Clinical Methicillin-resistant Staphylococcus Aureus Isolates Research Articles

Comparison of broth microdilution and E-test for susceptibility of MRSA to vancomycin

Introduction: Healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) developed soon after the introduction of methicillin. These bacteria have shown resistance to multiple drugs and therefore vancomycin became the antibiotic of choice for treatment of MRSA infections. Vancomycin is a bactericidal antibiotic that acts by inhibiting bacterial cell wall synthesis. This study aimed to compare broth microdilution (BMD) and E-test in determining vancomycin minimum inhibitory concentration (MIC) against MRSA.Methods: A total of 30 clinical isolates of MRSA were acquired from Colombo South Teaching Hospital, Sri Lanka. These MRSA strains were identified by cefoxitin disk diffusion and the vancomycin MIC was determined through BMD and E-test. The Kruskal-Wallis test was applied to test if the samples originated from the same distribution with post hoc determination of the correlation between methods using the Mann-Whitney U test.Results: All 30 MRSA isolates were 100% vancomycin susceptible (≤2 μg/mL) irrespective of methodology, according to the Clinical and Laboratory Standards Institute (CLSI) established breakpoints. However, the E-test MIC values were 1 to 2 dilutions higher than those of BMD. A statistically significant difference between vancomycin MICs of BMD and E-test (p = <0.00001) was calculated which indicated a difference in accuracy.Conclusion: Due to cost, convenience, and the ability to detect vancomycin intermediate Staphylococcus aureus (VISA), exploring the possibility of using E-test as an alternative to BMD is worthwhile.

Antimicrobial and Antibiofilm Activity of Highly Soluble Polypyrrole Against Methicillin-Resistant Staphylococcus aureus.

As a new promising therapeutic option, the activity of highly soluble polypyrrole (Hs-PPy), a specific formulation of a conducting polymer (polypyrrole - PPy) is evaluated against bacteria of clinical importance. The purpose was to evaluate the antimicrobial activity of Hs-PPy, alone or combined with oxacillin, as well as its antibiofilm potential against methicillin-resistant Staphylococcus aureus strains. Furthermore, the in silico inhibitory mechanism in efflux pumps was also investigated. 10 clinical isolates of MRSA and two reference strains were used. Antimicrobial activity was determined by broth microdilution, and the combination effect with oxacillin was evaluated by the checkerboard assay. The biofilm formation capacity of MRSA and the interference of Hs-PPy were evaluated. The inhibitory action of Hs-PPy on the efflux pump was evaluated in silico through molecular docking. Hs-PPy showed activity against the isolates, with inhibitory action between 62.5 and 125 μg/mL and bactericidal action at 62.5 μg/mL, as well as synergism in association with oxacillin. The isolates ranged from moderate to strong biofilm producers, and Hs-PPy interfered with the formation of this structure, but not with mature biofilm. There was no in silico interaction with the efflux protein EmrD, the closest homolog to NorA. Hs-PPy interferes with biofilm formation by MRSA, has synergistic potential, and is an efflux pump inhibitor.

Development of Membrane-Targeting Fluorescent 2-Phenyl-1H-phenanthro[9,10-d]imidazole-Antimicrobial Peptide Mimic Conjugates against Methicillin-Resistant Staphylococcus aureus.

The escalation of multidrug-resistant bacterial infections, especially infections caused by methicillin-resistant Staphylococcus aureus (MRSA), underscores the urgent need for novel antimicrobial drugs. Here, we synthesized a series of amphiphilic 2-phenyl-1H-phenanthro[9,10-d]imidazole-antimicrobial peptide (AMP) mimic conjugates (III1-30). Among them, compound III13 exhibited excellent antibacterial activity against G+ bacteria and clinical MRSA isolates (MIC = 0.5-2 μg/mL), high membrane selectivity, and low toxicity. Additionally, compared with traditional clinical antibiotics, III13 demonstrated rapid bactericidal efficacy and was less susceptible to causing bacterial resistance. Mechanistic studies revealed that III13 targets phosphatidylglycerol (PG) on bacterial membranes to disrupt membrane integrity, leading to an increase in intracellular ROS and leakage of proteins and DNA, ultimately causing bacterial cell death. Furthermore, III13 possessed good fluorescence properties with potential for further dynamic monitoring of the antimicrobial process. Notably, III13 showed better in vivo efficacy against MRSA compared to vancomycin, suggesting its potential as a promising candidate for anti-MRSA medication.

Subunit 76-kDa surface protein of Methicillin-Resistant Staphylococcus aureus (MRSA) is potentially useful for MRSA diagnostic tool

ObjectiveTo evaluate the diagnostic performance of the polyclonal antibody generated from the subunit surface protein of MRSA for MRSA detection. MethodsThe MRSA clinical isolates were identified by the cefoxitin disc diffusion test and confirmed by mecA PCR. The surface protein from the clinical isolates of MRSA was extracted and characterized with hemagglutination and adherence inhibition assays. Polyclonal antibody against the selected protein was produced in mice and then used for Western blot experiments. ResultsFour conserved surface protein bands (63, 76, 88, and 114-kDa) were found in each MRSA clinical isolate. Hemagglutination reaction was demonstrated by the subunit 76 and 114-kDa surface protein of MRSA at 1:32 dilution. Such proteins were identified as adhesive molecules in the enterocytes. The sensitivity and specificity of the polyclonal 76-kDa antibody in detecting MRSA were 94.59% and 85.14%, respectively, with the Kappa values fall under the interpretation of substantial agreement (0.752) with the gold standard, suggesting it is useful for MRSA detection. ConclusionSubunit 76 and 114-kDa surface proteins of MRSA exhibit adhesive properties in mediating MRSA infection. The polyclonal antibody of 76-kDa generated from the surface protein of MRSA could be used as an alternative for the identification of clinical isolates suspected with MRSA infection.

Detection of Novel spa Types in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Iraq

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive bacterium that is an opportunistic pathogen, causing infections in hospital settings and communities. MRSA has become a significant and increasing problem in Iraq. The aim of this study was to evaluate the genetic mutations of MRSA strains, especially in the spa gene, isolated from patients in Wassit, Iraq.Methods and Results: Biochemical tests were conducted to identify S. aureus isolates and then on MRSA. The MRSA was identified by the Chrome Agar method and confirmed by PCR with genotyping of the mecA gene. The disk diffusion method was used to detect antibiotic resistance to three different common antibiotics used at Wassit hospitals. The Vitek-2 compact system was utilized for the detection of the minimum inhibitory concentration (MIC) of vancomycin. All MRSA strains in this study were tested to screen the mecA gene, with 21 strains subjected to the molecular typing method for the spa gene. Out of 166 samples, 132(79.5%) contained S. aureus and 34(20.4%) were identified as MRSA. Genotyping showed that out of 34 MRSA, 31(91.2%) isolates were mecA-positive. The spa gene was detected in 21(61.8%) isolates out of 34 MRSA samples. The spa typing of 21 MRSA samples revealed four different spa types, as follows: t386 (3/14.3%), t3576 (1/4.8%), t10002 (1/4.8%), and t10234 (1/4.8%). High polymorphism rates were shown in isolates of spa type t386. Conclusion: Our data represent the first report to detect novel mutations in the spa gene in the MRSA clinical isolates from Wassit hospitals, Iraq.

Novel membrane-targeting isoxanthohumol-amine conjugates for combating methicillin-resistant Staphylococcus aureus (MRSA) infections

Methicillin-resistant Staphylococcus aureus (MRSA) is a widespread pathogen causing clinical infections and is multi-resistant to many antibiotics, making it urgent need to develop novel antibacterials to combat MRSA. Here, a series of novel isoxanthohumol-amine conjugates were synthesized as antibacterials. After bioactivity evaluation, a compound E2 was obtained, which showed excellent antibacterial activity against S. aureus and clinical MRSA isolates (MICs = 0.25−1 μg/mL), superior to vancomycin, and with negligible hemolysis and good membrane selectivity. Additionally, E2 exhibited fast bacterial killing, less susceptible to resistance, relatively low cytotoxicity, and good plasma stability. Mechanism investigation revealed that E2 can disrupt bacterial membranes by specifically binding to phosphatidylglycerol on the bacterial membrane, thus causing elevated intracellular ROS and leakage of DNA and proteins, and ultimately killing bacteria. Noticeably, E2 displayed a good in vivo safety profile and better in vivo therapeutic efficacy than the same dose of vancomycin, allowing it to be a potential antibacterial to conquer MRSA infections.

Sensitizing methicillin-resistant Staphylococcus aureus (MRSA) to cefuroxime: the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most β-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO3-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care β-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such "NaHCO3-responsive" isolates can be effectively cleared by β-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3-responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting β-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific β-lactam agents.

Whole genome sequencing of methicillin-resistant Staphylococcus aureus clinical isolates from Terengganu, Malaysia, indicates the predominance of the EMRSA-15 (ST22-SCCmec IV) clone

Despite the importance of methicillin-resistant Staphylococcus aureus (MRSA) as a priority nosocomial pathogen, the genome sequences of Malaysian MRSA isolates are currently limited to a small pool of samples. Here, we present the genome sequence analyses of 88 clinical MRSA isolates obtained from the main tertiary hospital in Terengganu, Malaysia in 2016–2020, to obtain in-depth insights into their characteristics. The EMRSA-15 (ST22-SCCmec IV) clone of the clonal complex 22 (CC22) lineage was predominant with a total of 61 (69.3%) isolates. Earlier reports from other Malaysian hospitals indicated the predominance of the ST239 clone, but only two (2.3%) isolates were identified in this study. Two Indian-origin clones, the Bengal Bay clone ST772-SCCmec V (n = 2) and ST672 (n = 10) were also detected, with most of the ST672 isolates obtained in 2020 (n = 7). Two new STs were found, with one isolate each, and were designated ST7879 and ST7883. From the core genome phylogenetic tree, the HSNZ MRSA isolates could be grouped into seven clades. Antimicrobial phenotype-genotype concordance was high (> 95%), indicating the accuracy of WGS in predicting most resistances. Majority of the MRSA isolates were found to harbor more than 10 virulence genes, demonstrating their pathogenic nature.

Bacterial Adaptive Memory in Methicillin-Resistant Staphylococcus aureus from Endotracheal Tubes.

To evaluate the expression dynamics of biofilm genes in methicillin-resistant Staphylococcus aureus (MRSA) retrieved from endotracheal tubes (ETT) and to determine how gene regulation is attenuated in vitro where host-environmental factors are no longer present. Biofilm was grown (24 h) in tryptic broth soy plus 0.25% glucose for a clinical MRSA isolate in planktonic state and after sessile growth named ETT-MRSA (S2, S3, S4, S5, S6, S7). Gene expression of five biofilm-related genes (icaC, clfB, ebps, fnbB, and RNA III) was assessed consecutively from day 1 to day 4 after ETT growth through real-time PCR. 16S rRNA was used as a control. The MRSA isolates retrieved from ETT were capable of producing biofilms dependent on ica. The gene expression dynamics of ETT-MRSA changed progressively compared to planktonic MRSA gene expression under both ambient air (p < 0.001) and ambient air with 5% CO2 (p < 0.001). Dynamic assessment of icaC expression in both atmospheric conditions showed progressive downregulation in vitro compared to in vivo ETT biofilms. The expression patterns of clfB and ebps genes were similar to icaC. In contrast, the expression of the RNA III gene showed progressive upregulation from day 1 to day 4 (p < 0.001). MRSA loses its biofilm gene expression in vitro, by adaptive features across multiple generations, as evidenced by the progressive downregulation of icaC and upregulation of RNA III. These findings underscore the significance of host-environment dependence in regulating bacterial biofilm genes, highlighting its importance in diagnostics. Bacterial strains lose their host-specific characteristics as they are cultured in vitro.

Synergistic Activity of 3-Hydrazinoquinoxaline-2-Thiol in Combination with Penicillin Against MRSA.

The growing resistance seen in various antibiotics, including those considered as last-resort options, underscores the pressing need for novel approaches and new substances to address MRSA infections. Combining antibiotics as a treatment approach can enhance effectiveness, expand the range of targeted bacteria, and minimize the likelihood of resistance emergence. This approach holds promise in addressing the escalating issue of antibiotic resistance. This study seeks to investigate the potential synergy between 3-hydrazinoquinoxaline-2-thiol and penicillin against a diverse array of MRSA isolates, thereby providing insights into their combined antimicrobial action. Twenty-two clinical MRSA isolates subjected to broth microdilution to determine the Minimum Inhibitory Concentrations (MICs) of 3-hydrazinoquinoxaline-2-thiol and penicillin. Subsequently, a checkerboard assay was employed to evaluate the interaction between 3-hydrazinoquinoxaline-2-thiol and penicillin, focusing on the Fractional Inhibitory Concentration Index (FICI). The MICs of penicillin and 3-hydrazinoquinoxaline-2-thiol were determined for 22 clinical MRSA strains. Penicillin exhibited MICs within a range of 1024 to 128 µg/mL, while 3-hydrazinoquinoxaline-2-thiol displayed MICs varying from 64 to 8 µg/mL. Remarkably, the combination of 3-hydrazinoquinoxaline-2-thiol and penicillin yielded a synergistic effect, resulting in a significant reduction of MICs by up to 64-fold. The potential of 3-hydrazinoquinoxaline-2-thiol in combination with penicillin as a viable solution against MRSA appears promising. However, to establish its practical utility, further extensive testing and experiments are essential.

Green synthesis of tryptanthrin appended 4-spiropiperidines and their anti-bacterial evaluation

Synthesis of 14 tryptanthrin appended 4-spiropiperidines and their detailed antibacterial evaluation are described. The synthesis was achieved by a green route employing ammonium acetate as a catalyst in the three-component reaction of tryptanthrin, malononitrile and m-phenylene diamine. The in vitro antibacterial activities of compounds were evaluated against ESKAPE pathogens and against clinical multidrug-resistant MRSA and VRSA isolates, from which the bromo- and chloro-substituted spiropiperidines 4b and 4c (MIC 4 μg/mL) was found to be most potent against S. aureus ATCC 29213 and 4c exhibited good selectivity index.

Investigation of Antimicrobial, Antioxidant, and Cytotoxic Activities of Boesenbergia rotunda rhizome extract

In recent years, antimicrobial resistance (AMR) has become a global threat to public health. In Hawai’i, there is an increasing rate of staph infections of methicillin-resistant Staphylococcus aureus (MRSA), and, hence, a need for new agents to combat the increase of AMR bacteria. This study evaluates the antimicrobial, antioxidant, and cytotoxic activities of Hawaiian-grown Boesenbergia rotunda. Test bacteria included clinical isolates of Gram-positive MRSA and methicillin-susceptible S. aureus (MSSA) as well as Gram-negative Serratia marcescens and Escherichia coli. Five compounds–cardamonin, pinostrobin, pinocembrin, pinostrobin chalcone, and isopanduratin A–were isolated from the ethyl acetate extract of B. rotunda rhizome, and their structures were identified by NMR spectroscopy. These samples exhibited antimicrobial activity against MRSA and MSSA strains, with minimum inhibitory concentration (MIC) values between 128 and 2 µg/mL, with isopanduratin A giving MIC values as low as 2 µg/mL. The antioxidant potential of samples was examined using ferric-reducing antioxidant power (FRAP) assay. At 1 mg/mL of tested samples, FRAP values ranged between 8.74 to 17.76 µM/µg, with pinostrobin chalcone exhibiting the highest FRAP value (17.76 ± 0.65 μM/μg). Moreover, cytotoxicity was measured via sulforhodamine B (SRB) assay. Cardamonin (IC50 of 19.43 ± 0.33 μM) and isopanduratin A (IC50 of 26.84 ± 1.06 μM) exhibited effectiveness against the lung cancer cell line A549. Compounds from B. rotunda showed potent antimicrobial effect against MRSA and MSSA strains as well as antioxidant and cytotoxic activities, and may have the potential for further evaluation and development for pharmaceutical applications.

Synergistic Potential of α-Melanocyte Stimulating Hormone Based Analogues with Conventional Antibiotic against Planktonic, Biofilm-Embedded, and Systemic Infection Model of MRSA.

The repotentiation of the existing antibiotics by exploiting the combinatorial potential of antimicrobial peptides (AMPs) with them is a promising approach to address the challenges of slow antibiotic development and rising antimicrobial resistance. In the current study, we explored the ability of lead second generation Ana-peptides viz. Ana-9 and Ana-10, derived from Alpha-Melanocyte Stimulating Hormone (α-MSH), to act synergistically with different classes of conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). The peptides exhibited prominent synergy with β-lactam antibiotics, namely, oxacillin, ampicillin, and cephalothin, against planktonic MRSA. Furthermore, the lead combination of Ana-9/Ana-10 with oxacillin provided synergistic activity against clinical MRSA isolates. Though the treatment of MRSA is complicated by biofilms, the lead combinations successfully inhibited biofilm formation and also demonstrated biofilm disruption potential. Encouragingly, the peptides alone and in combination were able to elicit in vivo anti-MRSA activity and reduce the bacterial load in the liver and kidney of immune-compromised mice. Importantly, the presence of Ana-peptides at sub-MIC doses slowed the resistance development against oxacillin in MRSA cells. Thus, this study highlights the synergistic activity of Ana-peptides with oxacillin advocating for the potential of Ana-peptides as an alternative therapeutic and could pave the way for the reintroduction of less potent conventional antibiotics into clinical use against MRSA infections.

Effect of Resveratrol and Curcumin on Gene Expression of Methicillin-Resistant Staphylococcus aureus (MRSA) Toxins.

Staphylococcus aureus is an opportunistic pathogen that can lead to a number of potentially terrible community- and hospital-acquired illnesses. Among the diverse set of virulence factors that S. aureus possesses, secreted toxins play a particularly preeminent role in defining its virulence. In this work, we aimed to facilitate the development of novel strategies utilizing natural compounds to lower S. aureus's toxin production and consequently enhance therapeutic approaches. Two natural polyphenols, resveratrol (RSV) and curcumin (CUR), were tested for their effect on reducing toxin gene production of MRSA isolates. Fifty clinical MRSA isolates were gathered from Riyadh and Jeddah. Molecular screening of toxin genes (sea, seb, sec, sed, seh, lukF, and lukS) harbored by MRSA was performed. Sub-inhibitory concentrations of RSV (50 μg/ml) and CUR (20 μg/ml) were determined to study their effect on the gene expression MRSA's toxin genes. Our findings revealed the presence of the tested genes in MRSA isolates, with lukF being the most prevalent gene and seh the least detected gene. We found that RSV reduced the relative expression of toxin genes, sea, seb, lukF, and lukS, respectively, while CUR decreased the relative expression of sea and seb genes in the examined isolates. Regarding lukF and lukS, CUR downregulated the expression of both genes in some isolates and upregulated the expression in other isolates. From these results, we concluded that RSV and CUR could be used as alternative therapeutic approaches to treat MRSA infections through reducing toxin production.

Profiling daptomycin resistance among diverse methicillin-resistant Staphylococcus aureus lineages in China.

Daptomycin (DAP) is effective against methicillin-resistant Staphylococcus aureus (MRSA). However, reduced susceptibility to DAP in MRSA may lead to treatment failures. We aim to determine the distribution of DAP minimum inhibitory concentrations (MICs) and DAP heteroresistance (hDAP) among MRSA lineages in China. A total of 472 clinical MRSA isolates collected from 2015 to 2017 in China were examined for DAP susceptibility. All isolates (n = 472) were found to be DAP susceptible, but 35.17% (166/472) of them exhibited a high DAP MIC (MIC >0.5 µg/mL). The high DAP MIC group contained a larger proportion of isolates with a higher vancomycin or teicoplanin MIC (>1.5 µg/mL) than the low DAP MIC group (19.3% vs 7.8%, P < 0.001; 22.3% vs 8.2%, P < 0.001). We compared the clonal complex (CC) distributions and clinical characteristics in MRSA isolates stratified by DAP MIC. CC5 isolates were less susceptible to DAP (MIC50 = 1 µg/mL) than CC59 isolates (MIC50 = 0.5 µg/mL, P < 0.001). Population analysis profiling revealed that 5 of 10 ST5 and ST59 DAP-susceptible MRSA isolates investigated exhibited hDAP. The results also showed that CC5 MRSA with an agrA mutation (I238K) had a higher DAP MIC than those with a wild-type agrA (P < 0.001). The agrA-I238K mutation was found to be associated with agr dysfunction as indicated by the loss of δ-hemolysin production. In addition, agr/psmα defectiveness was associated with hDAP in MRSA. Whole-genome sequencing analysis revealed mutations in mprF and walR/walK in DAP-resistant subpopulations, and most DAP-resistant subpopulations (6/8, 75%) were stable. Our study suggests that the increased DAP resistance and hDAP in MRSA may threaten the effectiveness against MRSA infections.

Antibacterial and Disinfecting Effects of Standardised Tea Extracts on More than 100 Clinical Isolates of Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are still a major problem in hospitals. The excellent safety profile, accessibility and anti-infective activity of tea extracts make them promising agents for the treatment of infected wounds. To investigate the possibility of sterilising MRSA-infected surfaces, including skin with tea extracts, we determined the MICs for different extracts from green and black tea (Camellia sinensis), including epigallocatechin gallate (EGCG), on a large number of clinical isolates of MRSA, selected to represent a high genetic diversity. The extracts were prepared to achieve the maximal extraction of EGCG from tea and were used as stable lyophilisate with a defined EGCG content. All extracts showed a complete inhibition of cell growth at a concentration of approx. 80 µg/mL of EGCG after a contact time of 24 h. Time-kill plots were recorded for the extract with the highest amount of EGCG. The reduction factor (RF) was 5 after a contact time of 240 min. EGCG and tea extracts showed an RF of 2 in methicillin-sensitive S. aureus. Extracts from green and black tea showed lower MICs than an aqueous solution with the same concentration of pure EGCG. To the best of our knowledge, we are the first to show a reduction of 99.999% of clinically isolated MRSA by green tea extract within 4 h.

LYZ2-SH3b as a novel and efficient enzybiotic against methicillin-resistant Staphylococcus aureus

BackgroundEnzybiotics are promising alternatives to conventional antibiotics for drug-resistant infections. Exolysins, as a class of enzybiotics, show antibacterial effects against methicillin-resistant Staphylococcus aureus (MRSA). This study evaluated a novel exolysin containing an SH3b domain for its antibacterial activity against MRSA.MethodsThis study designed a chimeric exolysin by fusing the Cell-binding domain (SH3b) from Lysostaphin with the lytic domain (LYZ2) from the gp61 enzyme. Subsequently, LYZ2-SH3b was cloned and expressed in Escherichia coli (E. coli). Finally, the antibacterial effects of LYZ2-SH3b compared with LYZ2 and vancomycin against reference and clinical isolates of MRSA were measured using the disc diffusion method, the minimal inhibitory concentration (MIC), and the minimal bactericidal concentration (MBC) assays.ResultsAnalysis of bioinformatics showed that LYZ2-SH3b was stable, soluble, and non-allergenic. Protein purification was performed with a 0.8 mg/ml yield for LYZ2-SH3b. The plate lysis assay results indicated that, at the same concentrations, LYZ2-SH3b has a more inhibitory effect than LYZ2. The MICs of LYZ2 were 4 µg/mL (ATCC 43,300) and 8 µg/mL (clinical isolate ST239), whereas, for LYZ2-SH3b, they were 2 µg/mL (ATCC 43,300) and 4 µg/mL (clinical isolate ST239). This suggests a higher efficiency of LYZ2-SH3b compared to LYZ2. Furthermore, the MBCs of LYZ2 were 4 µg/mL (ATCC 43,300) and 8 µg/mL (clinical isolate ST239), whereas, for LYZ2-SH3b, they were 2 µg/mL (ATCC 43,300) and 4 µg/mL (clinical isolate ST239), thus confirming the superior lytic activity of LYZ2-SH3b over LYZ2.ConclusionsThe study suggests that phage endolysins, such as LYZ2-SH3b, may represent a promising new approach to treating MRSA infections, particularly in cases where antibiotic resistance is a concern. But further studies are needed.

Molecular characterization of typing and subtyping of Staphylococcal cassette chromosome SCCmec types I to V in methicillin-resistant Staphylococcus aureus from clinical isolates from COVID-19 patients.

Methicillin resistance is acquired by the bacterium due to mecA gene which codes for penicillin-binding protein (PBP2a) having low affinity for β-lactam antibiotics. mecA gene is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). SCCmec genomic island comprises two site-specific recombinase genes namely ccrA and ccrB [cassette chromosome recombinase] accountable for mobility. Currently, SCCmec elements are classified into types I, II, III, IV and V based on the nature of the mec and ccr gene complexes and are further classified into subtypes according to variances in their J region DNA. SSCmec type IV has been found in community-acquired isolates with various genetic backgrounds. The present study was undertaken to categorize the types of SCCmec types and subtypes I, II, III, IVa, b, c, d, and V and PVL genes among clinical MRSA isolates from COVID-19 confirmed cases. Based on the Microbiological and Molecular (mecA gene PCR amplification) confirmation of MRSA isolated from 500 MRSA SCCmec clinical samples, 144 cultures were selected for multiplex analysis. The multiplex PCR method developed by Zhang et al. was adapted with some experimental alterations to determine the specific type of these isolates. Of the total 500 MRSA, 144 MRSA (60 were CA-MRSA and 84 were HA-MRSA) were selected for characterization of novel multiplex PCR assay for SSCmec Types I to V in MRSA. Molecular characterization of multiplex PCR analysis revealed results compare to the phenotypic results. Of the 60 CA-MRSA; in 56 MRSA strains type IVa was found and significantly defined as CA-MRSA while 4 strains showed mixed gens subtypes. Type II, III, IA, and V were present in overall 84 HA-MRSA. Molecular subtyping was significantly correlated to define molecularly as CA-MRSA and HA-MRSA however 15 (10%) strains showed mixed genes which indicates the alarming finding of changing epidemiology of CA-MRSA and HA-MRSA as well. We have all witnessed of COVID-19 pandemic, and its mortality was mostly associated with co-morbid conditions and secondary infections of MDR pathogens. Rapid detections of causative agents of these superbugs with their changing epidemiology by investing in typing and subtyping clones are obligatory. We have described an assay designed for targeting SSCmec types and subtypes I, II, III, IVa,V according to the current updated SCCmec typing system. Changing patterns of molecular epidemiology has been observed by this newly described assay.

Repurposing sunscreen as an antibiotic: zinc-activated avobenzone inhibits methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major healthcare concern with associated healthcare costs reaching over ${\$}$1 billion in a single year in the USA. Antibiotic resistance in S. aureus is now observed against last line of defense antibiotics, such as vancomycin, linezolid, and daptomycin. Unfortunately, high throughput drug discovery approaches to identify new antibiotics effective against MRSA have not resulted in much tangible success over the last decades. Previously, we demonstrated the feasibility of an alternative drug discovery approach, the identification of metallo-antibiotics, compounds that gain antibacterial activity only after binding to a transition metal ion and as such are unlikely to be detected in standard drug screens. We now report that avobenzone, the primary active ingredient of most sunscreens, can be activated by zinc to become a potent antibacterial compound against MRSA. Zinc-activated avobenzone (AVB-Zn) potently inhibited a series of clinical MRSA isolates [minimal inhibitory concentration (MIC): 0.62-2.5 µM], without pre-existing resistance and activity without zinc (MIC: >10 µM). AVB-Zn was also active against clinical MRSA isolates that were resistant against the commonly used zinc-salt antibiotic bacitracin. We found AVB-Zn exerted no cytotoxicity on human cell lines and primary cells. Last, we demonstrate AVB-Zn can be deployed therapeutically as lotion preparations, which showed efficacy in a mouse wound model of MRSA infection. AVB-Zn thus demonstrates Zn-activated metallo-antibiotics are a promising avenue for future drug discovery.

HptA Mutation May Mediate Fosfomycin Resistance in Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

Fosfomycin can be used alone or in combination to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. However, fosfomycin resistance has been observed in MRSA. In S. aureus, fosfomycin resistance is mediated by the fosfomycin-modifying enzyme FosB, or mutations in the target enzyme MurA. Mutations in the chromosomal glpT and uhpT genes, which encode fosfomycin transporters, also result in fosfomycin resistance. The three-component regulatory system HptRSA mediates the expression of uhpT and glpT in S. aureus. This study aimed to investigate the role of hptRSA mutation in fosfomycin resistance in MRSA clinical isolates. We found that hptRSA mutations were common in MRSA strains isolated from our hospital. Most mutations were amino acid substitutions and widely distributed in fosfomycin-sensitive and fosfomycin-resistant strains. However, HptA-truncated mutations were only found in fosB-negative fosfomycin-resistant strains with wild-type uhpT and glpT genes. Quantitative real-time PCR results showed that the transcription level of uhpT decreased by 13.7-25.6-fold in the HptA-truncated strains. Concordantly, the fosfomycin minimum inhibitory concentration (MIC) of HptA-truncated strains was 64-128 μg/mL, while SA240 was 2 μg/mL. The low transcription level of uhpT and high increase in MIC suggest that hptA mutation may lead to fosfomycin resistance in MRSA. We complemented hptA in one of the HptA-truncated clinical strains (SA179), showing reversal of fosfomycin resistance (from 128 to 32 μg/mL). Then we knocked out hptA in S. aureus Newman; fosfomycin MIC increased from 4 to 64 μg/mL, suggesting that HptA mutation may play an important role in fosfomycin resistance.