nose and skin. However, despite the widespread implementation and partial success of such measures, S aureus infection rates remain worrisome, and resistance to decolonisation agents is on the rise. In this Review we outline the epidemiology and mechanisms of S aureus colonisation, describe how colonisation underlies infection, and discuss current and novel approaches for S aureus decolonisation, with a focus on the latest findings on probiotic strategies and the intestinal S aureus colonisation site.

Probiotic for pathogen-specific Staphylococcus aureus decolonisation in Thailand: a phase 2, double-blind, randomised, placebo-controlled trial.

BACKGROUND: Decolonisation is considered a valuable means to reduce Staphylococcus aureus infection rates. However, previous topical strategies targeting the nose or skin had little success, and oral antibiotic-based decolonisation is ill advised because of eradication of the microbiota and development of antibiotic resistance. We previously showed that the probiotic Bacillus subtilis significantly diminished S aureus at the main intestinal colonisation site via specific bacterial interaction in mice; in this study, we tested this probiotic approach to control S aureus colonisation in humans. METHODS: We did a single-centre, phase 2, double-blind, randomised, placebo-controlled trial in adults from the Songkhla region of Thailand who were colonised by S aureus. Eligible participants were adults (aged ≥18 years) without history of intestinal disease, antibiotic treatment, or hospital admission within the previous 90 days. Participants were excluded if they were pregnant, breastfeeding, taking probiotics, or had diarrhoea. Participants were allocated (1:1) to groups by computer randomisation in blocks of four, and research coordinators were masked to group allocation. Participants received 250 mg of probiotic B subtilis MB40 or placebo once per day for 30 days and S aureus colonisation was determined after the last dose was received. The primary outcome was colonisation by S aureus (continuous, mean decrease in colony-forming-unit count) in the intestine (by faecal counts) and nares (by nasal swabs) after intervention (30-day regimen of B subtilis probiotic). This trial is registered with the Thai Clinical Trials Registry, TCTR20210128003. FINDINGS: The trial was done between Jan 29 and June 30, 2021, with enrolment taking place from Jan 29 to April 6, 2021. 115 participants were colonised by S aureus, either in the intestine (n=84), nose (n=50), or both (n=19), and were randomly assigned to treatment (n=55) and placebo groups (n=60). Oral probiotic B subtilis resulted in significant reduction of S aureus in stool (96·8%; p<0·0001) and nose (65·4%; p=0·0002). There were no differences in adverse effects or significant microbiome changes between the intervention and placebo groups. INTERPRETATION: B subtilis probiotic eliminated more than 95% of the total S aureus colonising the human body without altering the microbiota. This probiotic strategy offers several key advantages over presently used decolonisation strategies for potential use in people with chronic or long-term risk of S aureus infection. Furthermore, by establishing a defining role of the intestinal colonisation site, our findings call for revisiting fundamental notions about S aureus colonisation. FUNDING: National Research Council of Thailand and US National Institutes of Health.

Exacerbation of allergic rhinitis by the commensal bacterium Streptococcus salivarius.

Allergic rhinitis (AR)-commonly called hay fever-is a widespread condition that affects the quality of life of millions of people. The pathophysiology of AR remains incompletely understood. In particular, it is unclear whether members of the colonizing nasal microbiota contribute to AR. Here, using 16S ribosomal RNA sequencing, we show that the nasal microbiome of patients with AR (n = 55) shows distinct differences compared with that from healthy individuals (n = 105), including decreased heterogeneity and the increased abundance of one species, Streptococcus salivarius. Using ex vivo and in vivo models of AR, we demonstrate that this commensal bacterium contributes to AR development, promoting inflammatory cytokine release and morphological changes in the nasal epithelium that are characteristic of AR. Our data indicate that this is due to the ability of S. salivarius to adhere to the nasal epithelium under AR conditions. Our study indicates the potential of targeted antibacterial approaches for AR therapy.

Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

Characterization and immunomodulatory activity of sulfated galactan from the red seaweed Gracilaria fisheri.

Polysaccharides from the red seaweed Gracilaria fisheri possess many functions, which include antioxidant, antiviral, and antibacterial activities. However, detailed data on their immunomodulatory activities are scarce. Here, we isolated sulfated galactans (SG) from G. fisheri. We found that the predominant SG from G. fisheri, termed SG-1, had an estimated molecular mass of 100 kDa and activated murine J774A.1 macrophages via the dectin-1 signaling pathway. Furthermore, we observed enhancement of nitric oxide (NO) secretion, increased expression of inducible nitric oxide synthase (iNOS) mRNA, and increased mRNA levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukins IL-1ß and IL-6 by SG-1 in macrophages. Moreover, there was higher expression of intercellular adhesion molecule 1 (ICAM-1) and co-stimulatory molecules (B7-1 and B7-2) mRNA. Treatment with G. fisheri SG-1 at 50 µg/mL generally achieved or exceeded the pro-inflammatory activities of 100 ng/mL lipopolysaccharide. Our study demonstrates immune-stimulatory activities of G. fisheri SG that may be of value for immune-potentiating treatment in humans or livestock.

Enterococcal bacteremia in mice is prevented by oral administration of probiotic Bacillus spores.

Whether and how probiotics promote human health is a controversial issue. Their claimed benefit for counteracting gastrointestinal infection is linked predominantly to reducing pathogen abundance within the intestinal microbiota. Less understood mechanistically is the reported value that probiotics could have in reducing systemic infections. Enterococcus faecalis is an opportunistic pathogen that causes systemic infection after translocation through the intestinal epithelium, particularly in hospitalized and immune-depleted patients receiving antibiotic therapy. In this study, we used an E. faecalis mouse infection model with wild-type and isogenic mutant strains deficient in genes of the E. faecalis Fsr (fecal streptococci regulator) quorum-sensing system. We show that E. faecalis translocation from the mouse gut into the blood is mediated by the Fsr quorum-sensing system through production of the protease GelE, which compromises intestinal epithelium integrity. Furthermore, we demonstrate that orally administered probiotic Bacillus subtilis spores blocked E. faecalis translocation from the gut to the bloodstream and subsequent systemic infection in mice by inhibiting Fsr activity. These findings demonstrate that a key aspect of Enterococcus pathogenesis is controlled by quorum sensing, which can be targeted with probiotic Bacillus spores.

Probiotics to prevent Staphylococcus aureus disease?

There are a plethora of probiotic formulae that supposedly benefit human health on the market. However, the scientific underpinnings of the claimed benefits have remained poorly established. Scientific evidence is now increasingly being provided that explains those benefits, for example, by immune-stimulatory effects or inter-bacterial competition between beneficial and pathogenic bacteria. In our recent study (Piewngam et al. Nature 2018), we show that Bacillus colonization of the human intestine is negatively correlated with that of the human pathogen, Staphylococcus aureus. This type of colonization resistance is achieved by secretion of a class of lipopeptides by Bacillus species that inhibits S. aureus quorum-sensing signaling, which we found is crucial for S. aureus intestinal colonization. Here, we discuss what these findings imply for the general role of S. aureus intestinal colonization, the role of quorum-sensing in that process, and potential alternative ways to control S. aureus infection.

Alternative approaches to treat bacterial infections: targeting quorum-sensing.

Introduction: The emergence of multi- and pan-drug-resistant bacteria represents a global crisis that calls for the development of alternative anti-infective strategies. These comprise anti-virulence approaches, which target pathogenicity without exerting a bacteriostatic or bactericidal effect and are claimed to reduce the development of resistance. Because in many pathogens, quorum-sensing (QS) systems control the expression of virulence factors, interference with QS, or quorum-quenching, is often proposed as a strategy with a broad anti-virulence effect.Areas covered: We discuss the role and regulatory targets of QS control in selected Gram-positive and Gram-negative bacteria, focusing on those with clinical importance and QS control of virulence. We present the components of QS systems that form possible targets for the development of anti-virulence drugs and discuss recent research on quorum-quenching approaches to control bacterial infection.Expert opinion: While there has been extensive research on QS systems and quorum-quenching approaches, there is a paucity of in-vivo research using adequate animal models to substantiate applicability. In-vivo research on QS blockers needs to be intensified and optimized to use clinically relevant setups, in order to underscore that such drugs can be used effectively to overcome problems associated with the treatment of severe infections by antibiotic-resistant pathogens.

Composition of the intestinal microbiota in extended-spectrum ß-lactamase-producing Enterobacteriaceae carriers and non-carriers in Thailand.

There is increasing recognition that the intestinal microbiota govern human well-being and prevent diseases. Intestinal colonization by antibiotic-resistant pathogens, however, can lead to the spread of resistance as well as serious infections. Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) represent particularly dangerous pathogens, which are known to asymptomatically colonize the intestinal tract in the community. Here, we performed a 16S rRNA metagenomics sequence analysis to analyse differences in the microbiota composition between ESBL-E carriers and non-carriers in Thailand, where ESBL-E carriage rates are notoriously high. The most notable difference detected was that the phylum Bacteroidetes, and in particular, the species Bacteroides uniformis, were significantly more abundant in ESBL-E non-carriers than carriers. The Shannon diversity index in non-carriers (5.10 ± 0.69) was also lower than that in ESBL-E carriers (5.39 ± 0.48) without statistical significance (P=0.13). The overall beta diversity difference of the intestinal microbiota of ESBL-E carriers as compared to non-carriers was statistically significant (Adonis on weighted unifrac: R2=0.14, P=0.005). Furthermore, ESBL-E carriage was significantly lower in farmers than in those with other occupations. Our findings suggest that a dynamic interaction exists between microbiota diversity and ESBL-E carriage, which is possibly driven by dietary composition and may be exploited using probiotic approaches to control the spread of ESBL-E.

Antimicrobial susceptibility of Clostridium difficile isolated in Thailand.

BACKGROUND: Exposure to antimicrobials is the major risk factor associated with Clostridium difficile infection (CDI). Paradoxically, treatment of CDI with antimicrobials remains the preferred option. To date, only three studies have investigated the antimicrobial susceptibility of C. difficile from Thailand, two of which were published in the 1990s. This study aimed to investigate the contemporary antibiotic susceptibility of C. difficile isolated from patients in Thailand. METHODS: A collection of 105 C. difficile isolated from inpatients admitted at Siriraj Hospital in Bangkok in 2015 was tested for their susceptibility to nine antimicrobials via an agar incorporation method. RESULTS: All isolates were susceptible to vancomycin, metronidazole, amoxicillin/clavulanate and meropenem. Resistance to clindamycin, erythromycin and moxifloxacin was observed in 73.3%, 35.2% and 21.0% of the isolates, respectively. The in vitro activity of fidaxomicin (MIC50/MIC90 0.06/0.25 mg/L) was superior to first-line therapies vancomycin (MIC50/MIC90 1/2 mg/L) and metronidazole (MIC50/MIC90 0.25/0.25 mg/L). Rifaximin exhibited potent activity against 85.7% of the isolates (MIC ≤0.03 mg/L), and its MIC50 (0.015 mg/L) was the lowest among all antimicrobials tested. The prevalence of multi-drug resistant C. difficile, defined by resistance to ≥3 antimicrobials, was 21.9% (23/105). CONCLUSIONS: A high level of resistance against multiple classes of antimicrobial was observed, emphasising the need for enhanced antimicrobial stewardship and educational programmes to effectively disseminate information regarding C. difficile awareness and appropriate use of antimicrobials to healthcare workers and the general public.

Molecular Epidemiology of Clostridium difficile Infection in a Large Teaching Hospital in Thailand.

Clostridium difficile infection (CDI) is a leading cause of healthcare-associated morbidity and mortality worldwide. In Thailand, CDI exhibits low recurrence and mortality and its molecular epidemiology is unknown. CDI surveillance was conducted in a tertiary facility (Siriraj Hospital, Bangkok). A total of 53 toxigenic C. difficile strains from Thai patients were analyzed by multi-locus sequence typing (MLST), PCR ribotyping, and pulse-field gel electrophoresis (PFGE). The mean age of the cohort was 64 years and 62.3% were female; 37.7% of patients were exposed to > two antibiotics prior to a diagnosis of CDI, with beta-lactams the most commonly used drug (56.3%). Metronidazole was used most commonly (77.5%; success rate 83.9%), and non-responders were treated with vancomycin (success rate 100%). None of the isolates carried binary toxin genes. Most isolates (98.2-100%) were susceptible to metronidazole, vancomycin, tigecycline and daptomycin. There were 11 sequence types (STs), 13 ribotypes (RTs) and four PFGE types. Six previously identified STs (ST12, ST13, ST14, ST33, ST41 and ST45) and five novel STs unique to Thailand (ST66, ST67, ST68, ST69 and ST70) were identified. PCR RTs UK 017 (ST45) (45.3%) and UK 014/020 (ST33) (24.5%) were the most common. High concordance was observed between the MLST and ribotyping results (p<0.001). C. difficile isolates from Thai patients were highly susceptible to standard antimicrobial agents. In conclusion, the five STs indicate the high genetic diversity and unique polymorphisms in Thailand. Moreover, the emergence of antimicrobial resistance to vancomycin warranted continuous surveillance to prevent further spread of the toxigenic C. difficile isolates.

Comparative assessment of antimicrobial susceptibility testing for tigecycline and colistin against Acinetobacter baumannii clinical isolates, including multidrug-resistant isolates.

Acinetobacter baumannii has become a serious concern in clinical practice owing to its multiple resistance to antimicrobial agents. Tigecycline and colistin may be used as alternative therapies, although they lack practical susceptibility testing guidelines. This study assessed the reliability of commonly used methods (disc diffusion, Etest and VITEK(®) 2) for testing sensitivity to both agents compared with the reference broth microdilution (BMD) method against 290 A. baumannii clinical isolates, including multidrug-resistant isolates. For tigecycline, essential agreement and categorical agreement (CA) of minimum inhibitory concentration (MIC) testing were most correlated with BMD when using a breakpoint of susceptible (S)≤1/resistant (R)>2 mg/L; 94.8% and 84.5% (Etest) and 99.3% and 75.5% (VITEK 2), respectively. A disc diffusion zone diameter breakpoint of S≥17/R≤12 mm showed good agreement. All three methods did not show major errors or very major errors. For colistin, a BMD MIC breakpoint of S≤2/R>4 mg/L was proposed. The disc diffusion method was highly reproducible with a zone diameter breakpoint of S≥12/R≤9 mm. However, Etest results showed a different MIC range, and the MIC breakpoint should be modified to S≤0.5/R>2 mg/L, whilst a similar MIC breakpoint to BMD could be applied for VITEK 2. Both Etest and VITEK 2 showed a high CA for isolates with colistin-susceptible and -resistant results. We recommend that disc diffusion, Etest and VITEK 2 may be used with caution for testing tigecycline and colistin based on our proposed breakpoints. The reliability of individual methods will be discussed.