Antibacterial effect of Moringa oleifera on Staphylococcus aureus and Pseudomonas aeruginosa isolated from raw milk and some dairy products with special reference to biofilm gene expression.

Background: Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are well defined as food poisoning pathogens that are highly resistant and need continuous studies. Aim: The purpose of the work was to examine phenotypic and genotypic characteristics of both P. aeruginosa and S. aureus, and treatment trials with medicinal plants. Methods: Samples were examined for isolation of P. aeruginosa and S. aureus on selective media followed by biochemical confirmation, biofilm formation, genes detection, and expression of P. aeruginosa pslA biofilm gene was performed by quantitative real-time polymerase chain reaction after treatment with 0.312 mg/ml Moringa oleifera aqueous extract as a minimum inhibitory concentration. Results: The highest isolation rate of P. aeruginosa was 20% from both raw milk and Kariesh cheese, followed by 16% and 12% from ice cream and processed cheese, respectively, while the highest isolation rate of S. aureus was 36% from raw milk followed by 28% in ice cream and 16% in both Kariesh cheese and processed cheese. 30% of P. aeruginosa isolates were biofilm producers, while only 21% of S. aureus isolates were able to produce biofilm. The P. aeruginosa isolates harbor virulence-associated genes nan1, exoS, toxA, and pslA at 100%, 80%, 40%, and 40%, respectively. Staphylococcus aureus SEs genes were examined in S. aureus strains, where SEA and SEB genes were detected with 60%, but no isolate harbored SEC, SED, or SEE. The significant fold change of P. aeruginosa pslA expression was 0.40332 after treatment with M. oleifera aqueous extract. Conclusion: Pseudomonas aeruginosa and S. aureus harbor dangerous virulence genes that cause food poisoning, but M. oleifera extract could minimize their action.

Selenium nanoparticles coated bacterial polysaccharide with potent antimicrobial and anti-lung cancer activities.

Bacterial exopolysaccharides are homopolymeric or heteropolymeric polysaccharides with large molecular weights (10-1000 kDa). Exopolysaccharides' functional uses and potential have revolutionized the industrial and medicinal industries. Hence, the aim of the present study was to optimize the production of bacterial exopolysaccharide and apply it as a capping agent for selenium nanoparticles synthesis. Exopolysaccharide (EPS) producing Lactic acid bacteria (LAB) were isolated from dairy products then biochemically characterized and assessed for their potential antimicrobial effect. The most potent EPS producer was identified as Lactiplantibacillus plantarum strain A2 with accession number OP218384 using 16S rRNA sequencing. Overall, FTIR data of the extracted EPS revealed similarity with amylopectin spectrum. 1H NMR spectrum revealed an α-anomeric configuration of the glycosidic linkage pattern in the polysaccharides while the 13C NMR spectrum can also be separated into two main portions, the anomeric carbons region (δ 98-102 ppm) and the non-anomeric carbons region (δ 60-81 ppm). Antimicrobial activity of the produced EPS showed maximum activity against Staphylococcus aureus, MRSA, Enterobacter aerogenes, Klebsiella pneumoniae and Candida albicans respectively. The EPS capsule layer surrounding the bacterial cells was detected by TEM study. Optimization of EPS production was evaluated using Taguchi design, trial 23 reported the highest biomass yield and EPS output (6.5 and 27.12 g/L respectively) with 2.4 and 3.3 folds increase (from the basal media) respectively. The optimized exopolysaccharide was used as a capping and stabilizing agent for selenium nanoparticles (EPS-SeNPs) synthesis. Zeta potential, size and PDI of the synthesized nanoparticles were - 19.7 mV, 45-65 nm and 0.446 respectively with strong bactericidal and fungicidal effect against the tested pathogens. Complete microbial growth eradication was recorded after 6, 8 and 10 h against Staphylococcus aureus, Candida albicans and Klebsiella pneumoniae respectively. EPS-SeNPs showed a potent antioxidant effect reached 97.4% and anticancer effect against A549 lung cancer cell line (IC50 reached 5.324 µg/mL). EPS-SeNPs inhibited cancerous cell growth at S phase. Moreover, molecular studies revealed the anti-apoptotic activity of Bcl2's was inhibited and Bax was activated. The present investigation successfully synthesized selenium nanoparticles through bacterial EPS with significantly high antimicrobial and anticancer activity.

Treatment outcomes with benzylpenicillin and non-benzylpenicillin antibiotics, and the performance of the penicillin zone-edge test versus molecular detection of blaZ in penicillin-susceptible Staphylococcus aureus (PSSA) bacteraemia.

OBJECTIVES: The blaZ gene encodes penicillinase, which inactivates penicillin. As there were reports on suboptimal sensitivity for the penicillin zone-edge test, a phenotypic method for blaZ detection, we investigated treatment outcomes in patients with penicillin-susceptible Staphylococcus aureus (PSSA) bacteraemia (phenotypically negative for penicillinase), subjecting isolates to molecular testing for blaZ retrospectively. PATIENTS AND METHODS: A retrospective cohort study was conducted on 121 patients with a first episode of PSSA bacteraemia from 1 January 2012 to 31 October 2015 at Tan Tock Seng Hospital (TTSH), Singapore. Patients were grouped into IV benzylpenicillin and non-benzylpenicillin groups. The primary outcome was overall treatment failure, defined as either 30 day all-cause mortality and/or 90 day relapse. The penicillin (P10) zone-edge test was repeated on archived PSSA isolates, concurrently with penicillin MIC determination via gradient diffusion and PCR for blaZ. RESULTS: Among 121 patients, 57 patients (47.1%) received IV benzylpenicillin as the predominant antibiotic. There was no significant difference in overall treatment failure between treatment with the benzylpenicillin [7/57 (12.3%)] versus non-benzylpenicillin groups [12/64 (18.8%)] (P = 0.33) or cloxacillin/cefazolin [6/37 (16.2%)] (P = 0.59). For 112 PSSA isolates available for testing, repeat penicillin zone-edge testing was negative for penicillinase production, corroborating previous results. A single PSSA isolate with a negative penicillin zone-edge test was found to be positive for blaZ. CONCLUSIONS: We found no differences in overall treatment failure between patients with PSSA bacteraemia treated with benzylpenicillin, anti-staphylococcal ß-lactams cefazolin/cloxacillin and other antimicrobials, when using the penicillin zone-edge test as the phenotypic method for blaZ screening.

The fadXDEBA locus of Staphylococcus aureus is required for metabolism of exogenous palmitic acid and in vivo growth.

In Staphylococcus aureus, genes that should confer the capacity to metabolize fatty acids by ß-oxidation occur in the fadXDEBA locus, but their function has not been elucidated. Previously, incorporation into phospholipid through the fatty acid kinase FakA pathway was thought to be the only option available for S. aureus to metabolize exogenous saturated fatty acids. We now find that in S. aureus USA300, a fadX::lux reporter was repressed by glucose and induced by palmitic acid but not stearic acid, while in USA300ΔfakA basal expression was significantly elevated, and enhanced in response to both fatty acids. When cultures were supplemented with palmitic acid, palmitoyl-CoA representing the first metabolite in the ß-oxidation pathway was detected in USA300, but not in a fadXDEBA deletion mutant USA300Δfad, which relative to USA300 exhibited increased incorporation of palmitic acid into phospholipid accompanied by a rapid loss of viability. USA300Δfad also exhibited significantly reduced viability in a murine tissue abscess infection model. Our data are consistent with FakA-mediated incorporation of fatty acids into phospholipid as a preferred pathway for metabolism of exogenous fatty acids, while the fad locus is critical for metabolism of palmitic acid, which is the most abundant free fatty acid in human plasma.

Investigating a possible link between antiseptic treatment and the increased occurrence of daptomycin-resistant Staphylococcus aureus.

OBJECTIVES: Because of a steady increase in the detection of daptomycin-resistant (DAP-R) Staphylococcus aureus at three medical centres in Cologne, Germany, molecular surveillance was established from June 2016 to June 2018 to investigate the causes of the emergence and spread of respective isolates. Seventy-five S. aureus isolates, both DAP-R and DAP-susceptible, were collected from 42 patients for further analysis. METHODS: Broth microdilution was used to determine the MICs for DAP and polyhexamethylene biguanide/polyhexanide (PHMB). To investigate the effect of PHMB on the development of DAP resistance, we performed selection experiments with PHMB. All isolates studied were subjected to whole-genome sequencing. Epidemiological, clinical, microbiological and molecular data were analysed comparatively. RESULTS: Acquisition of DAP resistance was mainly observed in patients with acute and chronic wounds (40/42, 96.2%) treated with antiseptic (32/42, 76.2%) rather than systemic antibiotic therapy using DAP or vancomycin (7/42, 16.7%). DAP-R S. aureus had a diverse genetic background; however, within individual patients, isolates were closely related. At least three potential transmission events were detected. Most DAP-R isolates had concomitant elevated MICs for PHMB (50/54, 92.6%), and in vitro selection experiments confirmed that PHMB treatment is capable of generating DAP resistance. DAP resistance could be linked to 12 different polymorphisms in the mprF gene in the majority of clinical isolates (52/54, 96.3%) as well as in all in vitro selected strains. DISCUSSION: DAP resistance in S. aureus can occur independently of prior antibiotic therapy and can be selected by PHMB. Therefore, wound treatment with PHMB may trigger individual resistance development associated with gain-of-function mutations in the mprF gene.

A Clinically Selected Staphylococcus aureus clpP Mutant Survives Daptomycin Treatment by Reducing Binding of the Antibiotic and Adapting a Rod-Shaped Morphology.

Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations; however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and ß-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that inactivation of ClpP reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis, eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10× MIC daptomycin, resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression.

A short-chain acyl-CoA synthetase that supports branched-chain fatty acid synthesis in Staphylococcus aureus.

Staphylococcus aureus controls its membrane biophysical properties using branched-chain fatty acids (BCFAs). The branched-chain acyl-CoA precursors, utilized to initiate fatty acid synthesis, are derived from branched-chain ketoacid dehydrogenase (Bkd), a multiprotein complex that converts α-keto acids to their corresponding acyl-CoAs; however, Bkd KO strains still contain BCFAs. Here, we show that commonly used rich medias contain substantial concentrations of short-chain acids, like 2-methylbutyric and isobutyric acids, that are incorporated into membrane BCFAs. Bkd-deficient strains cannot grow in defined medium unless it is supplemented with either 2-methylbutyric or isobutyric acid. We performed a screen of candidate KO strains and identified the methylbutyryl-CoA synthetase (mbcS gene; SAUSA300_2542) as required for the incorporation of 2-methylbutyric and isobutyric acids into phosphatidylglycerol. Our mass tracing experiments show that isobutyric acid is converted to isobutyryl-CoA that flows into the even-chain acyl-acyl carrier protein intermediates in the type II fatty acid biosynthesis elongation cycle. Furthermore, purified MbcS is an ATP-dependent acyl-CoA synthetase that selectively catalyzes the activation of 2-methylbutyrate and isobutyrate. We found that butyrate and isovalerate are poor MbcS substrates and activity was not detected with acetate or short-chain dicarboxylic acids. Thus, MbcS functions to convert extracellular 2-methylbutyric and isobutyric acids to their respective acyl-CoAs that are used by 3-ketoacyl-ACP synthase III (FabH) to initiate BCFA biosynthesis.

20S-ginsenoside Rg3 inhibits the biofilm formation and haemolytic activity of Staphylococcus aureus by inhibiting the SaeR/SaeS two-component system.

Introduction. Staphylococcus aureus is a major cause of chronic diseases and biofilm formation is a contributing factor. 20S-ginsenoside Rg3 (Rg3) is a natural product extracted from the traditional Chinese medicine red ginseng.Gap statement. The effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial mechanism against S. aureus have not been reported.Aim. This study aimed to investigate the effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial action against clinical S. aureus isolates.Methodology. The effect of Rg3 on biofilm formation of clinical S. aureus isolates was studied by crystal violet staining. Haemolytic activity analysis was carried out. Furthermore, the influence of Rg3 on the proteome profile of S. aureus was studied by quantitative proteomics to clarify the mechanism underlying its antibacterial action and further verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).Results. Rg3 significantly inhibited biofilm formation and haemolytic activity in clinical S. aureus isolates. A total of 63 with >1.5-fold changes in expression were identified, including 34 upregulated proteins and 29 downregulated proteins. Based on bioinformatics analysis, the expression of several virulence factors and biofilm-related proteins, containing CopZ, CspA, SasG, SaeR/SaeS two-component system and SaeR/SaeS-regulated proteins, including leukocidin-like protein 2, immunoglobulin-binding protein G (Sbi) and fibrinogen-binding protein, in the S. aureus of the Rg3-treated group was downregulated. RT-qPCR confirmed that Rg3 inhibited the regulation of SaeR/SaeS and decreased the transcriptional levels of the biofilm-related genes CopZ, CspA and SasG.Conclusions. Rg3 reduces the formation of biofilm by reducing cell adhesion and aggregation. Further, Rg3 can inhibit the SaeR/SaeS two-component system, which acts as a crucial signal transduction system for the anti-virulence activity of Rg3 against clinical S. aureus isolates.

Multiple amplification-based fluorometric aptasensor for highly sensitive detection of Staphylococcus aureus.

Rapid and accurate detection and identification of Staphylococcus aureus (S. aureus) are of great significance for food safety, environmental monitoring, early clinical diagnosis, and prevention of the spread of drug-resistant bacteria. Herein, we design a fluorometric aptasensor for ultra-sensitive, specific, and rapid detection of S. aureus. The apasensor combines the enrichment and separation of magnetic nanoparticles (MNPs), the biotin-streptavidin conjugation system, and a single S. aureus can release four signaling probes for signal amplification. Aptamer acts as a specific biorecognition element of S. aureus. Four FAM-labeled partially complementary sequences (FAM-pcDNAs) were used as signaling probes. The aptamers were sequential hybridized with the four FAM-pcDNAs to form aptamer&pcDNAs, which were then bound to MNPs via the biotin-streptavidin. When the aptamer specifically recognizes and binds to S. aureus, the FAM-pcDNAs signaling probes are replaced and released into the supernatant. The concentration of S. aureus can be quantified by measuring the fluorescence intensity (λexc/em = 492/520 nm) of the replaced signaling probe FAM-pcDNAs. The results show that the proposed fluorometric aptasensor displays good specificity, ultra-high sensitivity (1.23 cfu/mL), wide linear range (1 ~ 108 cfu/mL), and fast detection speed (~ 1.5 h). The recovery test verifies further that the proposed fluorometric aptasensor can detect S. aureus in spiked blood samples. Since aptamers are easy to customize, we believe that fluorometric aptasensors based on multiple amplification have broad prospects in the construction of practical high-performance biosensors for bacterial detection. KEY POINTS: • Multiple amplification-based fluorometric aptasensor for S. aureus is developed • The aptasensor displays high specificity with a LOD of 1.23 CFU/mL • The aptasensor can directly detect S. aureus in spiked blood samples.

Genome analysis of secondary metabolite­biosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Persistence of Staphylococcus spp. in milk from cows undergoing homeopathy to control subclinical mastitis.

BACKGROUND: Mastitis is one of the major diseases in dairy cattle, as it causes great economic losses to producers due to the reduction of milk production and changes in the quality of the product. The disease is mainly caused by bacteria of the genus Staphylococcus spp., these microorganisms can express various virulence factors, such as biofilms for example. In herds with organic management, producers and technicians use unconventional ways to treat and control the disease, such as homeopathy. However, it is not known if this type of treatment is able to control pathogenic bacteria such as those of the genus Staphylococcus, of relevance to animal and human health. Thus, the objective of this study was to investigate the production of biofilm in vitro and its genes by Staphylococcus spp. isolated in the milk of cows treated with homeopathy, as well as the persistence of microorganisms in animals. METHODS: Ninety-nine isolates of Staphylococcus spp. from cows treated and not treated with homeopathy were identified by internal transcribed space-polymerase chain reaction and investigated for the presence of the icaABCD, bap, aap, atlE, and bhp genes and in vitro biofilm production using the adhesion method on polystyrene plates. The enzyme restriction profile was determined by Pulsed-Field Gel Electrophoresis. Clusters of S. aureus and S. epidermidis with three or more isolates had an isolate selected for Multilocus Sequence Typing. RESULTS: The frequency of S. aureus isolations was similar in treated and untreated cows, while 71.4% of the coagulase-negative identified were isolated in cows treated with homeopathy. The distribution of the operon ica genes was similar in animals with and without treatment, except for the icaD gene, more frequent in treated cows. Production of biofilm was associated with presence of one or more genes from the icaADBC operon. S. aureus revealed a greater diversity and greater dissemination in cows treated and not treated with homeopathy. Sequence Types ST1, ST5, and ST126 were identified in S. aureus. CONCLUSIONS: The presence of biofilm-associated genes and the in vitro production of biofilms, combined with the persistence of clonal profiles of Staphylococcus spp. demonstrate other forms of control for bovine mastitis should be researched for organic production herds.

A Structurally Characterized Staphylococcus aureus Evolutionary Escape Route from Treatment with the Antibiotic Linezolid.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance. IMPORTANCE Antibiotic resistance poses a critical problem to human health and decreases the utility of these lifesaving drugs. Of particular concern is the "superbug" methicillin-resistant Staphylococcus aureus (MRSA), for which treatment of infection requires the use of last-line antibiotics, including linezolid. In this paper, we characterize the atomic rearrangements which the ribosome, the target of linezolid, undergoes during its evolutionary journey toward becoming drug resistant. Using cryo-electron microscopy, we describe a particular molecular mechanism which MRSA uses to become resistant to linezolid.

Divergent Analyses of Genetic Relatedness and Evidence-Based Assessment of Therapeutics of Staphylococcus aureus from Semi-intensive Dairy Systems.

Use of antibiotics without following standard guidelines is routine practice in developing countries which is giving rise to genetic divergence and increased drug resistance. The current study analyzed genetic divergence and drug resistance by S. aureus and therapeutic efficacy of novel antibiotic combinations. The study revealed that 42.30% (minimum 20%-maximum 70%) of milk samples are positive for S. aureus. Study also revealed seven SNPs in the S. aureus nuc gene (c.53A>G, c.61A>G, c.73T>C, c.93C>A, c.217C>T, c.280T>C, and c.331T>A). Local isolates Staph-2 and Staph-3 were closely related to Bos taurus nuc gene (bovine S. aureus), while Staph-1 was closely related to Homo sapiens (human S. aureus) indicating shifting of host. Change of two amino acids and staphylococcal nuclease conserved domain was observed in all local isolates of S. aureus. The isoelectric points predicted by protParam of Staph-1, Staph-2, and Staph-3 proteins were 9.30, 9.20, and 9.20, respectively. The antibiotic susceptibility profile of S. aureus presented highest resistance against penicillin (46.67%) and glycopeptide (43.33%). When a single antibiotic regimen was adopted in a field trial, the highest efficacy was reported in the case of oxytetracycline (80%) while lowest was presented by azithromycin. Among antibiotics' combined regimen, the highest efficacy (80%) was presented by gentamicin with oxytetracycline: cefotaxime with vancomycin; and ciprofloxacin with vancomycin. The current study concluded rising percentages of S. aureus from dairy milk, proofs of genetic host shifts, and altered responses of in on field therapeutics.

Traditional Chinese Medicine Tanreqing Targets Both Cell Division and Virulence in Staphylococcus aureus.

Staphylococcus aureus has been recognized as an important human pathogen and poses a serious health threat worldwide. With the advent of antibiotic resistance, such as the increased number of methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to develop new therapeutical agents. In this study, Chinese traditional medicine Tanreqing (TRQ) has been used as an alternative treating agent against MRSA and we aim to unravel the mode of action of TRQ underlying MRSA inhibition. TRQ treatment affected numerous gene expression as revealed by RNA-seq analysis. Meanwhile, TRQ targeted cell division to inhibit cell growth as shown by illumination microscopy. Besides, we confirmed that TRQ downregulates the expression of virulence factors such as hemolysin and autolysin. Finally, we used a murine model to demonstrate that TRQ efficiently reduces bacterial virulence. Altogether, we have proved TRQ formula to be an effective agent against S. aureus infections.

Nigella sativa oil extract: A natural novel specific conjugal transfer inhibitor of vancomycin resistance from vanA/B-resistant Enterococcus faecium to Staphylococcus aureus.

AIM: The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) has been identified as one of the most challenging problems in healthcare settings worldwide. Specific conjugation inhibitors' development is critical in the fight against the spread of emerging VRSA. The impact of Nigella sativa oil on VR genes conjugal transfer from Enterococcus faecium (VREtfm) to vancomycin-sensitive S. aureus (VSSA) was investigated in this study. METHODS AND RESULTS: Enterococciwere isolated from retail broilers, fish, cows' milk, and human urine. VR E. faecalis and VREtfm VanA phenotypes were prevalent in retail broiler samples. The VREtfm isolates were dominant, exhibiting high levels of resistance to gentamycin and ciprofloxacin antibiotics, as well as the existence of both vanA and vanB genes and virulence traits (ESP+ , asa1+ ) as determined by PCR. Transconjugant VREtfm strains containing vanA/vabB and 20 kb plasmids (transfer frequency around 103 ) and carrying the Tn1546 transposon were identified. Tn1546 transposon transfer with its VR markers to VSSA was effectively inhibited in treated VREtfm donor strains with a sub-minimum inhibitory concentration of N. sativa oil. THE SIGNIFICANCE AND IMPACT OF THE STUDY: This work offers new insights for overcoming VR conjugal transfer utilizing natural N. sativa oil, as well as a suggestion for a novel specialized conjugation inhibitor that could effectively facilitate the difficulty of eliminating VR bacteria from healthcare settings.

Drug Resistance and Epigenetic Modulatory Potential of Epigallocatechin-3-Gallate Against Staphylococcus aureus.

Antimicrobial resistance of human pathogens, such as methicillin-resistant Staphylococcus aureus, is described by the World Health Organization as a health global challenge and efforts must be made for the discovery of new effective and safe compounds. This work aims to evaluate epigallocatechin-3-gallate (EGCG) epigenetic and modulatory drug potential against S. aureus in vitro and in vivo. S. aureus strains were isolated from commensal flora of healthy volunteers. Antibiotic susceptibility and synergistic assay were assessed through disk diffusion accordingly to EUCAST guidelines with and without co-exposure to EGCG at final concentrations of 250 µg/ml, 100 µg/ml, 50 µg/ml, and 25 µg/ml. Transcriptional expression of orfx, spdC, and WalKR was performed through qRT-PCR. A 90-day interventional study was performed with daily consumption of 225 mg of EGCG. Obtained data revealed a high prevalence of S. aureus colonization in healthcare workers and clearly demonstrated the antimicrobial and synergistic potential of EGCG as well as divergent resistant phenotypes associated with altered transcriptional expression of epigenetic and drug response modulators genes. Here, we demonstrate the potential of EGCG for antimicrobial treatment and/or therapeutic adjuvant against antibiotic-resistant microorganisms and report divergent patterns of epigenetic modulators expression associated with phenotypic resistance profiles.

Multi-methodological approach for the Quality assessment of Senecionis scandentis Herba (Qianliguang) in the herbal market.

We set forth to assess the quality of an herbal medicine sold in Hong Kong called Qianliguang by employing a multi-methodological approach. The quality is set by its identity, chemical composition, and bioactivities, among others. Qianliguang (Senecionis scandentis Herba, Senecio scandens Buch.-Ham. ex D.Don) has known antibacterial properties. However, it is poisonous and overconsumption can result in liver damage. Eighteen Qianliguang samples were purchased from herbal shops at various districts in Hong Kong. Samples were first authenticated organoleptically. DNA barcoding at the psbA-trnH, ITS2, and rbcL loci was then conducted to confirm the species. HPLC-UV was performed to screen for the presence of the chemical compounds and to quantify the flavonoid hyperoside. UPLC-MS was used to quantify the amount of the toxic pyrrolizidine alkaloid (PA) adonifoline. Microdilution assay was performed to show the antibacterial effect on Streptococcus aureus and S. pneumoniae. Results showed that five samples were found to be substituted by species belonging to the genus Lespedeza; four samples were mixtures containing not only Qianliguang but also Achyranthes aspera L., Lonicera confusa DC., or Solanum nigrum L. HPLC-UV showed that only ten contained enough hyperoside to meet the standard requirement. In addition, nine samples had adonifoline that exceeded the toxicity standard requirement. In the microdilution assay, samples containing Qianliguang showed inhibition on S. aureus and S. pneumoniae, while among the five Lespedeza sp. samples the antibacterial effects on S. aureus were not detectable; only one sample showed inhibition to S. pneumoniae. Our study illustrated the necessity of using a multi-methodological approach for herbal medicine quality assessment. We also showed that Qianliguang samples in the Hong Kong market were either toxic or adulterated. It is therefore essential to improve the quality control of Qianliguang and probably other herbs in the herbal market.

Catabolic Ornithine Carbamoyltransferase Activity Facilitates Growth of Staphylococcus aureus in Defined Medium Lacking Glucose and Arginine.

Previous studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC, encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1. Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH, encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1, but not argGH, rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes (proC and rocA) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine. IMPORTANCE Staphylococcus aureus can cause infection in virtually any niche of the human host, suggesting that it has significant metabolic versatility. Indeed, bioinformatic analysis suggests that it has the biosynthetic capability to synthesize all 20 amino acids. Paradoxically, however, it is conditionally auxotrophic for several amino acids, including arginine. Studies in our laboratory are designed to assess the biological function of amino acid auxotrophy in this significant pathogen. This study reveals that the metabolic block repressing arginine biosynthesis in media lacking glucose is the transcriptional repression of ornithine carbamoyltransferase encoded by arcB1 within the native arginine deiminase operon in addition to limited intracellular pools of ornithine. Surprisingly, approximately 50% of S. aureus clinical isolates can grow in media lacking arginine, suggesting that mutations are selected in S. aureus that allow growth in particular niches of the human host.

Evaluation of the Pathogenic-Mixed Biofilm Formation of Pseudomonas aeruginosa/Staphylococcus aureus and Treatment with Limonene on Three Different Materials by a Dynamic Model.

BACKGROUND: Biofilms have been found growing on implantable medical devices. This can lead to persistent clinical infections. The highly antibiotic-resistant property of biofilms necessitates the search for both potent antimicrobial agents and novel antibiofilm strategies. Natural product-based anti-biofilm agents were found to be as efficient as chemically synthesized counterparts with fewer side effects. In the present study, the effects of limonene as an antibiofilm agent were evaluated on Pseudomonas aeruginosa and Staphylococcus aureus biofilm formed on different surfaces using the CDC model system in continuous flow. The flgK gene and the pilA gene expression in P. aeruginosa, and the icaA gene and eno gene in S. aureus, which could be considered as efficient resistance markers, were studied. METHODS: Mono- and dual-species biofilms were grown on polycarbonate, polypropylene, and stainless-steel coupons in a CDC biofilm reactor (Biosurface Technologies, Bozeman, MT, USA). To evaluate the ability of limonene to inhibit and eradicate biofilm, a sub-MIC concentration (10 mL/L) was tested. The gene expression of P. aeruginosa and S. aureus was detected by SYBR Green quantitative Real-Time PCR assay (Meridiana Bioline, Brisbane, Australia). RESULTS: The limonene added during the formation of biofilms at sub-MIC concentrations works very well in inhibiting biofilms on all three materials, reducing their growth by about 2 logs. Of the same order of magnitude is the ability of limonene to eradicate both mono- and polymicrobial mature biofilms on all three materials. Greater efficacy was observed in the polymicrobial biofilm on steel coupons. The expression of some genes related to the virulence of the two microorganisms was differently detected in mono- and polymicrobial biofilm. CONCLUSIONS: These data showed that the limonene treatment expressed different levels of biofilm-forming genes, especially when both types of strains alone and together grew on different surfaces. Our findings showed that limonene treatment is also very efficient when biofilm has been grown under shear stress causing significant and irreversible damage to the biofilm structure. The effectiveness of the sanitation procedures can be optimized by applying antimicrobial combinations with natural compounds (e.g., limonene).

Hygromycin A derivatives isolated from Streptomyces sp. PC-22 in the rhizosphere soil of Pulsatilla chinensis.

On the basis of the one strain-many compounds (OSMAC) strategy, two new hygromycin A derivatives (3, 4), together with six known compounds were isolated from a medicinal plant inter rhizospheric Streptomyces in Pulsatilla chinensis. The structures of 3 and 4 were elucidated using NMR and HRESIMS analyses. A plausible biosynthetic pathway for these compounds was discussed. All the compounds were evaluated for their antimicrobial and cytotoxic activities. Compound 5 exhibited potent inhibitory activity against S. aureus and B. subtilis with the MICs of 16 and 8 µg ml-1, while 4 showed weak inhibitory activity against S. aureus.