Vancomycin-resistant Staphylococcus Aureus Research Articles

Eradication of vancomycin-resistant Staphylococcus aureus on a mouse model of third-degree burn infection by melittin: An antimicrobial peptide from bee venom

Third-degree burn infections caused by antibiotic-resistant bacteria are of high clinical concern. Chemical antibiotics are not promising in eradication of bacterial infections. In this challenging condition, antimicrobial peptides (AMPs) are recently introduced as novel promising agents to overcome the issue. Accordingly, our study aimed to evaluate the efficiency of ‘melittin’ as natural peptide in bee venom, in eradicating vancomycin resistant Staphylococcus aureus (VRSA) on a mouse model of third-degree burn infection.In vitro pharmacological value of melittin was determined by examining its inhibitory and killing activities on VRSA isolates at different doses and time periods. The action mechanism of ‘melittin’ was evaluated by fluorescent release assay and Field Emission Scanning Electron Microscopy (FE-SEM) analyses. In vivo activity and toxicity of melittin were also examined on a mouse model of third-degree burn infection.The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of melittin on all isolates ranged from ‘0.125–2 μg/mL’ and ‘0.125–4 μg/mL’, respectively. Rapid antibacterial activity of melittin on VRSA isolates was demonstrated by killing kinetics assays. Fluorometric and FE-SEM analyses indicated the membranolytic effects of melittin on VRSA isolates. The colonized VRSA bacteria were eradicated by melittin at 16 μg, in a single dose. No dermal toxicity and in vivo hemolysis were observed in the examined mice.The lack of in vivo toxicity of melittin along with its potent antibacterial activity indicated its promising therapeutic value as a topical drug against S. aureus associated third-degree burn infections.

Apt (Adenine Phosphoribosyltransferase) Mutation in Laboratory-Selected Vancomycin-Intermediate Staphylococcus aureus.

Comparative genomic sequencing of laboratory-derived vancomycin-intermediate Staphylococcus aureus (VISA) (MM66-3 and MM66-4) revealed unique mutations in both MM66-3 (in apt and ssaA6), and MM66-4 (in apt and walK), compared to hetero-VISA parent strain MM66. Transcriptional profiling revealed that both MM66 VISA shared 79 upregulated genes and eight downregulated genes. Of these, 30.4% of the upregulated genes were associated with the cell envelope, whereas 75% of the downregulated genes were associated with virulence. In concordance with mutations and transcriptome alterations, both VISA strains demonstrated reduced autolysis, reduced growth in the presence of salt and reduced virulence factor activity. In addition to mutations in genes linked to cell wall metabolism (ssaA6 and walK), the same mutation in apt which encodes adenine phosphoribosyltransferase, was confirmed in both MM66 VISA. Apt plays a role in both adenine metabolism and accumulation and both MM66 VISA grew better than MM66 in the presence of adenine or 2-fluoroadenine indicating a reduction in the accumulation of these growth inhibiting compounds in the VISA strains. MM66 apt mutants isolated via 2-fluoroadenine selection also demonstrated reduced susceptibility to the cell wall lytic dye Congo red and vancomycin. Finding that apt mutations contribute to reduced vancomycin susceptibility once again suggests a role for altered purine metabolism in a VISA mechanism.

Lessons Learned from an Experience with Vancomycin-Intermediate Staphylococcus aureus Outbreak in a Newly Built Secondary Hospital in Korea.

A vancomycin-intermediate Staphylococcus aureus (VISA) outbreak occurred in an intensive care unit (ICU) in South Korea. We aimed to investigate the condition that led to the VISA outbreak and seek measures to prevent further spread of the multidrug-resistant organism. A total of three VISA isolates were obtained from two patients and a health care worker (HCW) in a newly built 450-bed secondary hospital. Extensive screening of close contacts for VISA in terms of space sharing and physical contact, irrespective of contact time, was performed. Furthermore, multilocus sequence type, staphylococcal cassette chromosome mec type, and spa type profiles were determined for all VISA isolates. The relationship between vancomycin use and the minimum inhibitory concentration (MIC) of S. aureus was also investigated. Molecular typing showed that the strains of the three VISA isolates were identical, indicating horizontal hospital transmission. We assumed that VISA colonised in the HCW could have transmitted to the two patients, which resulted in one infection and one colonisation. The affected HCW was excused from work and was decolonised with mupirocin. Five weeks after the interventions, no additional VISA isolates were identified. No relationship between vancomycin use and MIC of S. aureus was identified. Extensive screening of contacts in addition to decolonisation is crucial in preventing the further spread of VISA.

Antimicrobial and antibiofilm photodynamic therapy against vancomycin resistant Staphylococcus aureus (VRSA) induced infection in vitro and in vivo

Biofilm mediated infection caused by multi-drug resistant bacteria are difficult to treat since it protects the microorganisms by host defense system, making them resistant to antibiotics and other antimicrobial agents. Combating such type of nosocomial infection, especially in immunocompromised patients, is an urgent need and foremost challenge faced by clinicians. Therefore, antimicrobial photodynamic therapy (aPDT) has been intensely pursued as an alternative therapy for bacterial infections. aPDT leads to the generation of reactive oxygen species (ROS) that destroy bacterial cells in the presence of a photosensitizer, visible light and oxygen. Here, we elucidated a possibility of its clinical application by reducing the treatment time and exposing curcumin to 20 J/cm2 of blue laser light, which corresponds to only 52 s to counteract vancomycin resistant Staphylococcus aureus (VRSA) both in vitro and in vivo. To understand the mechanism of action, the generation of total reactive oxygen species (ROS) was quantified by 2′-7′-dichlorofluorescein diacetate (DCFH-DA) and the type of phototoxicity was confirmed by fluorescence spectroscopic analysis. The data showed more production of singlet oxygen, indicating type-II phototoxicity. Different anti-biofilm assays (crystal violet and congo red assays) and microscopic studies were performed at sub-MIC concentration of curcumin followed by treatment with laser light against preformed biofilm of VRSA. The result showed significant reduction in the preformed biofilm formation. Finally, its therapeutic potential was validated in skin abrasion wistar rat model. The result showed significant inhibition of bacterial growth. Furthermore, immunomodulatory analysis with rat serum was performed. A significant reduction in expression of proinflammatory cytokines TNF-α and IL-6 were observed. Hence, we conclude that curcumin mediated aPDT with 20 J/cm2 of blue laser treatment (for 52 s) could be used against multi-drug resistant bacterial infections and preformed biofilm formation as a potential therapeutic approach.

A novel mutation of walK confers vancomycin-intermediate resistance in methicillin-susceptible Staphylococcus aureus

With the treatment failure by vancomycin and poor clinical outcomes, the emergence and spread of vancomycin intermediate-resistant Staphylococcus aureus (VISA) has raised more concerns in recent years. While most VISA strains are isolated from methicillin-resistant S. aureus (MRSA), the mechanism underlying the generation of VISA from methicillin-susceptible S. aureus (MSSA) is still largely unknown. Here, we identified a total of 10 mutations in 9 genes through comparative genome analysis from laboratory-derived VISA strain. We verified the role of a novel mutation of WalK (I237T) and our results further indicated that the introduction of WalK (I237T) by allelic replacement can confer vancomycin resistance in MSSA with common VISA characteristics, including thickened cell walls, reduced autolysis, and attenuated virulence. Consistent with these phenotypes, real-time quantitative reverse transcription-PCR revealed the altered expression of several genes associated with cell wall metabolism and virulence control. In addition, electrophoretic mobility shift assay indicated that WalR can directly bind to the promoter regions of oatA, sle1, and mgt, fluorescence-based promoter activity and β-galactosidase assays revealed WalK (I237T) can alter promoter activities of oatA, mgt, and sle1, thus regulating genes expression. These findings broaden our understanding of the regulatory network by WalKR system and decipher the molecular mechanisms of developmental VISA resistance in MSSA with point mutations.

Strain Improvement Program of Streptomyces roseosporus for Daptomycin Production.

Daptomycin is a cyclic lipopeptide antibiotic with potent activity against gram-positive bacteria. It has a calcium-dependent mechanism of action that disrupts multiple features of the bacterial membrane function. This antibiotic is highly demanded due to its effectiveness against to microorganisms resistant to other antibiotics, including vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant S. aureus (MRSA). Daptomycin is produced by fermentation of Streptomyces roseosporus, currently identified as Streptomyces filamentosus. However, low fermentation yields and high production costs are reported. This chapter describes a method of strain improvement involving random mutagenesis, rational screening by bioassay, and flask fermentation. The ultimate objective is to select mutants of S. roseosporus overproducing daptomycin in order to design a more cost-effective daptomycin production.

Vancomycin Resistance Among Clinical Isolates of Staphylococcus aureus Obtained from Selected Hospitals in Sokoto Metropolis

The decreased vancomycin susceptibility and subsequent emergence of vancomycin resistant Staphylococcus aureus (VRSA) strains is a major public health problem. This study was aimed at detecting the prevalence of vancomycin resistant Staphylococcus aureus among clinical isolates obtained from patients attending Specialist Hospital Sokoto and Maryam Abacha Women and Children Hospital Sokoto. A total of 80 S. aureus clinical isolates were obtained from the medical microbiology laboratories of the selected hospitals. Antibiotic sensitivity testing of the isolates was carried out using the agar dilution method and isolates were screened for vancomycin resistance using vancomycin agar screen method. Of the 80 S. aureus isolates studied, 69 (86.0%) were identified as vancomycin susceptible S. aureus (VSSA) with MIC value of ≤2 µg/ml, 11 (13.8%) were identified as vancomycin intermediate S. aureus (VISA) and had MIC value of 4-8 µg/mL (VISA) and none of the isolates was identified as vancomycin resistant aureus (VRSA). The study detects high prevalence rate of VISA in the study area and identifies the need for increased public awareness on the danger associated with the presence of drug resistant bacteria. Emphasis should be directed at discouraging practices such as the use of over the counter medications which increase the rate of development of drug resistant organisms.

Screening of Kunun-zaki for Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin-Resistant Staphylococcus aureus (VRSA) and Extended-Spectrum Beta-Lactamase (ESBL) Producing Salmonella spp.

Kunun-zaki is an indigenous, fermented, non-alcoholic cereal-based beverage produced and consumed primarily in Northern Nigeria. Due to its high nutritional and moisture content, it provides an ideal environment for the growth of foodborne pathogens. This study determined the presence of Methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Staphylococcus aureus (VRSA) and Extended spectrum beta lactamase (ESBL) producing Salmonella spp in Kunun-zaki sold in Umuahia metropolis, Nigeria. Out of the 40 Kunun-zaki samples examined, Salmonella spp was detected in 19 (47.5%), whereas S. aureus was detected in 22 (55%) of the samples. The total Salmonella count ranged from 1.90×105-9.70×106 (CFU/ml), whereas the total S. aureus count ranged from 3.40×104-7.50×106 (CFU/ml); these microbial counts do not conform to the standard limits of detectable microbes in ready-to-eat food samples as stipulated by the Center for Food Safety. All the isolates were subjected to antibiotic susceptibility testing using the modified Kirby Bauer disc diffusion method. On the one hand, most of the Salmonella spp were resistant to the beta-lactam class of antibiotics used (IMP 58%, MEM 90%, AMP 53%). On the other hand, the Salmonella isolates showed the highest sensitivities to ceftazidime (95%) and ofloxacin (90%). The S. aureus isolates revealed high susceptibility to ofloxacin (96%), and major resistance to oxacillin (41%) and the oxyimino-cephalosporin antibiotics used (CAZ 55%, CTR 41%). MRSA, VRSA and ESBL producing Enterobacteriaceae are amongst the most critical multidrug-resistant bacterial pathogens. In this study, 26% of the Salmonella spp isolates were confirmed to be ESBL producers, whereas 41% of the isolates were MRSA, 23% were VRSA, and 68% were Vancomycin intermediate Staphylococcus aureus (VISA). Hence, the Kunun-zaki could constitute a reservoir for the dissemination of multidrug-resistant foodborne pathogens if not correctly quality controlled.

Comparative Analysis of Virulence and Toxin Expression of Vancomycin-Intermediate and Vancomycin-Sensitive Staphylococcus aureus Strains.

Previous studies on vancomycin-intermediate Staphylococcus aureus (VISA) have mainly focused on drug resistance, the evolution of differences in virulence between VISA and vancomycin-sensitive S. aureus (VSSA) requires further investigation. To address this issue, in this study, we compared the virulence and toxin profiles of pair groups of VISA and VSSA strains, including a series of vancomycin-resistant induced S. aureus strains—SA0534, SA0534-V8, and SA0534-V16. We established a mouse skin infection model to evaluate the invasive capacity of VISA strains, and found that although mice infected with VISA had smaller-sized abscesses than those infected with VSSA, the abscesses persisted for a longer period (up to 9 days). Infection with VISA strains was associated with a lower mortality rate in Galleria mellonella larvae compared to infection with VSSA strains (≥ 40% vs. ≤ 3% survival at 28 h). Additionally, VISA were more effective in colonizing the nasal passage of mice than VSSA, and in vitro experiments showed that while VISA strains were less virulent they showed enhanced intracellular survival compared to VSSA strains. RNA sequencing of VISA strains revealed significant differences in the expression levels of the agr, hla, cap, spa, clfB, and sbi genes and suggested that platelet activation is only weakly induced by VISA. Collectively, our findings indicate that VISA is less virulent than VSSA but has a greater capacity to colonize human hosts and evade destruction by the host innate immune system, resulting in persistent and chronic S. aureus infection.

The combined antibacterial effects of sodium new houttuyfonate and berberine chloride against growing and persistent methicillin-resistant and vancomycin-intermediate Staphylococcus aureus

BackgroundInfections caused by drug-resistant Staphylococcus aureus, especially vancomycin-intermediate Staphylococcus aureus (VISA), leave clinicians with limited therapeutic options for treatment. Persister cells is a leading cause of recalcitrant infection and antibiotic treatment failure, and there is no drug in clinical use that specifically targets persister cells currently. Here, we report a promising combination therapy of sodium new houttuyfonate (SNH) and berberine chloride (BBR) which is able to eradicate both growing and persistent drug-resistant Staphylococcus aureus.ResultsThe susceptibility test showed SNH exhibited anti-MRSA activity with MIC90 at 64 μg/mL, while BBR showed weak anti-MRSA activity with MIC90 at 512 μg/mL. MICs of BBR in combination with 1/2 MIC SNH decreased by 4 to 64 folds compared with MICs of BBR alone. The results of time-killing assays revealed that the combined use of sub-MIC SNH and BBR offered an in vitro synergistic action against growing MRSA (including pathogenic MRSA) and VISA strains. More importantly, the combination of SNH and BBR was able to eradicate VISA Mu50 and pathogenic MRSA persister cells. The synergistic effect is likely related to the interruption of the cell membrane caused by SNH, which is confirmed by scanning electron microscope and membrane potential and permeability analysis.ConclusionsOur study provide a promising clinical curative strategy for combating drug-resistant S. aureus infections, especially for recalcitrant infections caused by persister cells.

In-depth characterization of antibacterial activity of melittin against Staphylococcus aureus and use in a model of non-surgical MRSA-infected skin wounds

Skin infections caused by methicillin-resistant Staphylococcus aureus (MRSA) require the development of new and effective topical antibiotics. In this context, melittin, the main component of apitoxin, has a potent antibacterial effect. However, little is known regarding the anti-inflammatory potential this peptide in infection models, or its ability to induce clinically important resistance. Here, we aimed to conduct an in-depth characterization of the antibacterial potential of melittin in vitro and evaluate the pharmaceutical potential of an ointment containing melittin for the treatment of non-surgical infections induced by MRSA. The minimum inhibitory concentration of melittin varied from 0.12 to 4 μM. The antibacterial effect was mainly bactericidal and fast (approximately 0.5 h after incubation) and was maintained even in stationary cells and mature MRSA biofilms. Melittin interacts synergistically with beta-lactams and aminoglycosides, and its ability to form pores in the membrane reverses the resistance of vancomycin-intermediate Staphylococcus aureus (VISA) to amoxicillin, and vancomycin. Its ability to induce resistance in vitro was absent, and melittin was stable in several conditions often associated with infected wounds. In vivo, aointment containing melittin reduced bacterial load and the content of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1 beta. Collectively, these data point to melittin as a potential candidate for topical formulations aimed at the treatment of non-surgical infections caused by MRSA.

New Antimicrobial Bioactivity against Multidrug-Resistant Gram-Positive Bacteria of Kinase Inhibitor IMD0354

Multidrug-resistant pathogens pose a serious threat to human health. For decades, the antibiotic vancomycin has been a potent option when treating Gram-positive multidrug-resistant infections. Nonetheless, in recent decades, we have begun to see an increase in vancomycin-resistant bacteria. Here, we show that the nuclear factor-kappa B (NF-κB) inhibitor N-[3,5-Bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD0354) was identified as a positive hit through a Caenorhabditis elegans–methicillin-resistant Staphylococcus aureus (MRSA) infection screen. IMD0354 was a potent bacteriostatic drug capable of working at a minimal inhibitory concentration (MIC) as low as 0.06 µg/mL against various vancomycin-resistant strains. Interestingly, IMD0354 showed no hemolytic activity at concentrations as high as 16 µg/mL and is minimally toxic to C. elegans in vivo with 90% survival up to 64 µg/mL. In addition, we demonstrated that IMD0354′s mechanism of action at high concentrations is membrane permeabilization. Lastly, we found that IMD0354 is able to inhibit vancomycin-resistant Staphylococcus aureus (VRSA) initial cell attachment and biofilm formation at sub-MIC levels and above. Our work highlights that the NF-κB inhibitor IMD0354 has promising potential as a lead compound and an antimicrobial therapeutic candidate capable of combating multidrug-resistant bacteria.

Superior antibacterial activity of reduced graphene oxide upon decoration with iron oxide nanorods

Iron oxide (Fe2O3) nanorods (NRs) decorated reduced graphene oxide (rGO) nanocomposite, endowed with high antibacterial activity, was prepared through the one-pot calcination of pre-synthesized Fe2O3 NRs in rGO aqueous dispersion. The Fe2O3 NRs were well infused between the rGO layers as revealed by the AFM, SEM, and TGA analysis, whereas the removal and distribution of oxygen-containing functional groups were distinguished with UV–vis and FT-IR spectroscopy. The antibacterial activity of Fe2O3/rGO nanocomposite towards methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and ciprofloxacin-resistant Staphylococcus aureus (CRSA) was investigated by disk diffusion method and growth curve analysis. The nanocomposite demonstrated excellent antibacterial activity as compared to bare rGO and inhibited nearly 87 % of the bacterial growth. The CRSA was more sensitive to nanocomposite compare MRSA and VRSA. More importantly, the nanocomposite obtained was found to achieve minimum inhibitory concentration (MIC) of ∼ 5 μg/mL. Besides, measurements of the charge transfer resistance, which is used to interpret bacterial response towards rGO or Fe2O3/rGO modified glassy carbon electrode, were performed using electrochemical impedance spectroscopy (EIS). The noteworthy antibacterial performance of the Fe2O3/rGO nanocomposite was probably attributed to the synergistic effect Fe2O3 NRs decorated rGO. The mechanism study suggested that nanocomposite can induce death to the bacteria by direct contact with the sharp edges of rGO and through the release of Fe3+ ions. The data, altogether, indicated that the Fe2O3 NRS decoration of rGO resulted in nanocomposite with enhanced antibacterial performance, hence, it may be used for developing new, safe, and effective antibiotics.

Characterization and in vitro Analysis of Probiotic-Derived Peptides Against Multi Drug Resistance Bacterial Infections.

An inexorable switch from antibiotics has become a major desideratum to overcome antibiotic resistance. Bacteriocin from Lactobacillus casei, a cardinal probiotic was used to design novel antibacterial peptides named as Probiotic Bacteriocin Derived and Modified (PBDM) peptides (PBDM1: YKWFAHLIKGLC and PBDM2: YKWFRHLIKKLC). The loop-shaped 3D structure of peptides was characterized in silico via molecular dynamics simulation as well as biophysically via spectroscopic methods. Thereafter, in vitro results against multidrug resistant bacterial strains and hospital samples demonstrated the strong antimicrobial activity of PBDM peptides. Further, in vivo studies with PBDM peptides showed downright recovery of balb/c mice from Vancomycin Resistant Staphylococcus aureus (VRSA) infection to its healthy condition. Thereafter, in vitro study with human epithelial cells showed no significant cytotoxic effects with high biocompatibility and good hemocompatibility. In conclusion, PBDM peptides displayed significant antibacterial activity against certain drug resistant bacteria which cause infections in human beings. Future analysis are required to unveil its mechanism of action in order to execute it as an alternative to antibiotics.

Global prevalence and distribution of vancomycin resistant, vancomycin intermediate and heterogeneously vancomycin intermediate Staphylococcus aureus clinical isolates: a systematic review and meta-analysis

Vancomycin-resistant Staphylococcus aureus (VRSA), Vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) are subject to vancomycin treatment failure. The aim of the present study was to determine their precise prevalence and investigate prevalence variability depending on different years and locations. Several international databases including Medline (PubMed), Embase and Web of Sciences were searched (data from 1997 to 2019) to identify studies that addressed the prevalence of VRSA, VISA and hVISA among human clinical isolates around the world. Subgroup analyses and meta-regression were conducted to indicate potential source of variation. Publication bias was assessed using Egger’s test. Statistical analyses were conducted using STATA software (version 14.0). Data analysis showed that VRSA, VISA and hVISA isolates were reported in 23, 50 and 82 studies, with an overall prevalence of 1.5% among 5855 S. aureus isolates, 1.7% among 22,277 strains and 4.6% among 47,721 strains, respectively. The overall prevalence of VRSA, VISA, and hVISA before 2010 was 1.2%, 1.2%, and 4%, respectively, while their prevalence after this year has reached 2.4%, 4.3%, and 5.3%. The results of this study showed that the frequency of VRSA, VISA and hVISA after 2010 represent a 2.0, 3.6 and 1.3-fold increase over prior years. In a subgroup analysis of different strain origins, the highest frequency of VRSA (3.6%) and hVISA (5.2%) was encountered in the USA while VISA (2.1%) was more prevalent in Asia. Meta-regression analysis showed significant increasing of VISA prevalence in recent years (p value ≤ 0.05). Based on the results of case reports (which were not included in the calculations mentioned above), the numbers of VRSA, VISA and hVISA isolates were 12, 24 and 14, respectively, among different continents. Since the prevalence of VRSA, VISA and hVISA has been increasing in recent years (especially in the Asian and American continents), rigorous monitoring of vancomycin treatment, it’s the therapeutic response and the definition of appropriate control guidelines depending on geographical regions is highly recommended and essential to prevent the further spread of vancomycin-resistant S. aureus.

Combinations of (lipo)glycopeptides with β-lactams against MRSA: susceptibility insights.

Increasing application of vancomycin due to the high prevalence of MRSA infections has led to the emergence of vancomycin intermediate-resistant Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA). Consequently, the need for alternative therapies that target MRSA has become evident. To evaluate the synergy between (lipo)glycopeptides (LGP/GPs) (vancomycin, teicoplanin, telavancin, dalbavancin and oritavancin) and β-lactams (ceftaroline, cefepime, cefazolin and oxacillin) against MRSA, hVISA, VISA and daptomycin non-susceptible (DNS) phenotypes. Twenty randomly selected clinical MRSA strains (i.e. 5 MRSA, 5 hVISA, 5 VISA and 5 DNS) were assessed versus LGP/GPs alone and LGP/GPs in combination with β-lactams for MICs. Although verification of antibiotic potency against bacterial strains is assessed by the microbroth dilution (MBD) MIC method recommended by the CLSI, some antibiotics need modified assay conditions in order to demonstrate their optimal activity. Addition of β-lactams reduced MIC values of LGP/GPs against all strains (up to 160-fold reduction). In general, LGPs (dalbavancin, oritavancin and telavancin) were more active (significant differences in MIC values, up to 8-fold) compared with vancomycin and teicoplanin. The majority of these combinations were bactericidal and superior to any single agent. This report has examined the susceptibility patterns of LGP/GPs and their combination with β-lactams. Of interest, the impact of susceptibility tests (in terms of MIC plates and their surface area) on the synergistic activity in 24 h time-kill experiments was apparent for LGPs. Further clinical research is required to investigate synergy with LGP/GPs and β-lactams against these Staphylococcus strains.

Single-nucleotide polymorphisms in a vancomycin-resistant Staphylococcus aureus strain based on whole-genome sequencing

The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) threatens global health. The mechanism of vancomycin resistance of VRSA without vanA gene acquisition was not fully elucidated. Therefore, we aimed to determine the mechanism of vancomycin resistance of VRSA besides that by vanA gene acquisition. In this study, we obtained vancomycin-resistant strains (V036-V64; MIC = 64 µg /ml) from susceptible strain (V036; MIC = 0.5 µg /ml) by exposure of vancomycin in vitro and examined the phenotypic characteristics and antibiotic susceptibility profiles of the resistant strain (V036-V64). To identify the genetic variations caused vancomycin resistance, we determined the complete genome sequences of V036 and V036-V64 and analyzed for single-nucleotide polymorphisms (SNPs) between two strains. Morphologically, V036-V64 had a twofold thicker cell wall compared with V036. Linezolid, rifampicin, and ceftaroline had similar MIC ranges against V036-V64 and V036, but V036-V64 showed lower susceptibilities to daptomycin and telavancin. We detected eight single-nucleotide polymorphisms differing between V036-V64 and V036: rimM (G16D), ssaA2 (G128A), rpsK (P60R), rpoB (R917C), walK (T492R), d-alanyl-d-alanine carboxypeptidase (L307I), vraT (A152V), and chromosome segregation ATPase (T440I). This study demonstrates that, under selective pressure, by the accumulation of mutations in genes related to cell wall synthesis, vancomycin-susceptible S. aureus can develop thicker cell walls and, hence, develop high vancomycin resistance. Thus, we highlight a novel vanA-negative mechanism for VRSA emergence.

Polyvalent Phage CoNShP-3 as a Natural Antimicrobial Agent Showing Lytic and Antibiofilm Activities against Antibiotic-Resistant Coagulase-Negative Staphylococci Strains.

Synthetic antimicrobials have a negative impact on food quality and consumer health, which is why natural antimicrobials are urgently needed. Coagulase-negative staphylococci (CoNS) has gained considerable importance for food poisoning and infection in humans and animals, particularly in biofilms. As a result, this study was conducted to control the CoNS isolated from food samples in Egypt. CoNS isolates were selected on the basis of their antibiotic susceptibility profiles and their biofilm-associated behavior. In this context, a total of 29 different bacteriophages were isolated and, in particular, lytic phages (6 isolates) were selected. The host range and physiological parameters of the lytic phages have been studied. Electron microscopy images showed that lytic phages were members of the families Myoviridae (CoNShP-1, CoNShP-3, and CoNSeP-2 isolates) and Siphoviridae (CoNShP-2, CoNSsP-1, and CoNSeP-1 isolates). CoNShP-1, CoNShP-2, and CoNShP-3 were found to be virulent to Staphylococcus haemolyticus, CoNSsP-1 to Staphylococcus saprophyticus and CoNSeP-1 and CoNSeP-2 to Staphylococcus epidermidis. Interestingly, the CoNShP-3 exhibited a typical polyvalent behavior, where not only lysis CoNS, but also other genera include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), Bacillus cereus and Bacillus subtilis. In addition, CoNShP-3 phage showed high stability at different temperatures and pH levels. Indeed, CoNShP-3 phage showed an antibiofilm effect against Staphylococcus epidermidis CFS79 and Staphylococcus haemolyticus CFS43, respectively, while Staphylococcus saprophyticus CFS28 biofilm was completely removed. Finally, CoNShP-3 phage demonstrated a high preservative efficacy over short and long periods of storage against inoculated CoNS in chicken breast sections. In conclusion, this study highlights the control of CoNS pathogens using a polyvalent lytic phage as a natural antibacterial and antibiofilm agent from a food safety perspective.

Citrus sinensis a potential solution against superbugs

The rate of drug resistance strains increased drastically over the past several decades, which forced researchers to develop new drugs to treat these pathogens. In the present study by the use of well diffusion method, the antimicrobial activity of Citrus sinensis and its synergism with chitosan modified compound (CMC) were checked. The multidrug resistant bacterial strains Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus (VRSA), Methicillin-resistant S. aureus (MRSA), Acinetobacter baumannii, VRSA and coagulase negative, and methicillin-resistant and vancomycin sensitive S. aureus were used against the different solvents of C. sinensis (seeds, leaves, stem, juice, fruit peel and C. sinensis peels) i.e. methanol and ethyl acetate to check its anti-MDR activity. Furthermore, methanolic and ethyl acetate extracts were then tagged with CMC and the results were compared. Out of two solvents, the highest activity was shown by ethyl acetate extracts followed by methanolic extracts and the formulation of the C. sinensis and CMC showed even more activity than simple extracts. Ethyl acetate extract of leaves of C. sinensis, tagged with CMC illustrates highest activity zone i.e. 53.3 mm against MDR E. coli while simple ethyl acetate C. sinensis leaf extract against A. baumannii showed the highest inhibition zone of 58.5 mm at the concentration of 100 mg/ml. Consequently, the C. sinensis could be a possible solution against superbugs.

Detection of mec A ,van A and van B genes of Staphylococcus aureus Isolated from Patients in Al Muthanna Province Hospitals

Background: Infections triggered by methicillin-resistant Staphylococcus aureus (MRSA) and onset Resistance to significant anxiety in healthcare environments worldwide from vancomycin-resistant Staphylococcus aureus (VRSA).. Aim:To discover Staphylococcus aureus resistant to methacillin and vancomycin by detecting (Mec A ,Van A and Van B) genes by PCR technique. Methods: A total of 250 samples from patients with different clinical cases whom admitted to Hospitals in Al Muthanna province during a period from January 2019 to July 2019. Staphylococcus aureus were isolated and identified by using cultural and biochemical testes.The extracted DNA of isolates were amplified by PCR to detect (mecA, vanA, and vanB) genes. Results: The results showed that 72/ 250 of Staphylococcus aureus isolates contained mecA gene, indicating that all isolates are Methicillin resistant S. aureus (MRSA), only five isolates contained van A gene and only nine isolates contained van B gene. Conclusion: Appropriate monitoring and control measures appear to be crucial to avoid the development and transmission of MRSA and VRSA strains in our nation.