Using UV/peracetic acid as pretreatment for subsequent bio-treatment of antibiotic-containing wastewater treatment: Mitigating microbial inhibition and antibiotic resistance genes proliferation.

UV/peracetic acid (PAA) treatment presents a promising approach for antibiotic removal, but its effects on microbial community and proliferation of antibiotic resistance genes (ARGs) during the subsequent bio-treatment remain unclear. Thus, we evaluated the effects of the UV/PAA on tetracycline (TTC) degradation, followed by introduction of the treated wastewater into the bio-treatment system to monitor changes in ARG expression and biodegradability. Results demonstrated effective TTC elimination by the UV/PAA system, with carbon-centered radicals playing a significant role. Crucially, the UV/PAA system not only eliminated antibacterial activity but also inhibited potential ARG host growth, thereby minimizing the emergence and dissemination of ARGs during subsequent bio-treatment. Additionally, the UV/PAA system efficiently removed multi-antibiotic resistant bacteria and ARGs from the bio-treatment effluent, preventing ARGs from being released into the environment. Hence, we propose a multi-barrier strategy for treating antibiotic-containing wastewater, integrating UV/PAA pre-treatment and post-disinfection with bio-treatment. The inhibition of ARGs transmission by the integrated system was verified through actual soil testing, confirming its effectiveness in preventing ARGs dissemination in the surrounding natural ecosystem. Overall, the UV/PAA treatment system offers a promising solution for tackling ARGs challenges by controlling ARGs proliferation at the source and minimizing their release at the end of the treatment process.

The synergistic effect of periodate/ferrate (VI) system on disinfection of antibiotic resistant bacteria and removal of antibiotic resistant genes: The dominance of Fe (IV)/Fe (V).

The proliferation of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) caused by antibiotic abuse has raised concerns about the global infectious-disease crisis. This study employed periodate (PI)/ferrate (VI) (Fe (VI)) system to disinfect Gram-negative ARB (Escherichia coli DH5α) and Gram-positive bacteria (Bacillus subtilis ATCC6633). The PI/Fe (VI) system could inactivate 1 × 108 CFU/mL of Gram-negative ARB and Gram-positive bacteria by 4.0 and 2.8 log in 30 min. Neutral and acidic pH, increase of PI dosage and Fe (VI) dosage had positive impacts on the inactivation efficiency of ARB, while alkaline solution and the coexistence of 10 mM Cl-, NO3-, SO42- and 20 mg/L humic acid had slightly negative impacts. The reactive species generated by PI/Fe (VI) system could disrupt the integrity of cell membrane and wall, leading to oxidative stress and lipid peroxidation. Intracellular hereditary substance, including DNA and ARGs (tetA), would leak into the external environment through damaged cells and be degraded. The electron spin resonance analysis and quenching experiments indicated that Fe (IV)/Fe (V) played a leading role in disinfection. Meanwhile, PI/Fe (VI) system also had an efficient removal effect on sulfadiazine, which was expected to inhibit the ARGs transmission from the source.

Effects of Chinese medicine herbal residues on antibiotic resistance genes and the bacterial community in chicken manure composting.

The use of livestock manure is an important way for antibiotic resistance genes (ARGs) to enter the environment, and composting is an effective method for removing ARGs from livestock manure. In this study, different volume ratios of Chinese medicinal herbal residues (CMHRs) were added to laboratory-scale chicken manure composting to evaluate their effects, if any, on the behavior of ARGs, mobile genetic elements (MGEs), and the bacterial community. At the end of the composting period, the composition of the microbial community changed. Firmicutes decreased and Bacteroidetes increased. The most striking effect was that the relative abundance of the 21 ARGs and 5 MGEs detected decreased by varying degrees in the different treatments (except for sulI and intI1). The removal rate of the ARGs increased with the increased addition of CMHRs. The correlations between transferase genes (tnpA and tnpA-02) and ARGs were significant (p < 0.05); therefore, transposons play an important role in the horizontal gene transfer of ARGs in chicken manure. The results imply that CMHRs would be an effective bulking agent for the removal of ARGs from chicken manure composting.

Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea.

The use of phenicol antibiotics in animals has increased. In recent years, it has been reported that the transferable gene mediates phenicol-oxazolidinone resistance. This study analyzed the prevalence and characteristics of phenicol-oxazolidinone resistance genes in Enterococcus faecalis and Enterococcus faecium isolated from food-producing animals and meat in Korea in 2018. Furthermore, for the first time, we reported the genome sequence of E. faecalis strain, which possesses the phenicol-oxazolidinone resistance gene on both the chromosome and plasmid. Among the 327 isolates, optrA, poxtA, and fexA genes were found in 15 (4.6%), 8 (2.5%), and 17 isolates (5.2%), respectively. Twenty E. faecalis strains carrying resistance genes belonged to eight sequence types (STs), and transferability was found in 17 isolates. The genome sequences revealed that resistant genes were present in the chromosome or plasmid, or both. In strains EFS17 and EFS108, optrA was located downstream of the ermA and ant(9)-1 genes. The strains EFS36 and EFS108 harboring poxtA-encoding plasmid cocarried fexA and cfr(D). These islands also contained IS1216E or the transposon Tn554, enabling the horizontal transfer of the phenicol-oxazolidinone resistance with other antimicrobial-resistant genes. Our results suggest that it is necessary to promote the prudent use of antibiotics through continuous monitoring and reevaluation.

Shotgun-metagenomics based prediction of antibiotic resistance and virulence determinants in Staphylococcus aureus from periprosthetic tissue on blood culture bottles.

Shotgun-metagenomics may give valuable clinical information beyond the detection of potential pathogen(s). Identification of antimicrobial resistance (AMR), virulence genes and typing directly from clinical samples has been limited due to challenges arising from incomplete genome coverage. We assessed the performance of shotgun-metagenomics on positive blood culture bottles (n = 19) with periprosthetic tissue for typing and prediction of AMR and virulence profiles in Staphylococcus aureus. We used different approaches to determine if sequence data from reads provides more information than from assembled contigs. Only 0.18% of total reads was derived from human DNA. Shotgun-metagenomics results and conventional method results were consistent in detecting S. aureus in all samples. AMR and known periprosthetic joint infection virulence genes were predicted from S. aureus. Mean coverage depth, when predicting AMR genes was 209 ×. Resistance phenotypes could be explained by genes predicted in the sample in most of the cases. The choice of bioinformatic data analysis approach clearly influenced the results, i.e. read-based analysis was more accurate for pathogen identification, while contigs seemed better for AMR profiling. Our study demonstrates high genome coverage and potential for typing and prediction of AMR and virulence profiles in S. aureus from shotgun-metagenomics data.

Effects of the Newly Isolated T4-like Phage on Transmission of Plasmid-Borne Antibiotic Resistance Genes via Generalized Transduction.

Bacteriophages are the most abundant biological entities on earth and may play an important role in the transmission of antibiotic resistance genes (ARG) from host bacteria. Although the specialized transduction mediated by the temperate phage targeting a specific insertion site is widely explored, the carrying characteristics of "transducing particles" for different ARG subtypes in the process of generalized transduction remains largely unclear. Here, we isolated a new T4-like lytic phage targeting transconjugant Escherichia coli C600 that contained plasmid pHNAH67 (KX246266) and encoded 11 different ARG subtypes. We found that phage AH67C600_Q9 can misload plasmid-borne ARGs and package host DNA randomly. Moreover, for any specific ARG subtype, the carrying frequency was negatively correlated with the multiplicity of infection (MOI). Further, whole genome sequencing (WGS) identified that only 0.338% (4/1183) of the contigs of an entire purified phage population contained ARG sequences; these were floR, sul2, aph(4)-Ia, and fosA. The low coverage indicated that long-read sequencing methods are needed to explore the mechanism of ARG transmission during generalized transduction.

An improved statistical method to identify chemical-genetic interactions by exploiting concentration-dependence.

Chemical-genetics (C-G) experiments can be used to identify interactions between inhibitory compounds and bacterial genes, potentially revealing the targets of drugs, or other functionally interacting genes and pathways. C-G experiments involve constructing a library of hypomorphic strains with essential genes that can be knocked-down, treating it with an inhibitory compound, and using high-throughput sequencing to quantify changes in relative abundance of individual mutants. The hypothesis is that, if the target of a drug or other genes in the same pathway are present in the library, such genes will display an excessive fitness defect due to the synergy between the dual stresses of protein depletion and antibiotic exposure. While assays at a single drug concentration are susceptible to noise and can yield false-positive interactions, improved detection can be achieved by requiring that the synergy between gene and drug be concentration-dependent. We present a novel statistical method based on Linear Mixed Models, called CGA-LMM, for analyzing C-G data. The approach is designed to capture the dependence of the abundance of each gene in the hypomorph library on increasing concentrations of drug through slope coefficients. To determine which genes represent candidate interactions, CGA-LMM uses a conservative population-based approach in which genes with negative slopes are considered significant only if they are outliers with respect to the rest of the population (assuming that most genes in the library do not interact with a given inhibitor). We applied the method to analyze 3 independent hypomorph libraries of M. tuberculosis for interactions with antibiotics with anti-tubercular activity, and we identify known target genes or expected interactions for 7 out of 9 drugs where relevant interacting genes are known.

An omics-based framework for assessing the health risk of antimicrobial resistance genes.

Antibiotic resistance genes (ARGs) are widespread among bacteria. However, not all ARGs pose serious threats to public health, highlighting the importance of identifying those that are high-risk. Here, we developed an 'omics-based' framework to evaluate ARG risk considering human-associated-enrichment, gene mobility, and host pathogenicity. Our framework classifies human-associated, mobile ARGs (3.6% of all ARGs) as the highest risk, which we further differentiate as 'current threats' (Rank I; 3%) - already present among pathogens - and 'future threats' (Rank II; 0.6%) - novel resistance emerging from non-pathogens. Our framework identified 73 'current threat' ARG families. Of these, 35 were among the 37 high-risk ARGs proposed by the World Health Organization and other literature; the remaining 38 were significantly enriched in hospital plasmids. By evaluating all pathogen genomes released since framework construction, we confirmed that ARGs that recently transferred into pathogens were significantly enriched in Rank II ('future threats'). Lastly, we applied the framework to gut microbiome genomes from fecal microbiota transplantation donors. We found that although ARGs were widespread (73% of genomes), only 8.9% of genomes contained high-risk ARGs. Our framework provides an easy-to-implement approach to identify current and future antimicrobial resistance threats, with potential clinical applications including reducing risk of microbiome-based interventions.

Evolution of Polymyxin Resistance Regulates Colibactin Production in Escherichia coli.

The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can promote colorectal cancer through DNA double stranded breaks and interstrand cross-links. While the structure and biosynthesis of colibactin have been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by E. coli are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC, NC101; the probiotic strain, Nissle 1917; and the antibiotic testing strain, ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb's by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with a heightened tolerance for polymyxin induced greater mammalian DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.

Additive quality influences the reservoir of antibiotic resistance genes during chicken manure composting.

Aerobic composting is commonly used to dispose livestock manure and is an efficient way to reduce antibiotic resistance genes (ARGs). Here, the effects of different quality substrates on the fate of ARGs were assessed during manure composting. Results showed that the total relative abundances of ARGs and intI1 in additive treatments were lower than that in control, and high quality treatment with low C/N ratio and lignin significantly decreased the relative abundance of tetW, ermB, ermC, sul1 and sul2 at the end of composting. Additionally, higher quality treatment reduced the relative abundances of some pathogens such as Actinomadura and Pusillimonas, and some thermotolerant degrading-related bacteria comprising Pseudogracilibacillus and Sinibacillus on day 42, probably owing to the change of composting properties in piles. Structural equation models (SEMs) further verified that the physiochemical properties of composting were the dominant contributor to the variations in ARGs and they could also indirectly impact ARGs by influencing bacterial community and the abundance of intI1. Overall, these findings indicated that additives with high quality reduced the reservoir of antibiotic resistance genes of livestock manure compost.

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes.

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.

Tracking antibiotic resistance gene transfer at all seasons from swine waste to receiving environments.

Antibiotic resistance genes (ARGs) in livestock farms have attracted a growing attention with potential effects on human health. As one of the most important organic fertilizer, swine waste provided an ideal environment for understanding the dissemination and accumulation of ARGs in agricultural ecosystems. Here we conducted a year-round follow-up trace from swine waste to receiving environments, with the purpose of revealing the contamination profiles and ecological risks of ARGs at different seasons. Results indicated that a variety of common ARGs and even high-risk ARGs (i.e., blaampC, blaOXA-1, blaTEM-1 and mcr-1) were prevalent from swine waste to farmland soil, with changing in various degrees from season to season. Regarding the occurrence pattern of ARGs, tetracycline resistance genes (tet-ARGs) were predominant genes at four seasons in all fresh pig feces, swine manure, manured soil and wastewater. The levels of most ARGs in solid waste were reduced at a different degree via natural composting, and the removal effect was best in summer, while ARGs decreased poorly after wastewater treatment, especially in winter (up to 10-1 copies/16S copies in the residual level), which increased the possibility of propagation to receiving environment. This concern was also validated by the investigation on farmland environment with long-term application of manure, where causing an increase in ARG abundances in soils (approximately 0.9-32.7 times). To our knowledge, this study is the first to demonstrate the distribution pattern of ARGs from swine waste to its receiving farmland environment at all seasons on this integrity chain.

Presence of Colistin- and Tigecycline-Resistant Klebsiella pneumoniae ST29 in Municipal Wastewater Influents in Japan.

The aim of this study was to investigate the presence of colistin- and/or tigecycline-resistant Klebsiella spp. in influents from four wastewater treatment plants (WWTPs), which partly reflect the gut microbiome of human populations. Colistin- and tigecycline-resistant Klebsiella pneumoniae isolates (K30/ST29) were detected four times from the WWTP A during a period of 3 months. Disruptions of the mgrB and ramR genes by ISEc68 and ISKpn21, respectively, were identified in those four isolates. They also shared the IncL/M 86,197-bp plasmids carrying a blaCTX-M-3 and Tn1548-associated armA [IS26-IntI1-dfrA12-gucF-aadA2-qacEΔ1-sul1-ISCR1-ISEc28-armA-ISEc29-msr(E)-mph(E)-IS26]. Those isolates formed a distinct cluster within wgMLST clusters of ST29 K30 public reference strains of human origin and were unique due to harboring of Tn21-like mercury resistance operon transposons in addition to silver, copper, and arsenic resistance determinants. Five K. pneumoniae strains with different STs and 1 Klebsiella quasipneumoniae strain, exhibiting colistin resistance, were detected in WWTPs B, C, and D. For these isolates, disruptions of mgrB by ISEc68 (three isolates) or ISEcl1 (one isolate), insertion of IS2 in the mgrB promoter region (one isolate), and inactivation of MgrB by a nonsense mutation (one isolate) were identified. Close monitoring of these mcr-negative colistin- and/or tigecycline-resistant bacteria in wastewater influents is imperative to avoid further limiting of treatment options.

Effectiveness of Fluoroquinolones with Difluoropyridine Derivatives as R1 Groups on the Salmonella DNA Gyrase in the Presence and Absence of Plasmid-Encoded Quinolone Resistance Protein QnrB19.

Aims: WQ-3810 has strong inhibitory activity against Salmonella and other fluoroquinolone-resistant pathogens. The unique potentiality of this is attributed to 6-amino-3,5-difluoropyridine-2-yl at R1 group. The aim of this study was to examine WQ-3810 and its derivatives WQ-3334 and WQ-4065 as the new drug candidate for wild-type Salmonella and that carrying QnrB19. Materials and Methods: The half maximal inhibitory concentrations (IC50s) of WQ-3810, WQ-3334 (Br atom in place of methyl group at R8), and WQ-4065 (6-ethylamino-3,5-difluoropyridine-2-yl in place of 6-amino-3,5-difluoropyridine-2-yl group at R1) in the presence or absence of QnrB19 were assessed by in vitro DNA supercoiling assay utilizing recombinant DNA gyrase and QnrB19. Results: IC50s of WQ-3810, WQ-3334, and WQ-4065 against Salmonella DNA gyrase were 0.031 ± 0.003, 0.068 ± 0.016, and 0.72 ± 0.39 µg/mL, respectively, while QnrB19 increased IC50s of WQ-3810, WQ-3334, and WQ-4065 to 0.44 ± 0.05, 0.92 ± 0.34, and 9.16 ± 2.21 µg/mL, respectively. Conclusion: WQ-3810 and WQ-3334 showed stronger inhibitory activity against Salmonella Typhimurium DNA gyrases than WQ-4065 even in the presence of QnrB19. The results suggest that 6-amino-3,5-difluoropyridine-2-yl group at R1 is playing an important role and WQ-3810 and WQ-3334 to be good candidates for Salmonella carrying QnrB19.

Exposure to Sublethal Ciprofloxacin Induces Resistance to Ciprofloxacin and Cross-Antibiotics, and Reduction of Fitness, Biofilm Formation, and Apx Toxin Secretion in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is increasingly resistant to antibiotics. However, little is known about the mechanisms of antibiotic resistance in this pathogen. In this study, we experimentally evolved the reference strain of both A. pleuropneumoniae serovar 1 and serovar 7, the most prevalent serovars worldwide, to quinolone resistance by sequential exposure to subinhibitory concentrations of ciprofloxacin. The adaptive ciprofloxacin-resistant mutants of A. pleuropneumoniae serovar 1 and serovar 7 had a minimum inhibitory concentration (MIC) increment from 0.004 to 1 or 2 µg/mL, respectively. Adaptation to ciprofloxacin was shown to confer quinolone resistance with a 32- to 512-fold increase (serovars 1 and 7, respectively) as well as cross-resistance to ampicillin with an increased MIC by 16,384- and 64-fold (serovars 1 and 7, respectively). The genetic analysis of quinolone resistance-determining region mutations showed that substitutions occurred in gyrA (S83A) and parC (D84N) of serovar 1, and gyrA (D87N) of serovar 7. The ciprofloxacin-resistant mutants showed significantly reduced bacterial fitness. The mutants also showed changes in efflux ability and biofilm formation. Notably, the transcription and secretion levels of Apx toxins were dramatically reduced in ciprofloxacin-resistant mutants compared with their wild-type strains. Altogether, these results demonstrated marked phenotypic changes in ciprofloxacin-resistant mutants of A. pleuropneumoniae. The results stress the need for further studies on the impact of both the genotypic and phenotypic characteristics of A. pleuropneumoniae following exposure to subinhibitory concentrations of antibiotics.

Swine growth promotion with antibiotics or alternatives can increase antibiotic resistance gene mobility potential.

Even though the use of antibiotics for food-producing animals may contribute to the emergence of antimicrobial resistance, antibiotics are still used as growth promoters. Due to consumer and regulatory pressures, the use of alternatives to antibiotics as growth promoters is increasing, thus more information is needed on their capability to disseminate antimicrobial resistance compared to antibiotics. We investigated the impacts of carbadox (antibiotic), copper sulfate and zinc oxide (metals) and mushroom powder (natural product) on the pig fecal resistome and microbiome. Antibiotic resistance gene (ARG) and mobile genetic element (MGE) abundances were measured using a high-throughput qPCR array with 382 primer pairs. Bacterial community composition was determined by 16S rRNA gene sequencing. More ARGs co-occurred with MGEs in the growth promoter group samples than in the control group samples. Community composition could not be linked to resistome in the growth promoter group samples, indicating a potential decoupling of ARGs and phylogeny. Additionally, machine-learning methods aided in defining the community and resistome differences in response to treatments. Since increased ARG mobility potential was the primary response to the dietary additives used in this study, we suggest that ARG mobility should be considered when designing antimicrobial use policies and antimicrobial resistance surveillances.

Eliciting the silent lucensomycin biosynthetic pathway in Streptomyces cyanogenus S136 via manipulation of the global regulatory gene adpA.

Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

In-vitro study of the effect of Centella asiatica on cholera toxin production and the gene expression level of ctxA gene in Vibrio cholerae isolates.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (L.) Urb or Indian pennywort is a plant of ethnopharmacological relevance, commonly called as Brahmi in South India known for its antimicrobial property in gut and for the treatment of other gut ailments. Natural anti-virulence drugs that disarm pathogens by directly targeting virulence factors or the cell viability and are thus preferred over antibiotics as these drugs impose limited selection pressure for resistance development. In this regard, an in-vitro experimental study was conducted to know the effect of extract of Centella asiatica(L.) Urb. on cholera toxin, gene expression and its vibriocidal effect on five standard strains of Vibrio cholerae; IDH03097 (El Tor variant), N16961 (El Tor), O395 (Classical) as well as five clinical strains (Haitian variant). AIM OF THE STUDY: To study the effect of extract of Centella asiatica on Vibrio cholerae. MATERIALS AND METHODS: Crude extract was prepared from the leaves and stem part of the plant. The vibriocidal concentration was tested at different concentrations of the extract. The amount of cholera toxin released from the strains before and after exposure to the extract of Centella asiatica to Vibrio cholerae was measured using Bead ELISA. ctxA gene expression in the strains before and after exposure to extract of Centella asiatica was measured using quantitative real time PCR. All the above assays were performed with commercially obtained asiaticoside as well. RESULTS: The vibriocidal activity was tested at the different concentration of the extract, where 1g/mL of crude extract and 12.5mg/mL of asiaticoside was found to be vibriocidal. The amount of cholera toxin released before and after the exposure to extract of C. asiatica was measured using Bead ELISA, showing a reduction of 70%, 89% and 93% toxin produced by classical, El Tor and variant respectively. ctxA gene expression before and after exposure to extract of Centella asiatica as well as asiaticoside was measured using qRT-PCR. We found a decrease in expression of ctxA gene transcription by 6.19 fold in classical strain, 4.29 fold in El Tor, 1.133 fold in variant strains and about 10.13-10.20 fold for the clinical strains of V. cholerae using the extract of C.asiatica while, the reduction with the exposure to the asiaticoside were 2.762 fold in classical strain, 4.809 in El Tor, 24.1 in variant strain and 34.77 - 34.8 for the clinical strains. CONCLUSION: Centella asiatica extract inhibited the CT production in Vibrio cholerae as well as decreased the transcription of ctxA gene expression.

Characterization of the human skin resistome and identification of two microbiota cutotypes.

BACKGROUND: The human skin microbiota is considered to be essential for skin homeostasis and barrier function. Comprehensive analyses of its function would substantially benefit from a catalog of reference genes derived from metagenomic sequencing. The existing catalog for the human skin microbiome is based on samples from limited individuals from a single cohort on reference genomes, which limits the coverage of global skin microbiome diversity. RESULTS: In the present study, we have used shotgun metagenomics to newly sequence 822 skin samples from Han Chinese, which were subsequently combined with 538 previously sequenced North American samples to construct an integrated Human Skin Microbial Gene Catalog (iHSMGC). The iHSMGC comprised 10,930,638 genes with the detection of 4,879,024 new genes. Characterization of the human skin resistome based on iHSMGC confirmed that skin commensals, such as Staphylococcus spp, are an important reservoir of antibiotic resistance genes (ARGs). Further analyses of skin microbial ARGs detected microbe-specific and skin site-specific ARG signatures. Of note, the abundance of ARGs was significantly higher in Chinese than Americans, while multidrug-resistant bacteria ("superbugs") existed on the skin of both Americans and Chinese. A detailed analysis of microbial signatures identified Moraxella osloensis as a species specific for Chinese skin. Importantly, Moraxella osloensis proved to be a signature species for one of two robust patterns of microbial networks present on Chinese skin, with Cutibacterium acnes indicating the second one. Each of such "cutotypes" was associated with distinct patterns of data-driven marker genes, functional modules, and host skin properties. The two cutotypes markedly differed in functional modules related to their metabolic characteristics, indicating that host-dependent trophic chains might underlie their development. CONCLUSIONS: The development of the iHSMGC will facilitate further studies on the human skin microbiome. In the present study, it was used to further characterize the human skin resistome. It also allowed to discover the existence of two cutotypes on the human skin. The latter finding will contribute to a better understanding of the interpersonal complexity of the skin microbiome. Video abstract.

Comparison of microbial communities and the antibiotic resistome between prawn mono- and poly-culture systems.

Antibiotic resistance genes (ARGs) in mariculture sediments pose a potential risk to public health due to their ability to transfer from environmental bacteria to human pathogens. Long term, this may reduce pathogen susceptibility to antibiotics in medical settings. In recent years, the poly-culture of multiple species has become a popular mariculture approach in China, thanks to its environmental and economic benefits. However, differences in microbial communities and antibiotic resistome between mono- and poly-culture systems are still unclear. In this study, microbial community composition and profiles of entire (microbial DNA) and mobile (plasmid and phage) ARGs in prawn mono- and poly-culture systems were investigated using metagenomics. The abundance of several viruses and human pathogens were enhanced in prawn poly-culture ponds, when compared to monoculture systems. In contrast, sediments from poly-culture systems had a lower diversity and ARG abundance when compared to mono-culture approaches. These ARG variations were predominantly related to mobile genetic elements. Prawn mariculture activities exerted a unique selectivity for ARGs in plasmids, and this selectivity was not influenced by culture methods. The findings of this study have important implications for the selection of mariculture systems in preventing pollution with ARGs.