The use of probiotics and prebiotics in decolonizing pathogenic bacteria from the gut; a systematic review and meta-analysis of clinical outcomes.

Repeated exposure to antibiotics and changes in the diet and environment shift the gut microbial diversity and composition, making the host susceptible to pathogenic infection. The emergence and ongoing spread of AMR pathogens is a challenging public health issue. Recent evidence showed that probiotics and prebiotics may play a role in decolonizing drug-resistant pathogens by enhancing the colonization resistance in the gut. This review aims to analyze available evidence from human-controlled trials to determine the effect size of probiotic interventions in decolonizing AMR pathogenic bacteria from the gut. We further studied the effects of prebiotics in human and animal studies. PubMed, Embase, Web of Science, Scopus, and CINAHL were used to collect articles. The random-effects model meta-analysis was used to pool the data. GRADE Pro and Cochrane collaboration tools were used to assess the bias and quality of evidence. Out of 1395 citations, 29 RCTs were eligible, involving 2871 subjects who underwent either probiotics or placebo treatment to decolonize AMR pathogens. The persistence of pathogenic bacteria after treatment was 22%(probiotics) and 30.8%(placebo). The pooled odds ratio was 0.59(95% CI:0.43-0.81), favoring probiotics with moderate certainty (p = 0.0001) and low heterogeneity (I2 = 49.2%, p = 0.0001). The funnel plot showed no asymmetry in the study distribution (Kendall'sTau = -1.06, p = 0.445). In subgroup, C. difficile showed the highest decolonization (82.4%) in probiotics group. Lactobacillus-based probiotics and Saccharomyces boulardii decolonize 71% and 77% of pathogens effectively. The types of probiotics (p < 0.018) and pathogens (p < 0.02) significantly moderate the outcome of decolonization, whereas the dosages and regions of the studies were insignificant (p < 0.05). Prebiotics reduced the pathogens from 30% to 80% of initial challenges. Moderate certainty of evidence suggests that probiotics and prebiotics may decolonize pathogens through modulation of gut diversity. However, more clinical outcomes are required on particular strains to confirm the decolonization of the pathogens. Protocol registration: PROSPERO (ID = CRD42021276045).

Global epidemiology of asymptomatic colonisation of methicillin-resistant Staphylococcus aureus in the upper respiratory tract of young children: a systematic review and meta-analysis.

OBJECTIVE: To estimate the global prevalence of asymptomatic colonisation, and determine the associated risk factors, antibiotic resistance and genotypes of methicillin-resistant Staphylococcus aureus (MRSA) in the upper respiratory tract of young children. DESIGN: Four bibliometric databases were searched for publications between 2010 and 2022 according to the protocol registered in PROSPERO. Cross-sectional or cohort studies describing the prevalence of asymptomatic colonisation of S. aureus and MRSA in young children were included. Data extraction and analysis were carried out by two reviewers independently according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 statement. Pooled prevalence was estimated using a random effects model. SETTING AND STUDIES: We included studies where children without respiratory tract infection or Staphylococcal infection were recruited from the community, children's institutions (ie, nurseries, kindergartens, daycare centres and preschools) and healthcare centre visits and assessed for asymptomatic colonisation with S. aureus and MRSA. MAIN OUTCOME MEASURES: The pooled prevalence of asymptomatic colonisation of S. aureus and MRSA of young children globally. RESULTS: In this systematic review and meta-analysis of 21 416 young children, the pooled global prevalence of asymptomatic S. aureus colonisation was 25.1% (95% CI 21.4 to 28.8) and MRSA colonisation was 3.4% (95% CI 2.8 to 4.1). The clones of MRSA strains included healthcare-associated MRSA, community-associated MRSA and livestock-associated MRSA. CONCLUSION: This study provides evidence of increased MRSA colonisation globally among young children, underlining the critical role of asymptomatic carriers in MRSA transmission and the need for control measures. PROSPERO REGISTRATION NUMBER: CRD 42022328385.

Enhancing Antibiotics Efficacy by Combination of Kuraridin and Epicatechin Gallate with Antimicrobials against Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is an opportunistic pathogen and a major cause of nosocomial and community-acquired infections. The alarming rise in Methicillin-resistant S. aureus (MRSA) infection worldwide and the emergence of vancomycin-resistant MRSA strains have created an urgent need to identify new and alternative treatment options. Triple combinations of antimicrobials with different antimicrobial mechanisms may be a good choice to overcome antimicrobial resistance. METHODS: In this study, we combine two natural compounds: kuraridin from Sophora flavescens and epicatechin gallate (ECG) from Camellia sinensis (Green tea), which could provide the best synergy with antibiotics against a selected panel of laboratory MRSA with known resistant mechanisms and clinical community-associated (CA) and hospital-associated (HA) MRSA as well. RESULTS: The combined use of ECG and kuraridin was efficacious in inhibiting the growth of a panel of tested MRSA strains. The antibacterial activities of gentamicin, fusidic acid and vancomycin could be further enhanced by the addition of ECG and kuraridin. In time-kill study, when vancomycin (0.5 µg/mL) was combined with ECG (2 µg/mL) and kuraridin (2 µg/mL), a very strong bactericidal growth inhibition against 3 tested strains ATCC25923, MRSA ST30 and ST239 was observed from 2 to 24 h. ECG and kuraridin both possess anti-inflammatory activities in bacterial toxin-stimulated peripheral blood mononuclear cells by suppressing the production of inflammatory cytokines (IL-1ß, IL-6 and TNFα) and are non-cytotoxic. In a murine pneumonia model infected with ATCC25923, MRSA ST30 or ST239, the combined use of ECG and kuraridin with vancomycin could significantly reduce bacterial counts. CONCLUSIONS: The present findings reveal the potential of ECG and kuraridin combination as a non-toxic herbal and antibiotics combination for MRSA treatment with antibacterial and anti-inflammatory activities.

Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a.

The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.

Mucin-degrading gut commensals isolated from healthy faecal donor suppress intestinal epithelial inflammation and regulate tight junction barrier function.

The intestinal epithelium surface is covered by a layer of mucus that harbors a complex and dynamic population of bacteria termed gut microbiota. In particular, some gut bacteria have the ability to degrade the mucin glycan for nutritional sources. However, the bacterial diversity of mucin-degrading bacteria in human gut microbiota and their role in the gut remains unclear. In this study, we characterized the diversity of mucin-degrading bacteria in the human gut microbiota by an established cultivation-based molecular profiling method. The results showed the gut commensals having the mucin degrading ability were widely distributed in the gut microbiota and were more abundant than previously thought. In addition, many previously uncharacterized mucin degraders were isolated from faecals samples, suggesting the mucin-degrading gut commensals were underappreciated. To gain a better understanding of the interaction between these mucin-degrading gut commensals and the host, the effect of the commensals on intestinal epithelial cells were examined, and the results revealed that the commensals (8 Bacteroides spp., 2 Parabacteroides spp, Akkermanisa muciniphila and Bifidobacterial dentium) incited low level of inflammatory response (IL-8 and TNF-α) but suppressed the inflammatory response induced by E. coli through downregulating the NF-κB pathway. The presence of gut commensals also showed potential in enhancing the epithelial tight junction (TJ) barrier function through regulating the mRNA expression of TJ protein genes such as Zo-1, Occludin, Claudin-1 and E-cadherin. Furthermore, the presence of commensal bacteria P. distasonis, B. thetaiotaomicron and A. muciniphila completely or partly restored the pro-inflammatory cytokine IL-1ß induced TJ barrier disruption. In conclusion, these findings indicate that mucin-degrading gut commensals were widely distributed in the gut microbiota and showed anti-inflammatory effect against pathogen infection and potential in modulating the epithelial barrier function.

Chimeric crRNA improves CRISPR-Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2.

Many CRISPR/Cas platforms have been established for the detection of SARS-CoV-2. But the detection platform of the variants of SARS-CoV-2 is scarce because its specificity is very challenging to achieve for those with only one or a few nucleotide(s) differences. Here, we report for the first time that chimeric crRNA could be critical in enhancing the specificity of CRISPR-Cas12a detecting of N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without compromising its sensitivity. This strategy could also be applied to detect other SARS-CoV-2 variants that differ only one or a few nucleotide(s) differences.

Longitudinal Genomic Characterization of Carbapenemase-producing Enterobacteriaceae (CPE) Reveals Changing Pattern of CPE Isolated in Hong Kong Hospitals.

An active, territory-wide, CPE surveillance program implemented from 2011 showed increasing levels of carbapenemase-producing Enterobacteriaceae (CPE) isolates from patients in Hong Kong hospitals. The molecular epidemiology of 567 CPE from patients of three of seven public hospital clusters in Hong Kong are described. During a 7-year period, the incidence of CPE isolation increased from 0.05 to 9.6/100 000 patient-days. The carbapenemase genes identified were polyclonal, including blaKPC, blaNDM and blaIMP, which were mainly associated with hospitalization overseas in previous years. However, increasing CPE isolation from patients without hospitalization overseas occurred in 2015, with blaNDM (52.6%) predominant followed by blaIMP (30.0%). Escherichia coli (46.4%) and Klebsiella spp. (38.3%) were the dominant species. Whole-genome sequencing was performed on 169 representative isolates with a combination of short and long reads using Illumina and Nanopore technology. Two distinct lineages of blaKPC-2-positive Klebsiella pneumoniae (ST11 and ST258) were identified with ST11 carrying yersiniabactin gene ybt-9 on ICEKp3. ST131 E. coli producing IMP-4 was present throughout the study period. The blaNDM and blaIMP genes were mainly carried in IncX3 and IncN-ST7 plasmids, respectively. blaOXA-48-like gene was carried in the IncX3 plasmid in E. coli and in the ColKP3 plasmid in K. pneumoniae. A lineage of K. pneumoniae with blaNDM-1 plus blaOXA-232 in distinct plasmids of IncF1B/IncHI1B was identified and associated with prior hospitalization overseas. This study highlights the threat of multiple types of CPE, with the predominance of blaNDM and blaIMP among CPE in our hospitals. Enhanced containment strategies are needed to mitigate the trend of rapidly rising CPE in healthcare settings.

Therapeutic Potential of Curcumin as an Antimycobacterial Agent.

Curcumin is the principal curcuminoid obtained from the plant Curcuma longa and has been extensively studied for its biological and chemical properties. Curcumin displays a vast range of pharmacological properties, including antimicrobial, anti-inflammatory, antioxidant, and antitumor activity. Specifically, curcumin has been linked to the improvement of the outcome of tuberculosis. There are many reviews on the pharmacological effects of curcumin; however, reviews of the antitubercular activity are comparatively scarcer. In this review, we attempt to discuss the different aspects of the research on the antitubercular activity of curcumin. These include antimycobacterial activity, modulation of the host immune response, and enhancement of BCG vaccine efficacy. Recent advances in the antimycobacterial activity of curcumin synthetic derivatives, the role of computer aided drug design in identifying curcumin targets, the hepatoprotective role of curcumin, and the dosage and toxicology of curcumin will be discussed. While growing evidence supports the use of curcumin and its derivatives for tuberculosis therapy, further preclinical and clinical investigations are of pivotal importance before recommending the use of curcumin formulations in public health.

Persistence of Pneumococcal Serotype 3 in Adult Pneumococcal Disease in Hong Kong.

The epidemiology of hospitalised pneumococcal disease in adults following the introduction of universal childhood pneumococcal immunisation in 2009 was assessed. Culture-confirmed Streptococcus pneumoniae (SP) from adults hospitalised between 2009 to 2017 were examined. The cases were categorised into invasive pneumococcal disease (IPD) and pneumonia (bacteraemic, non-bacteraemic, and that associated with other lung conditions). The isolates were serotyped and antimicrobial susceptibilities were determined by microbroth dilution. Patient characteristics, comorbidities, and outcomes were analysed. Seven hundred and seventy-four patients (mean age, 67.7 years, SD ± 15.6) were identified, and IPD was diagnosed in 110 (14.2%). The most prevalent serotype, 19F, was replaced by serotype 3 over time. Penicillin and cefotaxime non-susceptibilities were high at 54.1% and 39.5% (meningitis breakpoints), 19.9% and 25.5% (non-meningitis breakpoints), respectively. The overall 30-day mortality rate was 7.8% and 20.4% for IPD. Age ≥ 75 years (OR:4.6, CI:1.3-17.0, p < 0.02), presence of any complications (OR:4.1, CI:1.02-16.3, p < 0.05), pleural effusion (OR:6.7, CI:1.2-39.4, p < 0.03) and intensive care unit (ICU) admission (OR:9.0, CI:1.3-63.4, p < 0.03) were independent predictors of 30-day mortality. Pneumococcal disease by PCV 13 covered serotypes; in particular, 19F and 3 are still prominent in adults. Strengthening targeted adult vaccination may be necessary in order to reduce disease burden.

VraSR Regulatory System Contributes to the Virulence of Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) in a 3D-Skin Model and Skin Infection of Humanized Mouse Model.

The vancomycin-resistance associated sensor/regulator, VraSR two-component regulatory-system (VraSR), regulates virulence and the response of Staphylococcus aureus (SA) to environmental stress. To investigate the role of VraSR in SA skin and soft tissue infections (SSTI), we inactivated the VraSR of a clinical CA-MRSA ST30 strain by insertional mutation in vraR gene using the TargeTron-Gene Knockout System. We constructed an organotypic keratinocyte fibroblast co-culture (3D-skin model) and a humanized mouse as SSTI infection models. In the 3D-skin model, inactivation of VraSR in the strains ST30 and USA300 showed 1-log reduction in adhesion and internalization (p < 0.001) compared to the respective wildtype. The mutant strains of ST30 (p < 0.05) and USA300-LAC (p < 0.001) also exhibited reduced apoptosis. The wildtype ST30 infection in the humanized mouse model demonstrated increased skin lesion size and bacterial burden compared to BALB/c mice (p < 0.01). The response of the humanized mouse towards the MRSA infection exhibited human similarity indicating that the humanized mouse SSTI model is more suitable for evaluating the role of virulence determinants. Inactivation of VraSR in ST30 strain resulted in decreased skin lesion size in the humanized mouse SSTI model (p < 0.05) and reduction in apoptotic index (p < 0.01) when compared with the wildtype. Our results reveal that inactivating the VraSR system may be a potent anti-virulence approach to control MRSA infection.

Comparative Study of Two-Dimensional (2D) vs. Three-Dimensional (3D) Organotypic Kertatinocyte-Fibroblast Skin Models for Staphylococcus aureus (MRSA) Infection.

The invasion of skin tissue by Staphylococcus aureus is mediated by mechanisms that involve sequential breaching of the different stratified layers of the epidermis. Induction of cell death in keratinocytes is a measure of virulence and plays a crucial role in the infection progression. We established a 3D-organotypic keratinocyte-fibroblast co-culture model to evaluate whether a 3D-skin model is more effective in elucidating the differences in the induction of cell death by Methicillin-resistant Staphylococcus aureus (MRSA) than in comparison to 2D-HaCaT monolayers. We investigated the difference in adhesion, internalization, and the apoptotic index in HaCaT monolayers and our 3D-skin model using six strains of MRSA representing different clonal types, namely, ST8, ST30, ST59, ST22, ST45 and ST239. All the six strains exhibited internalization in HaCaT cells. Due to cell detachment, the invasion study was limited up to two and a half hours. TUNEL assay showed no significant difference in the cell death induced by the six MRSA strains in the HaCaT cells. Our 3D-skin model provided a better insight into the interactions between the MRSA strains and the human skin during the infection establishment as we could study the infection of MRSA in our skin model up to 48 h. Immunohistochemical staining together with TUNEL assay in the 3D-skin model showed co-localization of the bacteria with the apoptotic cells demonstrating the induction of apoptosis by the bacteria and revealed the variation in bacterial transmigration among the MRSA strains. The strain representing ST59 showed maximum internalization in HaCaT cells and the maximum cell death as measured by Apoptotic index in the 3D-skin model. Our results show that 3D-skin model might be more likely to imitate the physiological response of skin to MRSA infection than 2D-HaCaT monolayer keratinocyte cultures and will enhance our understanding of the difference in pathogenesis among different MRSA strains.

Systematic review of human gut resistome studies revealed variable definitions and approaches.

In this review, we highlight the variations of gut resistome studies, which may preclude comparisons and translational interpretations. Of 22 included studies, a range of 12 to 2000 antibiotic resistance (AR) genes were profiled. Overall, studies defined a healthy gut resistome as subjects who had not taken antibiotics in the last three to 12 months prior to sampling. In studies with de novo assembly, AR genes were identified based on variable nucleotide or amino acid sequence similarities. Different marker genes were used for defining resistance to a given antibiotic class. Validation of phenotypic resistance in the laboratory is frequently lacking. Cryptic resistance, collateral sensitivity and the interaction with repressors or promotors were not investigated. International consensus is needed for selecting marker genes to define resistance to a given antibiotic class in addition to uniformity in phenotypic validation and bioinformatics pipelines.

Design, synthesis and biological evaluation of antimicrobial diarylimine and -amine compounds targeting the interaction between the bacterial NusB and NusE proteins.

Discovery of antimicrobial agents with a novel model of action is in urgent need for the clinical management of multidrug-resistant bacterial infections. Recently, we reported the identification of a first-in-class bacterial ribosomal RNA synthesis inhibitor, which interrupted the interaction between the bacterial transcription factor NusB and NusE. In this study, a series of diaryl derivatives were rationally designed and synthesized based on the previously established pharmacophore model. Inhibitory activity against the NusB-NusE binding, circular dichroism of compound treated NusB, antimicrobial activity, cytotoxicity, hemolytic property and cell permeability using Caco-2 cells were measured. Structure-activity relationship and quantitative structure-activity relationship were also concluded and discussed. Some of the derivatives demonstrated improved antimicrobial activity than the hit compound against a panel of clinically important pathogens, lowering the minimum inhibition concentration to 1-2 µg/mL against Staphylococcus aureus, including clinical strains of methicillin-resistant Staphylococcus aureus at a level comparable to some of the marketed antibiotics. Given the improved antimicrobial activity, specific inhibition of target protein-protein interaction and promising pharmacokinetic properties without significant cytotoxicity, this series of diaryl compounds have high potentials and deserve for further studies towards a new class of antimicrobial agents in the future.

Bio-based hyperbranched poly(ester amide)-MWCNT nanocomposites: multimodalities at the biointerface.

There has been a growing interest in the use of nanomaterials featuring potent biocompatibility and biodegradability together with the added facet of antibacterial activity, particularly against drug-resistant bacterial species. Addressing these three features at the biointerface, we report the fabrication of multimodal bio-based hyperbranched poly(ester amide) (HBPEA)-microwave functionalized multiwalled carbon nanotube (f-MWCNT) nanocomposites by incorporation of various weight percentages (1, 2.5, and 5 wt%) of the f-MWCNTs into HBPEA by using an ex situ polymerization technique. Fourier transform infrared spectroscopy confirmed the structural changes upon interaction of the f-MWCNTs with HBPEA. The formation of thermosetting nanocomposites resulted in an acceptable improvement of the desired properties including their mechanical properties (∼170%), instrumental for providing mechanical integrity in cultured cells. The nanocomposite films were found to be biocompatible substrates for the in vitro adhesion and proliferation of peripheral blood mononuclear cells (PBMC) with enhanced cell viability correlating with the increase of the f-MWCNT content. The antibacterial results, monitored by a CFU count and the protein concentration, demonstrated that the prepared nanocomposites were more toxic towards Gram positive bacteria and Mycobacterium smegmatis than the Gram negative ones. The damage of bacterial cells upon interaction with the nanocomposites was validated by UV-visible spectroscopy and a SEM study. The antibacterial and biocompatibility studies suggested that these microporous nanocomposite films (3D interconnected porous structures with pore diameters of 5-105 µm and a porosity of 39.90%) possess concurrent long-term lethal activity against the bacterial cells and biocompatibility with PBMC. Thus, the prepared nanocomposites may find potential bio-medical applications, particularly as antimicrobial dressing materials for infected burn wounds.

DFT-based QSAR models to predict the antimycobacterial activity of chalcones.

In this study, antimycobacterial activity of a set of synthesized chalcone derivatives against Mycobacterium tuberculosis H37Rv was investigated by quantitative structure-activity relationship (QSAR) analysis using density functional theory (DFT) and molecular mechanics (MM+)-based descriptors in both gas and solvent phases. The best molecular descriptors identified were hardness, E(HOMO) , MR(A-4) and MR(B-4') that contributed to the antimycobacterial activity of the chalcones as independent factors. The correlation of these four descriptors with their antimycobacterial activity increases with the inclusion of solvent medium, indicating their importance in studying biological activity. QSAR models revealed that in gas phase, lower values of E(HOMO) , MR(A-4) and MR(B-4') increase the antimycobacterial activity of the chalcone molecules. However, in solvent phase, lower values of E(HOMO) and MR(B-4') and higher values of MR(A-4) increase their activity.