Comparative effectiveness of empirical antibiotic treatments in methicillin-susceptible Staphylococcus aureus infective endocarditis: A post hoc analysis of a prospective French cohort study.

OBJECTIVES: The empirical treatment of infective endocarditis is still debated. The aim of this study was to compare the impact of empirical treatment with antistaphylococcal penicillin (ASP) or cefazolin vs. other treatments in methicillin-susceptible Staphylococcus aureus (MSSA) endocarditis. METHODS: A post hoc analysis of a prospective cohort study of patients hospitalized in a French reference centre with MSSA endocarditis was conducted between 2013 and 2022. The primary outcome was the duration of bacteraemia under treatment. RESULTS: Of the 208 patients included, 101 patients (48.6%) were classified in the reference group (ASP or cefazolin) and 107 (52.4%) in the non-reference group. Empirical treatment with ASP/cefazolin was associated with a shorter duration of bacteraemia compared to other treatments (3.6 d vs. 4.6 d, P = 0.01). This difference was not corrected by the addition of an aminoglycoside (3.6 d vs. 4.7 d, P < 0.01). In multivariate analysis, empirical treatment with ASP/cefazolin was associated with a duration of bacteraemia ≤72 h (P = 0.02), whereas endocarditis on native valves (P = 0.01), and intracardiac abscess were associated with longer duration of bacteraemia (P = 0.01). CONCLUSIONS: Empirical treatment of endocarditis with ASP or Cefazolin is more effective than other treatments in MSSA endocarditis, even when the other treatments are combined with aminoglycosides.

Machine Learning-Based Virtual Screening of Antibacterial Agents against Methicillin-Susceptible and Resistant Staphylococcus aureus.

The application of computer-aided drug discovery (CADD) approaches has enabled the discovery of new antimicrobial therapeutic agents in the past. The high prevalence of methicillin-resistantStaphylococcus aureus(MRSA) strains promoted this pathogen to a high-priority pathogen for drug development. In this sense, modern CADD techniques can be valuable tools for the search for new antimicrobial agents. We employed a combination of a series of machine learning (ML) techniques to select and evaluate potential compounds with antibacterial activity against methicillin-susceptible S. aureus (MSSA) and MRSA strains. In the present study, we describe the antibacterial activity of six compounds against MSSA and MRSA reference (American Type Culture Collection (ATCC)) strains as well as two clinical strains of MRSA. These compounds showed minimal inhibitory concentrations (MIC) in the range from 12.5 to 200 µM against the different bacterial strains evaluated. Our results constitute relevant proven ML-workflow models to distinctively screen for novel MRSA antibiotics.

Ciprofloxacin-, Cefazolin-, and Methicilin-Soaked Graphene Paper as an Antibacterial Medium Suppressing Cell Growth.

This paper presents the results of research on the impact of graphene paper on selected bacterial strains. Graphene oxide, from which graphene paper is made, has mainly bacteriostatic properties. Therefore, the main goal of this research was to determine the possibility of using graphene paper as a carrier of a medicinal substance. Studies of the degree of bacterial inhibition were performed on Staphylococcus aureus and Pseudomonas aeruginosa strains. Graphene paper was analyzed not only in the state of delivery but also after the incorporation of the antibiotics ciprofloxacin, cefazolin, and methicillin into its structures. In addition, Fourier-Transform Infrared Spectroscopy, contact angle, and microscopic analysis of bacteria on the surface of the examined graphene paper samples were also performed. Studies have shown that graphene paper with built-in ciprofloxacin had a bactericidal effect on the strains of Staphylococcus aureus and Pseudomonas aeruginosa. In contrast, methicillin, as well as cefazolin, deposited on graphene paper acted mainly locally. Studies have shown that graphene paper can be used as a carrier of selected medicinal substances.

Levofloxacin susceptibility of Staphylococci from conjunctiva in patients with atopic dermatitis.

PURPOSE: The ocular surface in patients with atopic dermatitis (AD) is known to harbor an abundance of gram-positive cocci, particularly Staphylococcus aureus (S. aureus). This study reviewed the results of microbial cultures from the conjunctiva in AD patients, with special attention to the levofloxacin susceptibility of Staphylococci. STUDY DESIGN: Retrospective, single-center study. METHODS: This study involved 131 eyes of 112 Japanese patients with AD (87 men and 25 women, mean age: 40.4 ± 12.2 years) who underwent ocular surgery at Kyorin University Hospital. Bacterial isolates were collected from the conjunctival sacs in the preoperative period. Drug resistance to methicillin and levofloxacin was judged using the minimal inhibitory concentrations of oxacillin and levofloxacin determined by the broth dilution method. RESULTS: One hundred and fifty-seven strains were identified in 103 of the 131 eyes examined. S. aureus was isolated from 74 eyes (56.5%), followed by Staphylococcus epidermidis (S. epidermidis). In S. aureus, 11 strains (14.9%) were methicillin-resistant, and 18 (24.3%) were levofloxacin-resistant. In S. epidermidis, 15 strains (26.8%) were methicillin-resistant, and 17 (30.4%) were levofloxacin-resistant. No significant differences were observed in levofloxacin susceptibility with age, sex, previous ocular surgery, or duration of previous surgery. However, logistic multivariate analysis revealed that levofloxacin-resistant Staphylococci were concurrently resistant to methicillin, suggesting multidrug resistance. CONCLUSION: Distinctive bacterial distribution and drug resistance need consideration in the managing of ocular disorders among patients with AD.

In vitro evaluation of bactericidal effects of fluorescent light energy on Staphylococcus pseudintermedius and S. aureus.

BACKGROUND: Staphylococcus pseudintermedius and S. aureus are bacterial species of importance in veterinary medicine. The increasing incidence of antibiotic resistance necessitates the implementation of novel treatment modalities. Fluorescent light energy (FLE) is used as an adjunctive and primary treatment for canine pyoderma. However, no in vitro studies exist investigating its bactericidal effects against S. pseudintermedius or S. aureus. OBJECTIVES: To determine the bactericidal effects of FLE on S. pseudintermedius and S. aureus isolates. MATERIALS AND METHODS: Two meticillin-susceptible S. pseudintermedius (MSSP) isolates, three meticillin-resistant S. pseudintermedius (MRSP) isolates and one meticillin-resistant S. aureus (MRSA) isolate were studied. A commercially available blue light-emitting diode (bLED) lamp and photoconverting hydrogel FLE system was used. All bacteria were exposed to five conditions following inoculation: (i) no treatment (control); (ii) blue light (bLED) once; (iii) bLED twice consecutively; (iv) FLE (bLED and photoconverting hydrogel) once; and (v) FLE (bLED and photoconverting hydrogel) twice consecutively. Each individual exposure was 2 min long. RESULTS: No statistically significant differences (p < 0.05) were found for any treatment group when each bacterial isolate was evaluated individually, MSSP isolates were grouped, MRSP isolates were grouped, when all S. pseudintermedius isolates were combined, or when all isolates of both Staphylococcus species were combined. CONCLUSIONS AND CLINICAL RELEVANCE: While clinical success is reported when using FLE to treat Staphylococcus infections in animals, no in vitro antibacterial efficacy was identified for S. pseudintermedius or S. aureus under experimental conditions. The clinical success observed with FLE may be the result of a more complex in vivo response.

Does Topical Vancomycin Powder Use in Fracture Surgery Change Bacteriology and Antibiotic Susceptibilities? An Analysis of the VANCO Trial.

OBJECTIVE: To determine whether intrawound vancomycin changes the bacteriology of surgical site infection pathogens and investigate the emergence of antibiotic-resistant pathogens. DESIGN: Secondary analysis of phase III, prospective, randomized clinical trial. SETTING: Thirty-six US trauma centers. PATIENT SELECTION CRITERIA: Patients who became infected after fixation of tibial plateau or pilon fracture. OUTCOME MEASURES AND COMPARISONS: Pathogen types and bacterial susceptibilities as determined from routine clinical culture in the operating room. RESULTS: Seventy-four patients were studied who were 67.5% male with a mean age of 48.6 years. A lower proportion of gram-positive cocci was observed in the vancomycin powder compared with the standard-of-care group (3.7% vs. 8.0%, P = 0.01). Methicillin-resistant Staphylococcus aureus infection incidence was comparable in both the vancomycin powder and the standard-of-care groups, but rates of methicillin-susceptible S. aureus infections were lower in the treatment group (1.4% vs. 4.8%, P = 0.01). The incidence of coagulase-negative Staphylococci and gram-negative rod infections were similar in both groups. There was no significant difference in susceptibilities between groups in rates of vancomycin-resistant enterococcus. CONCLUSIONS: Topical vancomycin powder decreases the likelihood of gram-positive infections consistent with the biologic activity of vancomycin. Fewer methicillin-susceptible S. aureus and coagulase-negative Staphylococci infections were observed in the group treated with vancomycin powder. An effect of vancomycin powder on methicillin-resistant S. aureus infection risk was not detected given the low incidence in both the intrawound vancomycin and the standard-of-care groups. There was no emergence of gram-negative rod infections or increased resistance patterns observed. Use of topical vancomycin powder does not seem to produce infections in these patients with greater antibiotic resistance than would have occurred without its use. LEVEL OF EVIDENCE: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Novel small-molecule compound YH7 inhibits the biofilm formation of Staphylococcus aureus in a sarX-dependent manner.

The emergence of antibiotic-resistant and biofilm-producing Staphylococcus aureus isolates presents major challenges for treating staphylococcal infections. Biofilm inhibition is an important anti-virulence strategy. In this study, a novel maleimide-diselenide hybrid compound (YH7) was synthesized and demonstrated remarkable antimicrobial activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in both planktonic cultures and biofilms. The minimum inhibitory concentration (MIC) of YH7 for S. aureus isolates was 16 µg/mL. Quantification of biofilms demonstrated that the sub-MIC (4 µg/mL) of YH7 significantly inhibits biofilm formation in both MSSA and MRSA. Confocal laser scanning microscopy analysis further confirmed the biofilm inhibitory potential of YH7. YH7 also significantly suppressed bacterial adherence to A549 cells. Moreover, YH7 treatment significantly inhibited S. aureus colonization in nasal tissue of mice. Preliminary mechanistic studies revealed that YH7 exerted potent biofilm-suppressing effects by inhibiting polysaccharide intercellular adhesin (PIA) synthesis, rather than suppressing bacterial autolysis. Real-time quantitative PCR data indicated that YH7 downregulated biofilm formation-related genes (clfA, fnbA, icaA, and icaD) and the global regulatory gene sarX, which promotes PIA synthesis. The sarX-dependent antibiofilm potential of YH7 was validated by constructing S. aureus NCTC8325 sarX knockout and complementation strains. Importantly, YH7 demonstrated a low potential to induce drug resistance in S. aureus and exhibited non-toxic to rabbit erythrocytes, A549, and BEAS-2B cells at antibacterial concentrations. In vivo toxicity assays conducted on Galleria mellonella further confirmed that YH7 is biocompatible. Overall, YH7 demonstrated potent antibiofilm activity supports its potential as an antimicrobial agent against S. aureus biofilm-related infections. IMPORTANCE Biofilm-associated infections, characterized by antibiotic resistance and persistence, present a formidable challenge in healthcare. Traditional antibacterial agents prove inadequate against biofilms. In this study, the novel compound YH7 demonstrates potent antibiofilm properties by impeding the adhesion and the polysaccharide intercellular adhesin production of Staphylococcus aureus. Notably, its exceptional efficacy against both methicillin-resistant and methicillin-susceptible strains highlights its broad applicability. This study highlights the potential of YH7 as a novel therapeutic agent to address the pressing issue of biofilm-driven infections.

Fluorinated azoles as effective weapons in fight against methicillin-resistance staphylococcus aureus (MRSA) and its SAR studies.

The rapid spread of Methicillin-resistant Staphylococcus aureus (MRSA) and its difficult-to-treat skin and filmsy diseases are making MRSA a threat to human life. The most dangerous feature is the fast emergence of MRSA resistance to all recognized antibiotics, including vancomycin. The creation of novel, effective, and non-toxic drug candidates to combat MRSA isolates is urgently required. Fluorine containing small molecules have taken a centre stage in the field of drug development. Over the last 50 years, there have been a growing number of fluorinated compounds that have been approved since the clinical usage of fluorinated corticosteroids in the 1950 s and fluoroquinolones in the 1980 s. Due to its advantages in terms of potency and ADME (absorption, distribution, metabolism, and excretion), fluoro-pharmaceuticals have been regarded as a potent and useful tool in the rational drug design method. The flexible bioactive fluorinated azoles are ideal candidates for the development of new antibiotics. This review summarizes the decade developments of fluorinated azole derivatives with a wide antibacterial activity against diverged MRSA strains. In specific, we correlated the efficacy of structurally varied fluorinated azole analogues including thiazole, benzimidazole, oxadiazole and pyrazole against MRSA and discussed different angles of structure-activity relationship (SAR).

In vitro efficacy of a topical antimicrobial mousse and shampoo at proximal and distal hair locations against Staphylococcus pseudintermedius and Staphylococcus schleiferi.

BACKGROUND: Topical therapy is essential in assisting with the resolution of pyoderma. OBJECTIVES: (i) Evaluate the in vitro efficacy and residual activity of two different hair segments treated with shampoo and mousse against meticillin-sensitive and meticillin-resistant staphylococci; (ii) compare proximal and distal hair portions treated with the products and (iii) describe a new disc diffusion method for assessing residual efficacy. ANIMALS: Eleven privately owned, medium-haired dogs. MATERIALS AND METHODS: In this randomised, blinded and negatively controlled study, dogs were treated once with a 3% chlorhexidine digluconate-0.5% ophytrium shampoo on the lateral thorax, and the corresponding mousse on the opposite side. Hairs were plucked before treatment, two hours post-treatment, and day (D)2, D4, D7, D10 and D14. Hairs were weighed (0.01 g) and cut (1.0 cm) from the proximal portion, moistened with saline and placed on a sterile diffusion disc to absorb the solution. Proximal and distal hair bundles and diffusion discs were placed onto agar inoculated with an isolate of meticillin-sensitive or meticillin-resistant Staphylococcus pseudintermedius or Staphylococcus schleiferi. Inhibition zones were measured following incubation. RESULTS: Distal hairs had larger (p < 0.001) inhibition zones compared to proximal hairs. Mousse had significant differences (p < 0.05) between time points and locations for both the hair bundles and discs, while shampoo only had a significant difference (p < 0.001) between time points for the hairs. CONCLUSIONS AND CLINICAL RELEVANCE: Mousse was effective, and shampoo was only minimally effective in inhibiting bacterial growth in vitro, with the greatest effect occurring at the two hours time point. The distal hair shafts had greater inhibition.

Azalomycin F4a targets peptidoglycan synthesis of Gram-positive bacteria revealed by high-throughput CRISPRi-seq analysis.

Azalomycin F4a is a promising 36-membered polyhydroxy macrolide that shows antibacterial activity against drug-resistant Gram-positive bacteria, but its exact working mechanism remains to be elusive. Here, we isolated the azalomycin F4a product from a Streptomyces solisilvae and demonstrated its antibacterial activity against Gram-positive pathogens including Streptococcus pneumoniae, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). We further showed that combination of azalomycin F4a with methicillin has an additive antimicrobial effect on MRSA, where the minimal inhibitory concentrations (MIC) of methicillin to MRSA was decreased by 1000-fold in the presence of sublethal concentration of azalomycin F4a. A CRISPRi-seq based whole genome screen was employed to identify the potential targets of azalomycin F4a, which revealed that peptidoglycan synthesis (PGS) was inhibited by azalomycin F4a. Furthermore, azalomycin F4a treatment could significantly impair S. aureus biofilm formation. Our research highlights that cell wall synthesis is an additional target for novel classes of macrolide besides ribosome.

Epidemiology and impact of methicillin-sensitive Staphylococcus aureus with ß-lactam antibiotic inoculum effects in adults with cystic fibrosis.

Staphylococcus aureus is the most prevalent cystic fibrosis (CF) pathogen. Several phenotypes are associated with worsened CF clinical outcomes including methicillin-resistance and small-colony-variants. The inoculum effect (IE) is characterized by reduced ß-lactam susceptibility when assessed at high inoculum. The IE associates with worse outcomes in bacteremia and other high-density infections, and may therefore be relevant to CF. The prevalence of IE amongst a CF cohort (age ≥18 years), followed from 2013 to 2016, was investigated. Yearly methicillin-sensitive S. aureus (MSSA) isolates were screened at standard (5 × 105 CFU/mL) and high (5 × 107 CFU/mL) inoculum against narrow-spectrum anti-Staphylococcal ß-lactams and those with anti-pseudomonal activity common to CF. A ≥ 4-fold increase in minimum inhibitory concentration between standard and high inoculum defined IE. Isolates underwent blaZ sequencing and genotyping and were compared against published genomes. Fifty-six percent (99/177) of individuals had MSSA infection. MSSA was observed at ≥105 CFU/mL in 44.8% of entry sputum samples. The prevalence of the IE was 25.0%-cefazolin; 13.5%-cloxacillin; 0%-meropenem; 1.0%-cefepime; 5.2%-ceftazidime; and 34.4%-piperacillin-tazobactam amongst baseline MSSA isolates assessed. blaZ A associated with cefazolin IE (P = 0.0011), whereas blaZ C associated with piperacillin-tazobactam IE (P < 0.0001). Baseline demographics did not reveal specific risk factors for IE-associated infections, nor were long-term outcomes different. Herein, we observed the IE in CF-derived MSSA disproportionally for cefazolin and piperacillin-tazobactam and this phenotype strongly associated with underlying blaZ genotype. The confirmation of CF being a high density infection, and the identification of high prevalence of MSSA with IE in CF supports the need for prospective pulmonary exacerbation treatment studies to understand the impact of this phenotype.

The Controversial Effect of Antibiotics on Methicillin-Sensitive S. aureus: A Comparative In Vitro Study.

Methicillin-sensitive Staphylococcus (S.) aureus (MSSA) bacteremia remains a global challenge, despite the availability of antibiotics. Primary treatments include ß-lactam agents such as cefazolin and flucloxacillin. Ongoing discussions have focused on the potential synergistic effects of combining these agents with rifampicin or fosfomycin to combat infections associated with biofilm formation. Managing staphylococcal infections is challenging due to antibacterial resistance, biofilms, and S. aureus's ability to invade and replicate within host cells. Intracellular invasion shields the bacteria from antibacterial agents and the immune system, often leading to incomplete bacterial clearance and chronic infections. Additionally, S. aureus can assume a dormant phenotype, known as the small colony variant (SCV), further complicating eradication and promoting persistence. This study investigated the impact of antibiotic combinations on the persistence of S. aureus 6850 and its stable small colony variant (SCV strain JB1) focusing on intracellular survival and biofilm formation. The results from the wild-type strain 6850 demonstrate that ß-lactams combined with RIF effectively eliminated biofilms and intracellular bacteria but tend to select for SCVs in planktonic culture and host cells. Higher antibiotic concentrations were associated with an increase in the zeta potential of S. aureus, suggesting reduced membrane permeability to antimicrobials. When using the stable SCV mutant strain JB1, antibiotic combinations with rifampicin successfully cleared planktonic bacteria and biofilms but failed to eradicate intracellular bacteria. Given these findings, it is reasonable to report that ß-lactams combined with rifampicin represent the optimal treatment for MSSA bacteremia. However, caution is warranted when employing this treatment over an extended period, as it may elevate the risk of selecting for small colony variants (SCVs) and, consequently, promoting bacterial persistence.

Terpene-Functionalized Fluoroquinolones as Potential Antimicrobials: Synthesis and Properties.

This study was the first to synthesize terpene-containing conjugates of fluoroquinolones, ciprofloxacin and norfloxacin, and to evaluate their antibacterial activity against gram-positive methicillin sensitive (MSSA) and methicillin resistant (MRSA) S. aureus, gram-negative P. aeruginosa as well as antifungal activity against C. albicans. The ability of obtained fluoroquinolones to inhibit S. aureus growth was found to depend upon the presence of a linker separating the bulky terpene and fluoroquinolone fragments, and this activity diminished with increasing its length. The highest activity against MSSA was demonstrated by ciprofloxacin derivatives with campholenic (MIC 1 µg/mL) and 2-(isobornan-2-yl-sulfanyl)acetyl (MIC 0.5 µg/mL) substituents. The compound with the last fragment showed high activity against MRSA (MIC 8 µg/mL). The terpene-functionalized norfloxacin derivatives generally proved to be less active than those containing ciprofloxacin fragment. Camphor-10-sulfonylamide derivative with the ciprofloxacin fragment was the only one of all compounds that showed high antifungal activity against C. albicans (8 µg/mL). The study presents data on docking fluoroquinolones to S. aureus DNA gyrase to explain the reasons for manifestation or disappearance of antibacterial activity. The cytotoxicity of fluoroquinolones that showed any antimicrobial activity was investigated against bovine primary lung cells, and they were found to be not toxic in most cases.

Evaluation of Latex Agglutination Test for Rapid Identification of Staphylococcus aureus Isolated from Pyogenic Wound Infections at a Tertiary Care Hospital.

Background Staphylococcus aureus infections are increasingly reported worldwide. It is a major clinical problem and imposes significant morbidity and mortality due to widespread emergence of multidrug resistant pathogens like methicillin resistant Staphylococcus aureus. Thus, rapid and reliable identification of Staphylococcus aureus is essential for timely and effective management of patient. Objective The performance of Latex agglutination test (Staphaurex Plus) was compared to conventional method tube coagulase test which is gold standard too for the identification of Staphylococcus aureus. Method This study was conducted at B.P. Koirala Institute of Health Sciences. Following standard microbiological methods, isolation and identification was done in the Department of Microbiology. MRSA detection was performed following Clinical and Laboratory Standard Institute. All the isolates of Staphylococci were subjected for Latex agglutination test and was performed according to manufacturer's instructions using Staphaurex Plus kit. Result Out of 377 (methicillin sensitive Staphylococcus aureus - 142; methicillin resistant Staphylococcus aureus - 233; Coagulase Negative Staphylococci -2) isolates of Staphylococci, Latex agglutination test was found to be positive in 138 (97.1%) of methicillin sensitive Staphylococcus aureus (MSSA) and 220 (94.4%) of methicillin resistant Staphylococcus aureus (MRSA). Overall sensitivity, specificity, positive predictive value and negative predictive value of Latex agglutination test was found to be 95.46%, 100%, 100%, 10.52% respectively. Conclusion In conclusion, Latex agglutination test is a rapid and reliable test for the identification of Staphylococcus aureus.

A Review on Five and Six-Membered Heterocyclic Compounds Targeting the Penicillin-Binding Protein 2 (PBP2A) of Methicillin-Resistant Staphylococcus aureus (MRSA).

Staphylococcus aureus is a common human pathogen. Methicillin-resistant Staphylococcus aureus (MRSA) infections pose significant and challenging therapeutic difficulties. MRSA often acquires the non-native gene PBP2a, which results in reduced susceptibility to ß-lactam antibiotics, thus conferring resistance. PBP2a has a lower affinity for methicillin, allowing bacteria to maintain peptidoglycan biosynthesis, a core component of the bacterial cell wall. Consequently, even in the presence of methicillin or other antibiotics, bacteria can develop resistance. Due to genes responsible for resistance, S. aureus becomes MRSA. The fundamental premise of this resistance mechanism is well-understood. Given the therapeutic concerns posed by resistant microorganisms, there is a legitimate demand for novel antibiotics. This review primarily focuses on PBP2a scaffolds and the various screening approaches used to identify PBP2a inhibitors. The following classes of compounds and their biological activities are discussed: Penicillin, Cephalosporins, Pyrazole-Benzimidazole-based derivatives, Oxadiazole-containing derivatives, non-ß-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycles (ß-lactam antibiotics with 1,3-Bridges), Macrocycle-embedded ß-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-ß-lactam antibiotics, Carbapenem, novel Benzoxazole derivatives, Pyrazolylpyridine analogues, and other miscellaneous classes of scaffolds for PBP2a. Additionally, we discuss the penicillin-binding protein, a crucial target in the MRSA cell wall. Various aspects of PBP2a, bacterial cell walls, peptidoglycans, different crystal structures of PBP2a, synthetic routes for PBP2a inhibitors, and future perspectives on MRSA inhibitors are also explored.

Simple and Rapid Discrimination of Methicillin-Resistant Staphylococcus aureus Based on Gram Staining and Machine Vision.

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinical threat with high morbidity and mortality. Here, we describe a new simple, rapid identification method for MRSA using oxacillin sodium salt, a cell wall synthesis inhibitor, combined with Gram staining and machine vision (MV) analysis. Gram staining classifies bacteria as positive (purple) or negative (pink) according to the cell wall structure and chemical composition. In the presence of oxacillin, the integrity of the cell wall for methicillin-susceptible S. aureus (MSSA) was destroyed immediately and appeared Gram negative. In contrast, MRSA was relatively stable and appeared Gram positive. This color change can be detected by MV. The feasibility of this method was demonstrated in 150 images of the staining results for 50 clinical S. aureus strains. Based on effective feature extraction and machine learning, the accuracies of the linear linear discriminant analysis (LDA) model and nonlinear artificial neural network (ANN) model for MRSA identification were 96.7% and 97.3%, respectively. Combined with MV analysis, this simple strategy improved the detection efficiency and significantly shortened the time needed to detect antibiotic resistance. The whole process can be completed within 1 h. Unlike the traditional antibiotic susceptibility test, overnight incubation is avoided. This new strategy could be used for other bacteria and represents a new rapid method for detection of clinical antibiotic resistance. IMPORTANCE Oxacillin sodium salt destroys the integrity of the cell wall of MSSA immediately, appearing Gram negative, whereas MRSA is relatively stable and still appears Gram positive. This color change can be detected by microscopic examination and MV analysis. This new strategy has significantly reduced the time to detect resistance. The results show that using oxacillin sodium salt combined with Gram staining and MV analysis is a new, simple and rapid method for identification of MRSA.

Natural cordiaquinones as strategies to inhibit the growth and biofilm formation of methicillin-sensitive and methicillin-resistant Staphylococcus spp.

AIMS: The aim of this study was to investigate the antibacterial and antibiofilm potential of cordiaquinones B, E, L, N, and O against different Staphylococci strains, in addition to analyzing in silico the observed effect. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined according to CLSI guidelines. The inhibition of biofilm formation was investigated at sub-MICs. Atomic force microscopy (AFM) and density functional theory method were performed. The tested strains of Staphylococcus spp. were susceptible to cordiaquinones B, E, and L, among which cordiaquinone B exerted a bactericidal effect, confirmed by a bacterial growth curve study, against Staphylococcus saprophyticus. Cordiaquinones B and E showed lowest MBC values against S. saprophyticus. AFM revealed that cordiaquinone L reduced the mean cell size of S. saprophyticus. Cordiaquinones B and E inhibited the biofilm formation ability of S. aureus by ∼90%. The in silico analysis suggested that the antimicrobial activity of cordiaquinones is driven by their electron donation capability. CONCLUSIONS: Cordiaquinones inhibit the growth and biofilm formation (virulence factor) of both methicillin-sensitive and methicillin-resistant Staphylococci strains, indicating their antimicrobial potential.

Biologically-Relevant Staphylococcus Aureus Biofilm Phenotype Characterisation And Liability To Novel Antibiofilm Drugs.

Objectives: To characterise the biofilm matrix composition of a newly described Staphylococcus aureus biofilm phenotype. Method: This experimental study was conducted at the Faculty of Pharmacy, Helwan University, Cairo, Egypt, from January 2021 to March 2022, and comprised methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus biofilm-forming clinical isolates which were allowed to construct biofilms under two distinct culture conditions; one a commonly used condition, and the other one a novel, more biologically-relevant condition. The formed biofilms were analysed for matrix composition through treatment with proteinase,sodium meta-periodate, and streptokinase. The efficacy of Cis-2-Decenoic acid and hamamelitannin on the biologically-relevant biofilms was evaluated using biofilm viability assay based on a colorimetric assay for measuring cell metabolic activity and scanning electron microscope imaging. Data was analysed using GraphPad Prism 5.01. RESULTS: Of the 58 isolates, 45(77.6%) were methicillin-resistant Staphylococcus aureus and 13(22.4%) were methicillin susceptible Staphylococcus aureus. There was significant difference in responses to streptokinase, proteinase and sodium meta-periodate (p<0.05) among the differentially-developed biofilms in methicillin-resistant Staphylococcus aureus isolates. Regarding the methicillin-susceptible Staphylococcus aureus isolates, the differentially-developed biofilms showed significantly different liabilities to streptokinase only (p<0.05). Mean biofilm inhibition for Cis-2- Decenoic acid was 54.27±27.93% and mean biofilm dispersion was 71.92±11.59% while the corresponding valuesfor hamamelitannin were 83.03±13.95% and 70.48±7.116% against the newly described methicillin-resistant Staphylococcus aureus biofilm phenotype. CONCLUSIONS: Applying biologically-relevant culture conditions on staphylococci biofilms and antibiofilm drugs is recommended.

Thermal susceptibility and antibiotic synergism of methicillin-resistant Staphylococcus aureus biofilms.

Methicillin-Resistant Staphylococcus aureus (MRSA) biofilms are among the most dangerous infections on medical implants, typically requiring surgical explantation and replacement. This study investigated the thermal susceptibility of MRSA biofilms to thermal shocks from 60 to 80 °C for 1-30 min as well as the effect of various antibiotics (most notably methicillin) on thermal mitigation. Pre- and post-shock exposure to three different classes of antibiotics (ciprofloxacin, tobramycin, and methicillin) at concentrations ranging from 0.25 to 128 µg mL-1 were investigated. MRSA biofilms exhibited thermal susceptibility comparable to other common nosocomial pathogens, such as Pseudomonas aeruginosa, though with greater variability. Exposure to antibiotics of any class significantly decreased the degree of thermal shock required for reliable mitigation, including at subclinical concentration. These combined treatments reduced biofilm population more than the sum of thermal and chemical treatments alone, demonstrating synergism, while also indicating a critical population drop of ∼4.5 log10 beyond which the biofilms typically became non-viable.