A method of identifying microorganisms with reduced adaptability to a particular environment, wherein (1) each microorganism is independent by insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence. Mutating to provide a plurality of microorganisms, or clones of said microorganisms, such that each mutation has a different label sequence; (2) providing storage samples of each mutation produced by step (1), respectively, and providing storage nucleic acids each comprising a unique label sequence from each mutation; (3) introducing a plurality of mutations produced by step (1) into the specific environment such that the microorganisms capable of growing in the specific environment grow in the above environment; (4) recovering the microorganisms or selected portions thereof from the environment and separating nucleic acids from the recovered microorganisms; (5) comparing all label sequences in the nucleic acid isolated in step (4) with the unique label sequences of each of the stored mutations as in step (2); and (6) any label isolated in step (4). Screening for individual mutations that also do not have a sequence.

Identification of genes

Garlic (Allium sativum), a popular food spice and flavoring agent, has also been used traditionally to treat various ailments especially bacterial infections for centuries in various cultures around the world. The principal phytochemicals that exhibit antibacterial activity are oil-soluble organosulfur compounds that include allicin, ajoenes, and allyl sulfides. The organosulfur compounds of garlic exhibit a range of antibacterial properties such as bactericidal, antibiofilm, antitoxin, and anti-quorum sensing activity against a wide range of bacteria including multi-drug resistant (MDR) strains. The reactive organosulfur compounds form disulfide bonds with free sulfhydryl groups of enzymes and compromise the integrity of the bacterial membrane. The World Health Organization (WHO) has recognized the development of antibiotic resistance as a global health concern and emphasizes antibiotic stewardship along with the urgent need to develop novel antibiotics. Multiple antibacterial effects of organosulfur compounds provide an excellent framework to develop them into novel antibiotics. The review provides a focused and comprehensive portrait of the status of garlic and its compounds as antibacterial agents. In addition, the emerging role of new technologies to harness the potential of garlic as a novel antibacterial agent is discussed.

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Antibacterial Properties of Organosulfur Compounds of Garlic (Allium sativum).

The extensive usage of antibiotics and the rapid emergence of antimicrobial-resistant microbes (AMR) are becoming important global public health issues. Many solutions to these problems have been proposed, including developing alternative compounds with antimicrobial activities, managing existing antimicrobials, and rapidly detecting AMR pathogens. Among all of them, employing alternative compounds such as phytochemicals alone or in combination with other antibacterial agents appears to be both an effective and safe strategy for battling against these pathogens. The present review summarizes the scientific evidence on the biochemical, pharmacological, and clinical aspects of phytochemicals used to treat microbial pathogenesis. A wide range of commercial products are currently available on the market. Their well-documented clinical efficacy suggests that phytomedicines are valuable sources of new types of antimicrobial agents for future use. Innovative approaches and methodologies for identifying plant-derived products effective against AMR are also proposed in this review.

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Phytochemicals: A Promising Weapon in the Arsenal against Antibiotic-Resistant Bacteria.

Salmonella typhimurium (S typhimurium) represents an important global public health problem and has the ability to survive under desiccation conditions in foods and food processing facilities for years The aim of this study was to investigate the effects of Allium sativum (A sativum) and Cuminum cyminum (C cyminum) essential oils (EOs) against planktonic growth, biofilm formation and quorum sensing (QS) of S Typhimurium isolates, the strong biofilm producers The major components of EOs were determined by gas chromatography–mass spectrometry (GC–MS) Biofilm formation of S Typhimurium isolates was measured by crystal violet staining Then, the effects of the EOs on the planktonic cell growth (using determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)), measurement of the synergistic effects of EOs (using checkerboard method), biofilm formation (using microtiter-plate test and scanning electron microscope (SEM)), and expression of QS and cellulose synthesis genes (using quantitative real-time PCR) were assessed Finally, tetrazolium-based colorimetric (MTT) assay was used to examine EOs cytotoxicity on the Vero cell line GC–MS analysis showed that terpineol, carene and pinene in C cyminum EO and sulfur compounds in A sativum EO were the major components of the plant extract The Geometric mean of MIC values of the A sativum and C cyminum were 066 and 262 μL mL−1, respectively The geometric means of the fractional inhibitory concentration index (FICi) for both EOs were calculated as 105 The qPCR results showed that MIC/2 concentrations of both EOs significantly down-regulated of QS (sdiA and luxS) and cellulose synthesis (csgD and adrA) genes Scanning electron microscopy showed the EOs reduced the amount of S Typhimurium mature biofilm In general, we showed that C cyminum and A sativum EOs can be considered as the potential agents against planktonic and biofilm form of S Typhimurium without any concern of cytotoxic effect at 4 MIC concentrations on the eukaryotic Vero cells

Combined effects of Allium sativum and Cuminum cyminum essential oils on planktonic and biofilm forms of Salmonella typhimurium isolates.

Green synthesized nanoparticles (NPs) have attracted enormous attention for their clinical and non-clinical applications. A natural polyphenol, gallo-tannin (GT) was used to reduce and cap the Fe2O3-NPs. GT-Fe2O3-NPs were synthesized following co-precipitation of FeCl3 and FeSO4·7H2O with GT. Fe2O3-NPs absorbed light at 380 nm. Physicochemically, Fe2O3-NPs were spherical with slight aggregation and average diameter of 12.85 nm. X-ray diffraction confirmed crystallinity and EDX revealed the elemental percentage of iron and oxygen as 21.7% and 42.11%, respectively. FT-IR data confirmed the adsorption of gallo-tannin functional groups. Multiple drug-resistant (MDR) Escherichia coli (ESβL), Pseudomonas aeruginosa (ESβL), and Staphylococcus aureus were found susceptible to 500–1000 μg GT-Fe2O3-NPs per ml. In synergy, Fe2O3-NPs enhanced the efficiency of some antibiotics. GT-Fe2O3 NPs showed significant (P ≤ 0.05) inhibition of growth and biofilm against MDR E. coli, P. aeruginosa, and S. aureus causing morphological and biofilm destruction. Violacein production (quorum sensing mediated) by C. violaceum was inhibited by GT-Fe2O3-NPs in a concentration-dependent manner with a maximum decrease of 3.1-fold. A decrease of 11-fold and 2.32-fold in fungal mycelial growth and human breast cancer (MCF-7) cell viability, respectively was evident. This study suggests a plausible role of gallo-tannin capped Fe2O3-NPs as an alternative antibacterial, antiquorum sensing, antibiofilm, antifungal, and anti-proliferative agent.

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Synthesis of gallotannin capped iron oxide nanoparticles and their broad spectrum biological applications

Previously, we determined that diallyl disulfide (DADS) from garlic oil can inhibit Pseudomonas aeruginosa PAO1 pathogenic factors by inactivating the transcription of key genes from three quorum sensing (QS) systems (las, rhl, and pqs) based on the effects of DADS on growth, virulence factor production (elastase, pyocyanin, biofilm, and swarming motility), and RNA transcription (real-time q-PCR). To further investigate the mechanisms underlying the inhibition of the three P. aeruginosa QS systems by DADS, high-throughput RNA and proteome sequencing techniques were used to study differences in the transcriptional and proteome expression of P. aeruginosa PAO1 following treatment with DADS. The RNA-seq and proteomic data are available via NCBI Gene Expression Omnibus database with accession number GSE118801 and ProteomeXchange with identifier PXD011144, respectively. The experimental results indicated that all key genes of the three QS systems (las, rhl, and pqs) of P. aeruginosa PAO1 as well as the virulence factors (including exoprotease LasA, elastase LasB, lectin LecA and LecB, pyocyanin biosynthesis, and biofilm formation) regulated by these three QS systems were inhibited. This is consistent with our previous studies on the physiology, biochemistry, and RNA expression of P. aeruginosa treated with DADS. Additionally, our results also indicated that bacterial motility, chemotaxis, and two-component systems were inhibited by DADS treatment. All these changes abolish the sensitivity of P. aeruginosa PAO1 to environmental stimuli and cause the cells to be in a state of passivation. Further research is needed to determine how QS systems regulate these functions. Our findings could potentially contribute to the treatment and control of P. aeruginosa infection, virulence, and pathogenicity.

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Diallyl Disulfide From Garlic Oil Inhibits Pseudomonas aeruginosa Quorum Sensing Systems and Corresponding Virulence Factors.

The invention discloses a neutralizing agent used for detecting microorganisms in cosmetics. The neutralizing agent comprises 1.0-1.5% of lecithin, 1.0-3.0% of Tween-80, 0.1-0.5% of histidine, 0.1-0.5% of sodium thiosulfate, 0.1-0.5% of peptone, 0.05-0.1% of sodium thioglycollate, 0.85% of sodium chloride and the balance of water according to 100% mass fraction. The neutralizing agent used for detecting microorganisms in cosmetics or reaction products of the neutralizing agent and the cosmetics do not have the effect on killing or inhibiting growth of experimental microorganisms. The neutralizing agent does not have the effect on destroying nutritional ingredients and does not affect the physical characteristics of the nutritional ingredients, has a practical and reliable neutralization effect on preservatives in the cosmetics, and has quite good effects and quite huge market prospects.

Neutralizing agent used for detecting microorganisms in cosmetics and preparation method thereof

Many outbreaks of Burkholderia cepacia complex (Bcc) infections are associated with contaminations in personal care products (PCPs). This study aimed to analyze a collection of Bcc isolates in PCPs and assess the susceptibility of preservatives, including dimethoxy dimethyl hydantoin (DMDMH), methylisothiazolinone-chloromethylisothiazolinone (MIT/cMIT), and methyl 4-hydroxybenzoate (MH). The Bcc isolates collected during the 3-year (2015-2017) study period were further examined by biochemical identification system, phylogenetic analysis based on recA nucleotide sequences, and multilocus sequence typing analysis. Preservatives susceptibility testing of Bcc bacteria were evaluated by minimum inhibitory concentration and minimum bactericidal concentration. A total of seven distinct sequence types (STs) were identified, which belonged to four different Bcc species: Burkholderia cenocepacia (ST621, ST258, and novel ST), Burkholderia lata (ST339 and ST336), Burkholderia contaminans (ST482), Burkholderia cepacia (ST922). For DMDMH and MH, the maximum permitted concentrations according to the safety specification of cosmetics (0.6% and 0.4%) were able to inhibit or kill all Bcc strains, but 40% of Bcc isolates could survive at higher than maximum permitted concentrations of MIT/cMIT (of a mixture in the ratio 3:1 of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one). The PCPs contamination of Bcc strains should be given more attention by manufacturers because of its diversity in molecular epidemiology and its low susceptibility to preservatives such as MIT/cMIT.

Burkholderia Cepacia Complex in Personal Care Products: Molecular Epidemiology and Susceptibility to Preservatives.

The invention discloses a pyocyanine culture medium and method for detecting Pseudomonas aeruginosa in cosmetics. Every liter of the pyocyanine culture medium contains 0.8-1.2g of lecithin, 6.0-8.0g of Tween-80, 18.0-20.0g of peptone, 1.4-1.6g of magnesium chloride, 8.0-16.0g of potassium sulfate, 0.05-0.1g of triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether), 25.0-30.0g of gelatin, 13.0-15.0g of agar and the balance of water. The pH value is 6.9-7.1. The method comprises the following steps: carrying out enrichment culture on a cosmetic sample by using an SCDLP (soya casein digest lecithin polysorbate broth) enrichment fluid, directly carrying out a 42-DEG C growth test by using the pyocyanine culture medium, carrying out screening detection on Pseudomonas aeruginosa, observing the screening culture result, and if bacterial colonies appear after the screening culture, further carrying out identification in combination with a gelatin liquefaction test. The method simplifies the operation steps in the Pseudomonas aeruginosa detection process, enhances the detection efficiency, and can be used for daily detection on Pseudomonas aeruginosa in cosmetics.

Pyocyanine culture medium and method for detecting Pseudomonas aeruginosa in cosmetics

The invention discloses multiple PCR detection primers of escherichia coli, pseudomonas aeruginosa and staphylococcus aureus, a kit and a detection method. Specific primers are designed by means of combined utilization of escherichia coli alkaline phosphatase gene, pseudomonas aeruginosa outer membrance proteins gene and staphylococcus aureus heat-resistant nuclease gene sequences, multiple PCR detection is conducted on a sample by means of the specific primers according to the method, and specific detection of the escherichia coli, the pseudomonas aeruginosa and the staphylococcus aureus in the sample can be achieved at a time; the detection time is short, operation is easy and convenient, the detection efficiency can be effectively improved, the requirement of rapid detection is met, the sensitivity is high, the specificity is high, and the multiple PCR detection primers of the escherichia coli, the pseudomonas aeruginosa and the staphylococcus aureus, the kit and the detection method can be applied to microbiological detection work of cosmetics.

Xie Xiaobao

Papers

Diallyl Disulfide From Garlic Oil Inhibits Pseudomonas aeruginosa Quorum Sensing Systems and Corresponding Virulence Factors.

Neutralizing agent used for detecting microorganisms in cosmetics and preparation method thereof

Burkholderia Cepacia Complex in Personal Care Products: Molecular Epidemiology and Susceptibility to Preservatives.

Pyocyanine culture medium and method for detecting Pseudomonas aeruginosa in cosmetics

Multiple PCR detection primers of escherichia coli, pseudomonas aeruginosa and staphylococcus aureus, kit and detection method