Oxacillin Minimum Inhibitory Concentration Research Articles

Antibacterial efficacy of antimicrobial peptide-functionalized hydrogel particles combined with vancomycin and oxacillin antibiotics

The rise of antibiotic resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), requires novel approaches to combat infections. Medical devices like implants and wound dressings are frequently used in conjunction with antibiotics, motivating the development of antibacterial biomaterials capable of exhibiting combined antibacterial effects with conventional antibiotics. This study explores the synergistic antibacterial effects of combining antimicrobial peptide (AMP) functionalized hydrogel particles with conventional antibiotics, vancomycin (VCM) and oxacillin (OXA), against Staphylococcus aureus and MRSA. The AMP employed, RRPRPRPRPWWWW-NH2, has previously demonstrated broad-spectrum activity and enhanced stability when attached to hydrogel substrates. Here, checkerboard assays revealed additive and synergistic interactions between the free AMP and both VCM and OXA against Staphylococcus aureus and MRSA. Notably, the AMP-OXA combination displayed a significant synergistic effect against MRSA, with a 512-fold reduction in OXA’s minimum inhibitory concentration (MIC) when combined with free AMP. The observed synergism against MRSA was retained upon covalent AMP immobilization onto the hydrogel particles; however, at a lower rate with a 64-fold reduction in OXA MIC. Despite this, the OXA-AMP hydrogel particle combinations retained considerable synergistic potential against MRSA, a strain resistant to OXA, highlighting the potential of AMP-functionalized materials for enhancing antibiotic efficacy. These findings underscore the importance of developing antimicrobial biomaterials for future medical devices to fight biomaterial-associated infections and reverse antimicrobial resistance.

Susceptibility of Staphylococcus aureus to Anti-Inflammatory Drugs with a Focus on the Combinatory Effect of Celecoxib with Oxacillin In Vitro.

Musculoskeletal infections (MIs) are among the most difficult-to-treat staphylococcal diseases due to antibiotic resistance. This has encouraged the development of innovative strategies, such as combination therapy, to combat MI. The aim of this study was to investigate the in vitro antistaphylococcal activity of anti-inflammatory drugs and the combined antimicrobial effect of celecoxib and oxacillin. The minimum inhibitory concentrations (MICs) of 17 anti-inflammatory drugs against standard strains and clinical isolates of S. aureus, including methicillin-resistant strains (MRSAs), were determined using the broth microdilution method. The fractional inhibitory concentration indices (FICIs) were evaluated using checkerboard assays. Celecoxib produced the most potent antistaphylococcal effect against all tested strains (MICs ranging from 32 to 64 mg/L), followed by that of diacerein against MRSA3 and MRSA ATCC 33592 (MIC 64 mg/L). Several synergistic effects were observed against the tested S. aureus strains, including MRSA (FICI ranging from 0.087 to 0.471). The strongest synergistic interaction (FICI 0.087) was against MRSA ATCC 33592 at a celecoxib concentration of 2 mg/L, with a 19-fold oxacillin MIC reduction (from 512 to 26.888 mg/L). This is the first report on the combined antistaphylococcal effect of celecoxib and oxacillin. These findings suggest celecoxib and its combination with oxacillin as perspective agents for research focused on the development of novel therapies for MI caused by S. aureus. This study further indicates that celecoxib could resensitize certain MRSA strains, in some cases, to be susceptible to β-lactams (e.g., oxacillin) that were not previously tested. It is essential to mention that the in vitro concentrations of anti-inflammatory drugs are higher than those typically obtained in patients. Therefore, an alternative option for its administration could be the use of a drug delivery system for the controlled slow release from an implant at the infection site.

Evaluation of minimum inhibitory concentration data in National Healthcare Safety Network’s Antimicrobial Resistance Option

Background: Clinical laboratories perform antimicrobial susceptibility testing (AST) primarily by determining the minimum inhibitory concentration (MIC) for an organism–antimicrobial combination and comparing it with established breakpoints to generate interpretations. The Antimicrobial Resistance (AR) Option of CDC’s National Healthcare Safety Network (NHSN) permits hospitals to submit clinical isolate AST data, including test values and interpretations (Figure 1). The Clinical and Laboratory Standards Institute (CLSI) periodically revises breakpoints, but their adoption by clinical laboratories can be delayed, potentially affecting national AR surveillance data accuracy. Using MIC values, instead of clinical laboratory interpretations, can improve surveillance data accuracy and overcome misclassification due to delayed uptake of revised breakpoints. We evaluated the completeness and consistency of MIC data submitted to the AR Option for fluoroquinolone-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Methods: We included data on (1) E. coli isolates tested for ciprofloxacin or levofloxacin susceptibility and (2) S. aureus isolates tested for oxacillin or cefoxitin susceptibility in 2022 and reported by October 1, 2023. We evaluated completeness among isolates reporting a final AST interpretation as the proportion of isolates reporting both an MIC value and interpretation. We evaluated consistency using percent agreement comparing the laboratory’s MIC interpretation (classified as resistant or not resistant) with the interpretation derived by applying 2021 CLSI M100 breakpoints to the MIC values reported for the same isolate. Results: Across 974 hospitals, fluoroquinolone MICs and interpretations were reported for 172,012/393,359 E. coli isolates (43.7%), and oxacillin or cefoxitin MICs and interpretations were reported for 38,519/79,372 S. aureus isolates (48.5%). Of isolates with both MIC values and interpretations, 157,902 (91.8%) E. coli and 7,808(79.7%) S. aureus isolates had MICs that could be classified as resistant or non-resistant (i.e., intermediate or susceptible) per CLSI breakpoints (Figure 2). The remaining MICs were unclassifiable (reported as intervals spanning CLSI breakpoints, e.g., ≤1 μg/ml ciprofloxacin for E. coli). Among isolates with classifiable MICs, the agreement between the clinical laboratory and CLSI-based interpretation was 99.5% for E. coli and 99.7% for S. aureus. Conclusion: MIC values and interpretations were available for

Doxycycline post-exposure prophylaxis could theoretically select for resistance to various antimicrobials in 19 pathobionts: an in silico analysis

ObjectivesDoxycycline post exposure prophylaxis (PEP) has been shown to reduce the incidence of bacterial STIs. However, if there is genetic linkage between resistance to tetracycline and other antimicrobials, then it could also select for resistance to these other antimicrobials. We therefore undertook to evaluate if there is an association between the minimum inhibitory concentrations (MICs) of tetracycline and other antimicrobials in 19 clinically important bacterial species. MethodsMixed-effects linear regression was used to assess if minocycline MICs were associated with the MICs of eight other antimicrobials (ceftriaxone, ampicillin, oxacillin, vancomycin, erythromycin, levofloxacin, amikacin, and trimethoprim-sulfamethoxazole) in 19 bacterial species in the Antimicrobial Testing Leadership and Surveillance (ATLAS) database. ResultsWith the notable exception of vancomycin, where no association was found, strong positive associations were typically found between the MICs of minocycline and each of the eight antimicrobials in each of the species assessed. For example, the minocycline MICs of all the Gram-positive species were positively associated with ampicillin, ceftriaxone, oxacillin and erythromycin MICs (all P-values < 0.001). The only exceptions were ampicillin for Streptococcus pyogenes and ceftriaxone for S. dysgalactiae, where no significant associations were found. Similarly in the Gram-negative species, the minocycline MICs of all the species except Haemophilus influenzae and Stenotrophomonas maltophilia were positively associated with the MICs of ceftriaxone, ampicillin, levofloxacin and amikacin (all P-values < 0.001). ConclusionsThere is a theoretical risk that doxycycline PEP could select for resistance not only to tetracyclines but to a range of other antimicrobials in each of the 19 pathobionts assessed.

Correlation between oxacillin MIC values with those of linezolid against clinical isolates of MRSA

Infection by methicillin-resistant (MRSA) is a great threat to medical care facilities and also in communities, due to its multidrug resistance to commonly used antimicrobial agents. The overuse of glycopeptide antibiotics, such as vancomycin and linezolid, has led to the emergence of reduced susceptibility to these anti-MRSA agents, which in turn may lead to therapeutic failure. This study has been conducted to explore the correlation if any, of MIC of oxacillin to the MIC of linezolid in clinically significant isolates of MRSA in a tertiary care hospital.: The study was carried out over the period of two months after obtaining a waiver of consent from the Institute Ethics Committee. Seventy-five clinically significant MRSA isolates were included in the study. All MRSA isolates were subjected to cefoxitin and linezolid antibiotic disk susceptibility testing. Minimum inhibitory concentration (MIC) to oxacillin and linezolid was performed by the agar dilution method. The MIC and MIC were also recorded both for oxacillin and linezolid MIC among these MRSA isolates. Correlation between oxacillin MIC and linezolid MIC was estimated using the Pearson correlation coefficient, r.Themajority of MRSA isolates (41%) were isolated from skin and soft tissue infections (SSTIs) (40%). MIC values for oxacillin ranged from 4 μg/mL to &amp;#62;32μg/mL and MIC values for linezolid ranged from ≤ 0.25µg/mL to 4 µg/mL. The majority of these isolates (40%) had linezolid MIC of ≤ 0.25µg/mL. All the isolates were uniformly susceptible to linezolid. Pearson correlation coefficient, r was 0.41, between oxacillin MICs and linezolid MICs, which indicated poor correlation. Although we did not observe any resistance to linezolid among the MRSA isolates, we should monitor carefully the antibiotic selection pressure and creeping MIC, to aid in the early detection of the emergence of resistance.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

It's Not You, It's SOSA: A Case Study on Breaking Up With an FDA-Cleared Susceptibility Testing System's Oxacillin Results for Staphylococcus spp. Other Than S. aureus and S. lugdunensis.

BackgroundIn 2021, the Clinical and Laboratory Standards Institute revised its susceptible oxacillin minimum inhibitory concentration (MIC) breakpoint for Staphylococcus spp. other than S. aureus and S. lugdunensis (SOSA) from ≤0.25 to ≤0.5 µg/mL. Here, we describe the response to this breakpoint change, which at the time of this study was not yet recognized by the US Food and Drug Administration (FDA), in our laboratory, where the primary method for antimicrobial susceptibility testing (AST) of SOSA is VITEK 2. VITEK 2 uses the Automated Expert System (AES) to integrate the results of oxacillin MIC and cefoxitin screen tests into a final interpretation; our laboratory also adjudicates discordant oxacillin and cefoxitin results using a PBP2a test.MethodsWe retrospectively reviewed and assessed the yield of PBP2a testing for 189 SOSA isolates with discordant (when applying the FDA susceptible oxacillin breakpoint of ≤0.25 µg/mL) VITEK 2 oxacillin and cefoxitin results, and then prospectively incorporated PBP2a testing for isolates with oxacillin MICs of 0.5 µg/mL and positive cefoxitin screens into our algorithm.ResultsCompared with accepting the VITEK 2 AES interpretation, PBP2a testing substantially improved the accuracy of mecA-mediated resistance classification in both scenarios, especially for the ∼4.7% of isolates with oxacillin MICs ≤0.5 µg/mL and positive cefoxitin screens.ConclusionsAlthough detection of mecA or PBP2a is the gold standard for assessment of β-lactam resistance in staphylococci, targeting a subset of isolates for mecA or PBP2a testing based on phenotypic AST results that predict an increased risk of misclassification may be a pragmatic, labor- and cost-saving approach.

Mutations in the gdpP gene are a clinically relevant mechanism for β-lactam resistance in meticillin-resistant Staphylococcus aureus lacking mec determinants

In Staphylococcus aureus , resistance to β-lactamase stable β-lactam antibiotics is mediated by the penicillinbinding protein 2a, encoded by mecA or by its homologues mecB or mecC. However, a substantial number of meticillin-resistant isolates lack known mec genes and, thus, are called meticillin resistant lacking mec (MRLM). This study aims to identify the genetic mechanisms underlying the MRLM phenotype. A total of 141 MRLM isolates and 142 meticillin-susceptible controls were included in this study. Oxacillin and cefoxitin minimum inhibitory concentrations were determined by broth microdilution and the presence of mec genes was excluded by PCR. Comparative genomics and a genome-wide association study (GWAS) approach were applied to identify genetic polymorphisms associated with the MRLM phenotype. The potential impact of such mutations on the expression of PBP4, as well as on cell morphology and biofilm formation, was investigated. GWAS revealed that mutations in gdpP were significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme involved in the degradation of the second messenger cyclic-di-AMP in S. aureus . A total of 131 MRLM isolates carried truncations, insertions or deletions as well as amino acid substitutions, mainly located in the functional DHH-domain of GdpP. We experimentally verified the contribution of these gdpP mutations to the MRLM phenotype by heterologous complementation experiments. The mutations in gdpP had no effect on transcription levels of pbp4; however, cell sizes of MRLM strains were reduced. The impact on biofilm formation was highly strain dependent. We report mutations in gdpP as a clinically relevant mechanism for β-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, since MRLM are easily misclassified as MSSA (meticillin-susceptible S. aureus ), which may lead to unnoticed spread of β-lactam-resistant isolates and subsequent treatment failure.

Action of methicillin on the "in vitro" growth of bacteria Staphyloccocus aureus methicillin-resistance previously treated with homeopathic dilutions

Background: Methicillin-resistant Staphylococcus aureus (MRSA) causes nosocomial infections, and it has been considered as a worldwide epidemic. The medical system seeks new strategies to fight against MRSA that do not generate resistant strains to antibiotics. Homeopathy has been explored as one of these new strategies, which may play a pivotal role. In this context, we conducted studies on the action of homeopathy on growth of MRSA bacteria in vitro. The results showed a decrease in growth of bacterial strains with homeopathic dilutions of Belladonna and the S. aureus nosode. Now we have proposed to evaluate the minimum inhibitory concentration (MIC) of the antibiotic methicillin or oxacillin on S. aureus MRSA, previously incubated with the homeopathic dilutions of Belladonna or S. aureus nosode.&#x0D; Methods: The Clinical and Laboratory Standards Institute (CLSI 2014) standards were followed according to the determination of the minimum inhibitory concentration (MIC). In 5 mL of cation adjusted Mueller Hinton (CAMH) broth, it was added 420 µl of 30% alcohol or Belladonna and S. aureus’ nosode in the dilutions 6cH, 12cH and 30cH. Then a 20µl of bacterial suspension of MRSA was added to 0.5 McFarland range and diluted to 1/10. The tubes were incubated in an oven at 37⁰C for three hours. The plates were previously prepared with 50µl per well of serial dilutions of the antibiotic oxacillin in concentrations of 128 µg/mL to 0.5 µg/mL in CAMH broth. Then it was added 50 µl per well of bacterial cultures. The plate was incubated in an oven at 37⁰C for 24 hours and the bacterial growth measured in a spectrophotometer 600nm. The point of the MIC of oxacillin for S. aureus is 4 µg/mL, according to CLSI 2014 criteria.&#x0D; Results: We did not observe the total inhibition of bacterial growth when incubated with the homeopathic medicine and oxacillin. In evaluation of the spectrophotometer culture, we observed significant changes in the growth, compared to the control (30% alcohol). Cultures treated with Belladonna 6cH and the antibiotic in the dilution 4 µg/mL showed a decrease of 40% of the growth, while in the 30cH the drop was of 75%. Cultures treated with the S. aureus nosode 30cH and the antibiotic at 4 µg/mL dilution, showed a decrease of 60% in bacterial growth in vitro.&#x0D; Conclusion: The results suggest that bacterial cultures the S. aureus (MRSA) incubated with the homeopathic medicines would be more susceptible to oxacillin’s antimicrobial action.

Antibiotic resistance of Staphylococcus aureus isolated from raw cow and goat milk produced in the Tiaret and Souk Ahras areas of Algeria.

Background and Aim:Staphylococcus aureus is a leading cause of infection in both humans and animals. Most livestock strains have shown antibiotic resistance to the many molecules used in veterinary therapeutics. This study aimed to assess the resistance patterns of these bacteria, we carried out our study in the Tiaret and Souk Ahras areas of Algeria.Materials and Methods:We collected 116 samples of bovine and goat milk to detect S. aureus. We used a selective media to isolate the strains, followed by biochemically identifying the isolates. We determined the susceptibility of the strains to antibiotic molecules using the disk diffusion method and confirmed the methicillin-resistant S. aureus (MRSA) with oxacillin minimum inhibitory concentration (MIC).Results:Our results showed that 26.72% of the samples were contaminated with S. aureus, and we recovered 31 isolates from the positive samples. We ascribed a high resistance profile to penicillin G (96.77%), fusidic acid (67.74%), and tobramycin (45.16%) and isolated 4MRSA strains.Conclusion:The presence of S. aureus, including MRSA strains in raw milk, can present a public health hazard, because these strains can cause widespread food poisoning. This finding will be useful to the veterinarians to choose an adequate treatment and to sensitize livestock breeders and milk producers to ensure the health of consumers.

Isolation, characterisation and antibiotic susceptibility of staphylococcal isolates with special reference to methicillin-resistant Staphylococcus aureus from the anterior nares of healthcare workers in A tertiary healthcare centre

Background: Staphylococcus aureus (S. aureus) and its resistant form methicillinresistant S. aureus (MRSA) is one of the most common nosocomial pathogens causing a wide range of infections in humans. The anterior nares are the main ecological niche for S. aureus. Nasal carriage of S. aureus acts as an important reservoir of infection among the colonised healthcare workers and they transmit the infection to the community. The aim of the present study was to estimate the nasal colonisation of S. aureus (with special reference to MRSA) in healthcare workers (doctors and nursing staff) and its antibiotic susceptibility pattern. Methods: A descriptive study was planned in the Department of Microbiology, JLN Medical College, Ajmer (Rajasthan, India) after due approval from the institutional ethics committee. A total of 170 healthcare workers of either sex aged between 18 to 60 years were screened for S. aureus. Identification was done using standard microbiological techniques, by studying their morphology, colony and biochemical characteristics. MRSA was detected by cefoxitin disc diffusion test, oxacillin disc diffusion test, minimum inhibitory concentration (MIC) of oxacillin by E-test and oxacillin screen agar test. The observations were described in proportions and Chisquared test was used to find independence. Statistical significance was considered at 5 %. Results: Among 170 samples, 159 (93.53 %) samples (50 doctors and 109 nursing staff) had staphylococci colonisation. Among these 159 isolates, 34 (21.38 %) were S. aureus. Further, 8 (5.03 %) S. aureus isolates were resistant to both cefoxitin and oxacillin and had oxacillin MIC values ≥ 4 µg/mL and were considered MRSA. All the MRSA were detected in the nursing staff (males: 5.50 %, females: 1.83 %). All S. aureus and MRSA isolates were found sensitive to linezolid, vancomycin and mupirocin (minimum inhibitory concentration ≤ 4 µg/mL). Conclusion: Screening and treatment of healthcare workers colonised with MRSA should be an important component of hospital infection control policy. These measures will prevent spread of infection to patients and the community and thereby reduce the morbidity, mortality and healthcare costs associated with nosocomial infections.

Prevalence of multidrug resistance in the Egyptian methicillin-resistant Staphylococcus aureus aureus isolates

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major health hazards and became of greater public health concern. This work aimed to shed substantial light on prevalence of MRSA in different clinical isolates and their resistance to different antibiotics. Among 258 Staphylococcus aureus isolates recovered from different clinical sources (urine, pus, throat swab, blood, seminal fluid, prostatic fluid, sputum swab, ascetic fluid, skin swab, nipple discharge and urinary catheter). 70 isolates were identified as MRSA. The highest percentage of MRSA was recorded from pus samples (57.1%) followed by urine (30%). Antimicrobial susceptibility test using 14 antibiotics showed that all MRSA were resistant to amoxicillin and cefuroxime, while only 50% were sensitive to vancomycin. High minimum inhibitory concentration of oxacillin (256 µg/ml) was detected in 12.9% of MRSA isolates.

Combination Therapy Using Low-Concentration Oxacillin with Palmitic Acid and Span85 to Control Clinical Methicillin-Resistant Staphylococcus aureus.

The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). MRSA is difficult to kill with a single antibiotic because it has evolved to be resistant to various antibiotics by increasing the PBP2a (mecA) expression level, building up biofilm, introducing SCCmec for multidrug resistance, and changing its membrane properties. Therefore, to overcome antibiotic resistance and decrease possible genetic mutations that can lead to the acquisition of higher antibiotic resistance, drug combination therapy was applied based on previous results indicating that MRSA shows increased susceptibility to free fatty acids and surfactants. The optimal ratio of three components and the synergistic effects of possible combinations were investigated. The combinations were directly applied to clinically isolated strains, and the combination containing 15 μg/mL of oxacillin was able to control SCCmec type III and IV isolates having an oxacillin minimum inhibitory concentration (MIC) up to 1024 μg/mL; moreover, the combination with a slightly increased oxacillin concentration was able to kill SCCmec type II. Phospholipid analysis revealed that clinical strains with higher resistance contained a high portion of 12-methyltetradecanoic acid (anteiso-C15:0) and 14-methylhexadecanoic acid (anteiso-C17:0), although individual strains showed different patterns. In summary, we showed that combinatorial therapy with a low concentration of oxacillin controlled different laboratory and highly diversified clinical MRSA strains.

Development and in vitro evaluation of microparticles of fluoxetine in galactomannan against biofilms of S. aureus methicilin resistant

The emergence of multidrug-resistant (MDR) bacteria is a global problem, by reducing the effectiveness of traditional antibiotics and decreasing the therapeutic arsenal to treat bacterial infections. This has led to an increase in researches about how to overcome this resistance to antibiotics. One strategy is the repositioning (or repurposing) of existing drugs not previously used to combat microorganisms, rather than the development of new drugs. Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRIs) and is considered one of the first highly selective antidepressants of the monoamine neurotransmitter serotonin (5-HT). The objective of this study is to prepare and physically characterize fluoxetine microparticles with galactomannan and evaluate their efficacy against strains of Staphylococcus aureus sensitive and resistant to methicillin. The microparticles were analyzed by differential scanning calorimetry (DSC), infrared analysis (IR) and X-ray diffraction (XRD). In addition, the percentage of encapsulation efficiency (EE%) and drug release kinetics were determined in vitro, along with the determination of the minimum inhibitory concentration (MIC) and evaluation of the action against biofilms. Physical tests were conducted to characterize galactomannan (GAL), FLX, oxacillin (OXA) and the galactomannan/fluoxetine microparticles (GFM). The EE% value was 98 % and, in regard the release, tests with the microparticles released about 60 % of the drug in 200 min. The isolated MIC results for FLX (255 μg/mL) and OXA MIC (1.97–15.62 μg/mL) showed that the strains were resistant. Furthermore, in the biofilms, microparticles showed statically significant improvement for all concentrations used. The study revealed that fluoxetine encapsulated in microparticles has the potential to act as an effective antimicrobial agent.

Screening an Established Natural Product Library Identifies Secondary Metabolites That Potentiate Conventional Antibiotics.

Health organizations worldwide have warned that we are on the cusp of a "post-antibiotic era," necessitating new approaches to combat antibiotic resistant infections. One such approach is the development of antibiotic adjuvants, which have little or no inherent antibiotic activity at their active concentrations but instead potentiate the activity of antibiotics against antibiotic-resistant bacteria. Recently, we demonstrated that meridianin D, a natural product originally reported to have activity against Staphylococcus aureus and Mycobacterium tuberculosis, possesses the ability to reverse colistin resistance in colistin resistant bacteria. As most natural product screens typically involve screening for only certain activities (anticancer, antiviral, and antimicrobial are typical), we posited that the meridianin D discovery was not unique and there are potentially many natural products that have adjuvant activity. To explore this, the National Cancer Institute (NCI) Natural Product Library Set IV was screened for adjuvant activity using four classes of antibiotics (β-lactams, aminoglycosides, macrolides, and polymyxins) against three bacterial pathogens (methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, and Klebsiella pneumoniae). Sixteen compounds suppressed β-lactam resistance in MRSA, five of which effected a 16-fold reduction in the oxacillin minimum inhibitory concentration (MIC). Two natural products effectively suppressed aminoglycoside resistance in both of the Gram-negative species tested, and no hits were observed with macrolides. In contrast, a larger number of natural product adjuvants were identified when screening against colistin-resistant strains of A.baumannii and K.pneumoniae. Nine compounds reduced the colistin MIC to its breakpoint or lower (up to a 1024-fold reduction). Clorobiocin, novobiocin, and prodigiosin were most effective, reducing the colistin MIC in K.pneumoniae strain B9 to 2 μg/mL at concentrations as low as 0.625, 2.5, and 1.25 μM, respectively. Restored sensitivity to colistin with these compounds does not appear to coincide with known mechanisms of colistin resistance.

Borderline resistance to oxacillin in Staphylococcus aureus after treatment with sub-lethal sodium hypochlorite concentrations

Surface disinfectants are regularly used in prophylactic and infection control measures. Concern has been raised whether residues of sub-inhibitory disinfectant concentrations may constitute a selective pressure and could contribute to the development of strains which are tolerant and/or resistant to biocides including antibiotics. The current study investigated whether Staphylococcus (S.) aureus ATCC® 29213™ and ATCC® 6538™ would change their growth characteristics and antimicrobial susceptibility profiles after prolonged treatment with sub-inhibitory concentrations of sodium hypochlorite (NaOCl). NaOCl is a fast-acting disinfectant with a broad-spectrum activity, inexpensive and widely used in healthcare and the food production industry. Minimum inhibitory concentration (MIC) for NaOCl was determined by broth macrodilution according to the guidelines for disinfectant efficacy testing provided by the German Veterinary Medical Society. Serial passages after 24 h and 72 h, respectively, in defined sub-inhibitory concentrations of NaOCl resulted in a number of phenotypic variants. Two of these variants, derived from S. aureus ATCC® 29213™, showed elevated MICs of oxacillin and were considered as in vitro-generated borderline oxacillin-resistant S. aureus (BORSA). Transmission electron microscopy revealed a significantly thickened cell wall in these isolates, a phenomenon that has also been described for Listeria monocytogenes after low-level exposure to NaOCl. Whole genome sequencing revealed an early stop codon in the gene coding for the GdpP protein and thereby abolishing the function of this gene. GdpP represents a phosphodiesterase that regulates gene expression, and loss of function of the GdpP protein has been described in association with borderline oxacillin resistance. Our findings suggest that a mutation in the GdpP protein gene and morphological changes of the cell wall were induced by repeated exposure to sub-lethal NaOCl concentrations, and most likely accounted for a BORSA phenotype in two variants derived from S. aureus ATCC® 29213™.

Hospital epidemiology and antimicrobial susceptibility of isolated methicillin-resistant Staphylococcus aureus: a one-year retrospective study at a tertiary care center in Thailand

ABSTRACT Background: Increased rates of Staphylococcus aureus resistance and its morbidity and mortality have raised concern about the strategy of antibiotic use. Objectives: This study aimed to determine the prevalence of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) isolates among Thai patients with S. aureus infection and to identify risk factors and appropriate antibiotics for these resistant strains. Methods: Data of culture-proven S. aureus isolates from clinical specimens during 2017 in King Chulalongkorn Memorial Hospital, Thailand, were retrospectively collected and classified as methicillin-sensitive S. aureus or MRSA by cefoxitin screening and oxacillin minimum inhibitory concentration by the Vitek 2 system. Each isolate was also tested for susceptibility to teicoplanin, erythromycin, clindamycin, linezolid, trimethoprim/sulfamethoxazole, ciprofloxacin, moxifloxacin, tetracycline, doxycycline, and vancomycin by Vitek 2. Demographic information and comorbidities from medical records were reviewed to identify risk factors for S. aureus infection. Results: MRSA isolates were identified in 147 (17%) of 890 patients with no different ratio in adults or children. A higher proportion of MRSA in hospital-acquired settings was observed (27% vs. 12%; p < 0.001). Comorbidities significantly associated with MRSA were chronic lung, cardiovascular, and neurological diseases. Atrial fibrillation, dementia, and benign prostatic hyperplasia were independently associated with MRSA isolation. Vancomycin was still susceptible to all kinds of infection. One VRSA isolate was from colonization. Conclusion: The prevalence of MRSA in our facility seemed to be comparatively low. Vancomycin is still an appropriate option for MRSA coverage since all S. aureus isolates in our center were sensitive to vancomycin. However, careful attention is warranted since one colonization isolate was VRSA.

Evaluation of various phenotypic methods with genotypic screening for detection of methicillin-resistant Staphylococcus aureus

Abstract Background Staphylococcus aureus is one of the common opportunistic gram-positive pathogens which are often associated with nosocomial infections. Detection of methicillin-resistant S. aureus (MRSA) has become complicated due to the complex phenotypic and genomic pattern. Objective To evaluate the sensitivity and specificity pattern of various phenotypic methods used in screening mec genes harboring MRSA. Methods Clinical isolates of S. aureus were collected from diagnostic centers in Tamil Nadu. Phenotypic identification methods such as Minimal Inhibitory Concentration for oxacillin, oxacillin screen agar (OSA), oxacillin disk diffusion, and cefoxitin disk diffusion (CFD) tests were compared. The clinical isolates were classified into MRSA and methicillin-susceptible S. aureus (MSSA) based on the polymerase chain reaction (PCR) amplification of the mecA gene. Result Out of 50 S. aureus, 21 were found to be MRSA based on the presence of the mecA gene. All 21 mecA-positive isolates were found to be resistant through minimum inhibitory concentration (MIC) and CFD test, having a sensitivity of 100% and specificity of 52% and 62%, respectively. OSA and oxacillin disk tests were found to have a sensitivity of 86% and specificity of 48% and 52%, respectively. Conclusion The combination of two phenotypic methods, CFD and oxacillin MIC, can be used for the detection of MRSA in clinical laboratories.

Characterization of an Oxacillin-Susceptible mecA-Positive Staphylococcus aureus Isolate from an Imported Meat Product.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates that carry mecA but are oxacillin susceptible (OS-MRSA; oxacillin minimum inhibitory concentration [MIC] ≤2 μg/mL) are increasingly reported worldwide in the past decade. In this study, an OS-MRSA isolate FC1102921 from an imported meat sample was characterized and the oxacillin resistance mutation frequency and mutant prevention concentration (MPC) to oxacillin was determined. Isolate FC1102921 was pvl negative and contained SCCmec V (5C2&5), the sequence type and spa type were identified as ST398-t034. The MPCs of isolate FC1102921 to oxacillin was 16 μg/mL. The resistance mutation frequency of isolate FC1102921 to oxacillin was 0.036%. The expression of mecA in isolate FC1102921 was detectable, but the mecA expression in the mutant isolate FC1102921m increased 13.8-folds. Our data indicated that S. aureus FC1102921 was not a hypermutator, and the high-resistance mutation frequency of S. aureus FC1102921 was specific to oxacillin and penicillin. SCCmec V region of isolate FC1102921 and FC1102921m was identical, functional blaI and blaR1 were identified in SCCmec region of both isolates, and no plasmid loss was identified in mutant isolate FC1102921m. To the best of our knowledge, this is the first report of the resistance mutation frequency and MPC study of an OS-MRSA ST398-t034 isolate. The high oxacillin MPC value and high oxacillin resistance mutation frequency was not related to a curable plasmid as previously reported, which further highlighted that an in-depth study should be carried out to clarify the mechanism and role of the OS-MRSA isolates in the emergence and transmission of MRSA ST398-t034.

Antibiotic hypersensitivity in MRSA induced by special protein aggregates

Emergence of multidrug-resistant bacteria is a major global concern. According to WHO, methicillin-resistant Staphylococcus aureus (MRSA) is a threatening pathogen resistant to a wide spectrum of antibiotics. Herein, to overcome drug resistance in MRSA, we successfully integrated traditional antibacterial methods but with a novel trick that included use of hen egg-white lysozyme's special aggregates generated by fibrillization. The minimum inhibitory concentration of oxacillin (Ox) for MRSA declined from 600 μM to <20 μM when using aggregates. Scanning and transition electron micrographs showed completely disrupted cells when treated with aggregated protein/Ox (20 μM). The assisting role of aggregates to induce antibiotic hypersensitivity was continuous and stable, but sub-inhibitory antibiotic concentration (20 μM) was required again after 8 h. Investigations regarding mechanism of antibiotic hypersensitivity revealed that aggregates were oligomers but not mature fibrils. Furthermore, reactive oxygen species levels rose significantly after treating bacteria with aggregated protein/Ox. Study of resistance mechanisms indicated that in response to wall structure alterations, mecA expression dropped significantly in the presence of aggregated protein/Ox (20 μM) relative to Ox (20 μM). This observation can be a breakthrough in finding alternatives where antibiotic dosage can be significantly reduced, thereby preventing emergence of new multidrug-resistant bacteria.