Oxacillin MIC Research Articles

Potential antivirulence and antibiofilm activities of sub-MIC of oxacillin against MDR S. aureus isolates: an in-vitro and in-vivo study

BackgroundMulti-drug resistant Staphylococcus aureus is one of the most common causes of nosocomial and community-acquired infections, with high morbidity and mortality. Treatment of such infections is particularly problematic; hence, it is complicated by antibiotic resistance, and there is currently no reliable vaccine. Furthermore, it is well known that S. aureus produces an exceptionally large number of virulence factors that worsen infection. Consequently, the urgent need for anti-virulent agents that inhibit biofilm formation and virulence factors has gained momentum. Therefore, we focused our attention on an already-approved antibiotic and explored whether changing the dosage would still result in the intended anti-virulence effect.MethodsIn the present study, we determined the antibiotic resistance patterns and the MICs of oxacillin against 70 MDR S. aureus isolates. We also investigated the effect of sub-MICs of oxacillin (at 1/4 and 1/8 MICs) on biofilm formation using the crystal violet assay, the phenol-sulphuric acid method, and confocal laser scanning microscopy (CLSM). We examined the effect of sub-MICs on virulence factors and bacterial morphology using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electron microscopy, respectively. Moreover, we studied the effect of sub-MICs of oxacillin (OX) in-vivo using a wound infection model.ResultsOxacillin at 1/2 MIC showed a significant decrease in bacterial viability, while 1/4 and 1/8 MICs had negligible effects on treated bacterial isolates. Treatment of MDR isolates with 1/4 or 1/8 MICs of oxacillin significantly reduced biofilm formation (64% and 40%, respectively). The treated MDR S. aureus with sub-MICs of OX exhibited a dramatic reduction in several virulence factors, including protease, hemolysin, coagulase, and toxic shock syndrome toxin-1 (TSST-1) production. The sub-MICs of OX significantly decreased (P < 0.05) the gene expression of biofilm and virulence-associated genes such as agrA, icaA, coa, and tst. Furthermore, oxacillin at sub-MICs dramatically accelerated wound healing, according to the recorded scoring of histological parameters.ConclusionThe treatment of MDR S. aureus with sub-MICs of oxacillin can help in combating the bacterial resistance and may be considered a promising approach to attenuating the severity of S. aureus infections due to the unique anti-biofilm and anti-virulence activities.

Role of Oxacillin Susceptibility Testing Strategy in Changing Scenario of mecA Positive Staphylococcus aureus Isolates (OS-MRSA) Detection

Staphylococcus aureus strains that are mecA and PBP2a positive but phenotypically susceptible to oxacillin are becoming more and more abundant, according to research from all around the world. The oxacillin susceptibility of Staphylococcus aureus (OS-MRSA) contributes to consequent treatment-failure due to misidentification by conventional susceptibility tests. Therefore, the objective of the current study was to ascertain the prevalence of OSMRSA in a tertiary care facility located in Mysore, South India. 395 MRSA isolates collected from diverse clinical samples were included in this lab-based prospective investigation. These isolates were tested using an oxacillin 1μg disc phenotypically by standard disc diffusion test, and simultaneously MIC to Oxacillin was determined from Vitek2 systems. Additionally, MRSA specific mecA gene detection was applied to these isolates in order to confirm their MRSA status genotypically. PCR findings demonstrate that 65% of the isolates were MRSA. The vitek2 system detected 4.06% OS-MRSA isolates with an oxacillin MIC of ≤2µg/ml. The disc diffusion method identified a total of 13.75% isolates as oxacillin sensitive and 10% isolates were oxacillin intermediately sensitive. Oxacillin sensitivity was shown for 1.87% of the mecA-positive MRSA isolates using the VITEK2 and disc diffusion techniques. This analysis found isolates with lower oxacillin MICs but relatively reduced OS-MRSA incidence. Using an oxacillin disc for routine laboratory MRSA detection might occasionally produce false negative results, which can result in improper antibiotic administration and treatment failure. In order to distinguish OS-MRSA from MRSA, it is crucial to combine phenotypic and genotypic techniques.

In vitro and in vivo susceptibility to cefalexin and amoxicillin/clavulanate in canine low-level methicillin-resistant Staphylococcus pseudintermedius.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) lineages harbouring staphylococcal cassette chromosome (SCC) mec types IV, V and ΨSCCmec57395 usually display low oxacillin MICs (0.5-2 mg/L). To evaluate how oxacillin MICs correlate with PBP mutations and susceptibility to β-lactams approved for veterinary use. Associations between MICs and PBP mutations were investigated by broth microdilution, time-kill and genome sequence analyses in 117 canine MRSP strains harbouring these SCCmec types. Clinical outcome was retrospectively evaluated in 11 MRSP-infected dogs treated with β-lactams. Low-level MRSP was defined by an oxacillin MIC <4 mg/L. Regardless of strain genotype, all low-level MRSP isolates (n = 89) were cefalexin susceptible, whereas no strains were amoxicillin/clavulanate susceptible according to clinical breakpoints. Exposure to 2× MIC of cefalexin resulted in complete killing within 8 h. High (≥4 mg/L) oxacillin MICs were associated with substitutions in native PBP2, PBP3, PBP4 and acquired PBP2a, one of which (V390M in PBP3) was statistically significant by multivariable modelling. Eight of 11 dogs responded to systemic therapy with first-generation cephalosporins (n = 4) or amoxicillin/clavulanate (n = 4) alone or with concurrent topical treatment, including 6 of 7 dogs infected with low-level MRSP. Oxacillin MIC variability in MRSP is influenced by mutations in multiple PBPs and correlates with cefalexin susceptibility. The expert rule recommending that strains with oxacillin MIC ≥0.5 mg/L are reported as resistant to all β-lactams should be reassessed based on these results, which are highly clinically relevant in light of the shortage of effective antimicrobials for systemic treatment of MRSP infections in veterinary medicine.

Role of the NaHCO3 Transporter MpsABC in the NaHCO3-β-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care β-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO3 responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to β-lactams such as cefazolin and oxacillin in the presence of NaHCO3. A bicarbonate transporter, MpsAB (membrane potential-generating system), was recently found in S. aureus, where it plays a role in concentrating NaHCO3 for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype. Radiolabeled NaH14CO3 uptake profiling revealed significantly higher accumulation in NaHCO3-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO2 conditions, NaHCO3-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO3 supplementation under 5% CO2 conditions. NaHCO3-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO3-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO3 transporter MpsABC is a key contributor to the NaHCO3-β-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most β-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO3 responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to β-lactams in the presence of NaHCO3. A recently described S. aureus NaHCO3 transporter, MpsAB, is involved in intracellular NaHCO3 concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO3-β-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using β-lactams.

Retrospective Evaluation of the Association of Oxacillin MIC on Acute Treatment Outcomes with Cefazolin and Antistaphylococcal Penicillins in Methicillin-Susceptible Staphylococcus aureus Bacteremia.

Antistaphylococcal penicillins (ASP) and cefazolin are first-line treatment of methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. Borderline oxacillin resistance (i.e., oxacillin MICs 1-8 μg/mL) is observed in strains hyperproducing beta-lactamases. This mechanism is also behind the proposed inoculum effect. Minimal data exists on the comparative efficacy of cefazolin or ASP in qualitatively susceptible strains that demonstrate MICs of oxacillin of 1 to 2 μg/mL compared to strains with MIC of oxacillin < 1 μg/mL. We performed a retrospective cohort study of acute treatment outcomes in adult patients with community-acquired MSSA bacteremia treated with cefazolin or ASP, stratified by oxacillin MIC. The primary outcome was a composite of all-cause mortality during the index inpatient admission, failure to clear blood cultures within 72 h after initiating definitive therapy, and change in therapy due to perceived lack of efficacy. A total of 402 patients were included in this study, including 226 isolates with an oxacillin MIC ≥ 1 μg/mL and 176 isolates with an MIC < 1 μg/mL. There were no differences in the rate of the primary outcome occurrence between patients with an oxacillin MIC ≥ 1 μg/mL and an MIC < 1 μg/mL (16.4% versus 15.9%, P = 0.90). There was no difference in the primary outcome between high versus low oxacillin MIC groups among those who received ASP (22.9% versus 24.1%, P = 0.86) or cefazolin (10.3% versus 11.9%, P = 0.86). In our cohort of patients with MSSA bacteremia, oxacillin MIC (i.e., ≥ 1 versus < 1 μg/mL) was not associated with acute treatment outcomes, regardless of the beta-lactam selected as definitive therapy.

The Lysin Exebacase Has a Low Propensity for Resistance Development in Staphylococcus aureus and Suppresses the Emergence of Resistance to Antistaphylococcal Antibiotics.

Exebacase (CF-301) belongs to a novel class of protein-based antibacterial agents, called lysins (peptidoglycan hydrolases). Exebacase exhibits potent antistaphylococcal activity and is the first lysin to initiate clinical trials in the United States. To support clinical development, the potential for resistance development to exebacase was assessed over 28 days of serial daily subculture in the presence of increasing concentrations of the lysin performed in its reference broth medium. Exebacase MICs remained unchanged over serial subculture for three replicates each of methicillin-susceptible Staphylococcus aureus (MSSA) strain ATCC 29213 and methicillin-resistant S. aureus (MRSA) strain MW2. For comparator antibiotics also tested, oxacillin MICs increased by 32-fold with ATCC 29213 and daptomycin and vancomycin MICs increased by 16- and 8-fold, respectively, with MW2. Serial passage was also used to examine the capacity of exebacase to suppress selection for increased oxacillin, daptomycin, and vancomycin MICs when used together in combination, wherein daily exposures to increasing concentrations of antibiotic were performed over 28 days with the added presence of fixed sub-MIC amounts of exebacase. Exebacase suppressed increases in antibiotic MICs over this period. These findings are consistent with a low propensity for resistance to exebacase and an added benefit of reducing the potential for development of antibiotic resistance. IMPORTANCE To guide development of an investigational new antibacterial drug, microbiological data are required to understand the potential for development of resistance to the drug in the target organism(s). Exebacase is a lysin (peptidoglycan hydrolase) that represents a novel antimicrobial modality based on degradation of the cell wall of Staphylococcus aureus. Exebacase resistance was examined here using an in vitro serial passage method that assesses the impact of daily exposures to increasing concentrations of exebacase over 28 days in medium approved for use in exebacase antimicrobial susceptibility testing (AST) by the Clinical and Laboratory Standards Institute (CLSI). No changes in susceptibility to exebacase were observed over the 28-day period for multiple replicates of two S. aureus strains, indicating a low propensity for resistance development. Interestingly, while high-level resistance to commonly used antistaphylococcal antibiotics was readily obtained using the same method, the added presence of exebacase acted to suppress antibiotic resistance development.

Phenotypic and genotypic characterization of oxacillin-susceptible and mecA positive Staphylococcus aureus strains isolated in Uruguay

The aim of this study was to characterize phenotypically and genotypically 27 mecA positive Staphylococcus aureus strains with oxacillin MICs of ≤2μg/ml by Vitek 2, isolated in different regions of Uruguay. Susceptibility to oxacillin and cefoxitin was studied by gradient diffusion, disk diffusion to cefoxitin, and Phoenix and MicroScan systems. PBP2a was determined. SCCmec typing was performed and the isolates were compared by PFGE. Twenty-six isolates were susceptible to oxacillin; one strain was susceptible to cefoxitin by disk diffusion and 3 strains by gradient diffusion. Phoenix and MicroScan panels detected methicillin resistance in 25 and 27 strains, respectively. Twenty-six strains tested positive for PBP2a. Twenty-six strains carried SCCmec V and 24 belonged to pulsotype A. One strain carried SCCmec IV and did not belong to pulsotype A. Cefoxitin disk diffusion test and PBP2a detection correctly identified 26 of these 27 strains as MRSA. PFGE results suggest the dissemination of a cluster of MRSA carrying SCCmec V.

O03 ST97 Staphylococcus aureus and oxacillin resistance: an emerging challenge for microbiologists?

BackgroundThe presence of cefoxitin and oxacillin resistance in Staphylococcus aureus isolates is suggestive of methicillin resistance (MRSA) with molecular detection of the mecA or mecC genes providing confirmation. The UK SMI and EUCAST guidelines for MRSA detection describe a subset of strains that exhibit reduced susceptibility to oxacillin and cefoxitin, though negative for mecA and mecC genes—frequently named borderline oxacillin-resistant S. aureus (BORSA). Local data noted a number of S. aureus isolates that matched the above phenotype leading to laboratory, clinical and epidemiological questions.MethodsRoutine samples were collected between August 2020 and April 2021 at West Midlands Health Security Agency Laboratory, Birmingham. If provisional results suggested an MRSA isolate, the following confirmatory tests were undertaken. Locally, clinical samples underwent susceptibility testing with cefoxitin disc diffusion and oxacillin gradient diffusion (MRSA screens) or Vitek 2 (non-MRSA screens). Isolates with discrepant results [susceptible to cefoxitin; resistant to oxacillin (MIC >2 mg/L) or vice versa] were sent to the reference laboratory at Colindale, UK for further testing, which included mecA or mecC gene detection by PCR and serotyping with Illumina sequencing for all isolates.ResultsIn total, 53 isolates were sent to the reference lab of which 22 were confirmed to have an ST97 serotype. All 53 were negative by PCR for mecA and mecC genes. For the 22 isolates of ST97, local oxacillin MIC values ranged from 2 to 6 mg/L. Cefoxitin susceptibility was confirmed locally in all isolates. The 22 isolates were from 15 patients; wound swabs (n = 18); blood culture (n = 1), sputum (n = 1) and MRSA screen (n = 2). Median patient age was 48 years (IQR 38–59) with 5 being female and 10 male. A history of injecting drug use was documented in 53% (8/15).ConclusionsBORSA remains a problem from a laboratory, clinical and infection control perspective. This work raises two important questions: what is the most appropriate local laboratory testing pathway and what is the clinical relevance of these isolates (i.e. can flucloxacillin be relied upon in treatment)? The ST97 serotype appears to be associated with skin and soft tissue infection and may be linked to people who inject drugs.

Prevalence, Mechanism, Genetic Diversity, and Cross-Resistance Patterns of Methicillin-Resistant Staphylococcus Isolated from Companion Animal Clinical Samples Submitted to a Veterinary Diagnostic Laboratory in the Midwestern United States.

Methicillin-resistant Staphylococcus (MRS) is a leading cause of skin and soft tissue infections in companion animals, with limited treatment options available due to the frequent cross-resistance of MRS to other antibiotics. In this study, we report the prevalence, species distribution, genetic diversity, resistance mechanism and cross-resistance patterns of MRS isolated from companion animal (mostly dog and cat) clinical cases submitted to Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) between 2012 and 2019. The majority of isolates were identified as Staphylococcus pseudintermedius (68.3%; 2379/3482) and coagulase-negative Staphylococcus (CoNS) (24.6%; 857/3482), of which 23.9% and 40.5% were phenotypically resistant to methicillin, respectively. Cross resistance to other β-lactams (and to a lesser extent to non-β-lactams) was common in both methicillin-resistant S. pseudintermedius (MRSP) and CoNS (MRCoNS), especially when oxacillin MIC was ≥4 μg/mL (vs. ≥0.5–<4 μg/mL). The PBP2a protein was detected by agglutination in 94.6% (521/551) MRSP and 64.3% (146/227) MRCoNS. A further analysis of 31 PBP2a-negative MRS isolates (all but one MRCoNS) indicated that 11 were mecA gene-positive while 20 were negative for mecA and other mec genes by PCR. The resistance to last-resort anti-staphylococcal human drugs (e.g., tigecycline, linezolid, vancomycin) among the MRS tested was none to very low. Even though genotyping indicated an overall high level of genetic diversity (87 unique PFGE patterns and 20 MLST types) among a subset of MRSP isolates tested (n = 106), certain genotypes were detected from epidemiologically connected cases at the same or different time points, suggesting persistence and/or nosocomial transmission. These results indicate a relatively high prevalence of MRS from companion animals in the Midwestern US; therefore, it is important to perform routine susceptibility testing of Staphylococcus in veterinary clinical settings for the selection of appropriate antimicrobial therapy.

Clerodane diterpenoids with anti-inflammatory and synergistic antibacterial activities from Tinospora crispa

Compounds 5 and 7 exhibited nitric oxide release inhibitory activities with IC50 values of 7.5 and 10.6 μM, respectively, and compounds 9 and 14 at the sub-MIC level significantly decreased the MIC of oxacillin against MRSA from 32.0 to 1.0 and 0.5 μg mL−1, respectively.

Regulation of bla system in ST59-related oxacillin-susceptible mecA-positive Staphylococcus aureus.

Oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) is clinically significant and isolated globally but the mechanism of its occurrence remains indistinct. We sought to assess the mechanism of regulating oxacillin susceptibility in OS-MRSA isolates by evaluating the evolutionary dynamics of OS-MRSA and the discrepancies of mecA-regulating genes in OS-MRSA and oxacillin-resistant MRSA (OR-MRSA). Nine OS-MRSA isolates and 77 OR-MRSA isolates were sequenced using next-generation sequencing (NGS) platforms. Two representative OS-MRSA isolates (ET-13, ET-16) were induced to be oxacillin resistant and sequenced also. OS-MRSA ET-16 and its counterpart isolate with induced oxacillin resistance, ET-16I, and their mutants were used to confirm the role of the bla system in regulating methicillin susceptibility. Oxacillin MICs were determined using Etests. Expression of mecA and blaR1 was quantified by quantitative RT-PCR. A deletion in blaR1 in most OS-MRSA isolates (7/9; 77.78%) was found using NGS data, and overexpression of OR-blaR1 in OS-MRSA isolate ET-16 restored its oxacillin resistance. OS-MRSA could be induced to be oxacillin resistant, while growth was suppressed in the induced isolates. Plasmid containing the bla locus was lost in most induced isolates during the induction process and complementation of blaR1-blaI from OS-MRSA ET-16 to the induced isolate ET-16I converted its oxacillin susceptibility. Deletion in blaR1 resulted in oxacillin susceptibility in OS-MRSA, and loss of the bla regulator in OS-MRSA restored oxacillin resistance. The bla system played a crucial role in regulating oxacillin susceptibility in OS-MRSA isolates.

1240. Ceftobiprole Activity against Drug-Resistant Staphylococcus aureus Clinical Isolates Collected in the United States from 2016 through 2020

BackgroundMultidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA) present significant treatment challenges and can cause serious morbidity and mortality. Ceftobiprole, the active moiety of the prodrug ceftobiprole medocaril, is an advanced cephalosporin approved in many European and other countries for the treatment of adults with community- and hospital-acquired pneumonia, excluding ventilator-associated pneumonia. Ceftobiprole is currently in phase 3 clinical development to support a New Drug Application in the United States for acute bacterial skin and skin structure infections and S. aureus bacteremia. Here, the activity of ceftobiprole and comparators was evaluated against recent MDR S. aureus and MRSA clinical isolates.Methods13,868 S. aureus isolates were collected from patients with various infection types at 34 US medical centers from 2016–2020. Susceptibility to ceftobiprole and comparator agents was tested by CLSI methods. Current CLSI and EUCAST interpretive criteria were applied (Table). Isolates were categorized as MDR if they were non-susceptible (NS; CLSI criteria) to ≥3 of the following antimicrobials: clindamycin (CM), daptomycin (DAP), erythromycin (ERY), gentamicin (GM), levofloxacin (LEV), linezolid (LZD), tetracycline (TET), tigecycline (TGC), trimethoprim-sulfamethoxazole (TMP-SMX), or vancomycin (VAN). Isolates displaying oxacillin MIC values ≥4 mg/L were categorized as MRSA.ResultsCeftobiprole was more active than ceftaroline (CPT) against MRSA (99.2% susceptible [S] versus 94.0% S, respectively) (Table). Ceftobiprole maintained activity against 88.0% of the CPT-NS isolates, but CPT was only active against 6.5% of the ceftobiprole-NS isolates. Ceftobiprole was also highly active (97.7–100.0% S) against isolates NS to CM, DAP, ERY, GM, LEV, LZD, TET, TGC, or TMP-SMX. No VAN-NS isolates were detected. Importantly, ceftobiprole was more active (97.7% S) than CPT (83.0% S) against the subset of MDR-MRSA isolates.Conclusion Conclusions: Ceftobiprole was highly active in vitro against MRSA and MDR S. aureus collected at US medical centers during 2016–2020. These results support the further development of ceftobiprole to treat S. aureus infections in the US. Disclosures Leonard R. Duncan, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services (Research Grant or Support, Contract no. HHSO100201600002C)Shionogi (Research Grant or Support) Kamal Hamed, MD, MPH, Basilea Pharmaceutica International, Ltd (Employee)Department of Health and Human Services (Research Grant or Support, Contract no. HHSO100201600002C) Jennifer Smart, PhD, Basilea Pharmaceutica International, Ltd. (Employee)Department of Health and Human Services (Research Grant or Support, Contract no. HHSO100201600002C) Michael A Pfaller, MD, Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Department of Health and Human Services (Research Grant or Support, Contract no. HHSO100201600002C)Pfizer, Inc. (Research Grant or Support) Helio S. Sader, MD, PhD, FIDSA, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services (Research Grant or Support, Contract no. HHSO100201600002C)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)

Inducible Resistance to β-Lactams in Oxacillin-Susceptible mecA1-Positive Staphylococcus sciuri Isolated From Retail Pork.

Most isolated strains of Staphylococcus sciuri contain mecA1, the evolutionary origin of mecA, but are sensitive to β-lactams (OS-MRSS, oxacillin-susceptible mecA1-positive S. sciuri). In order to improve the efficacy of antibiotic treatment, it is important to clarify whether the resistance of OS-MRSS to β-lactams is an inducible phenotype. In this study, three OS-MRSS strains with oxacillin MIC = 1 μg/ml were isolated from 29 retail pork samples. The resistance of OS-MRSS to β-lactams (MIC > 256 μg/ml) was found to be induced by oxacillin, and the induced resistance was observed to remain stable within a certain period of time. Interestingly, the induced β-lactam resistance was not caused by mecA1, heterogeneous resistance, or any genetic mutation, but mainly due to increased wall teichoic acid (WTA) synthesis that thickened the cell wall. The induced strains also showed slower growth rate, as well as decreased adhesion ability and biofilm thickness. These phenotypes were found to be achieved through altered gene expression in associated pathways, such as the citrate cycle and pentose phosphate pathway. The results challenge the traditional antibiotic sensitivity test. In the presence of β-lactam antibiotics, OS-MRSS that was initially sensitive to β-lactams was observed to gradually develop β-lactam resistance in several days. This often-neglected phenomenon in antibiotic sensitivity tests requires further research attention.

Evaluation of cefoxitin disc diffusion test as a rapid phenotypic method for detection of methicillin resistance

: Accuracy and promptness in the detection of methicillin resistance are of key importance in ensuring correct antibiotic treatment in infected patients and control of MR staphylococci in the hospital environment. The aim of this study was to detect MRSA phenotypically by oxacillin screen agar and Oxacillin MIC method and to evaluate cefoxitin disc diffusion test as a screening tool for MRSA detection. In the present study, a total of 50 isolates of from various clinical samples collected were used for the detection of Methicillin resistant (MRSA). Methicillin resistance was determined by oxacillin disc diffusion, cefoxitin disc diffusion the oxacillin screen agar test and MIC. Out of 50 isolates 21 (42%) isolates were detected as MRSA based on MIC method, which is considered as gold standard method for the detection of MRSA. All the isolates of MRSA were 100% susceptible to vancomycin and linezolid. rnIn the present study, cefoxitin diffusion method has given 100% sensitivity and specificity in concordance with MIC method. However, the oxacillin screen agar method showed 95.24% sensitivity and 96.55% specificity. As per our study and previous reports elsewhere on phenotypic detection of MRSA, cefoxitin is more potent inducer of the regulatory system and an accurate surrogate marker for the detection of MRSA in the routine susceptibility testing.

Swine as reservoirs of zoonotic borderline oxacillin-resistant Staphylococcus aureus ST398

Methicillin resistance mediated by the mecA gene in Staphylococcus aureus, also known as “true MRSA”, is typically associated with high oxacillin MIC values (≥8 mg/L). Because non-mecA-mediated oxacillin resistant S. aureus phenotypes can also cause hard-to-treat diseases in humans, their misidentification as methicillin-susceptible S. aureus strains (MSSA) can compromise the efficiency of the antimicrobial therapy. These strains have been refereed as Borderline Oxacillin-Resistant S. aureus (BORSA) but their characterization and role in clinical microbiology have been neglected. Considering the increasing importance of livestock-associated methicillin-resistant S. aureus ST398 (LA-MRSA) as an emerging zoonotic pathogen worldwide, this study aimed to report the genomic context of oxacillin resistance in porcine S. aureus ST398 strains. S. aureus isolates were recovered from asymptomatic pigs from three herds. Oxacillin MIC values ranged from 4 to 32 mg/L. MALDI-TOF-confirmed isolates were screened for mecA and mecC by PCR and genotyped by means of PFGE and Rep-PCR. Seven isolates were whole genome sequenced. None of the isolates harbored the mecA gene or its variants. Although all seven sequenced isolates belonged to one sequence type (ST398), two different spa types (t571 and t1471) were identified. All isolates harbored conserved blaZ gene operon and no mutations on genes encoding for penicillin-binding-proteins were detected. Genes conferring resistance against other drugs such as aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin (MLS), tetracycline and trimethoprim were also detected. Isolates also harbored virulence genes encoding for adhesins (icaA; icaB; icaC; icaD; icaR), toxins (hlgA; hlgB; hlgC; luk-PV) and protease (aur). Pigs can serve as reservoirs of non-mecA-mediated oxacillin-resistant ST398 strains potentially pathogenic to humans. Considering that mecA has been the main target to screen methicillin-resistant staphylococci, the occurrence of BORSA phenotypes is probably underestimated in livestock.

Identification of Staphylococcus pseudintermedius Isolates from Wound Cultures by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Improves Accuracy of Susceptibility Reporting at an Increase in Cost.

Staphylococcus pseudintermedius can easily be mistaken for Staphylococcus aureus using phenotypic and rapid biochemical methods. We began confirming the identification of all coagulase-positive staphylococci isolated from human wound cultures at our centralized laboratory, servicing both community and inpatients, with matrix-assisted laser desorption ionization-time of flight mass spectrometry instead of using phenotypic and rapid biochemical tests, and determined the prevalence of S. pseudintermedius since the change in identification procedure and at what cost. A retrospective review was performed on all wound swab cultures from which coagulase-positive staphylococci were isolated 7 months before and after the change in identification procedure. A total of 49 S. intermedius/pseudintermedius (SIP) isolates were identified, including 7 isolates from 14,401 wound cultures in the before period and 42 isolates from 14,147 wound cultures in the after period. The number of SIP isolates as a proportion of isolated coagulase-positive staphylococci increased significantly from the before, 7/6,351 (0.1%), to the after, 42/5,435 (0.7%), period (difference, 0.6% [95% confidence interval, 0.037 to 0.83%, P < 0.0001]). Antibiotic susceptibility testing was performed in 42 isolates; none had an oxacillin MIC of 1.0 to 2.0 μg/ml, the range in which, if the isolate was misidentified as S. aureus, a very major error in susceptibility interpretation would occur. The increase in cost of the change in identification procedure was Can$17,558 per year in our laboratory, performing microbiology testing for community and acute-care patients in a zone servicing nearly 1.7 million people. While we will only continue to learn more about this emerging pathogen if we make attempts to properly identify it in clinical cultures, the additional time and cost involved may be unacceptably high in some laboratories. .

Improved Detection of mecA-Mediated β-Lactam Resistance in Staphylococcus lugdunensis Using a New Oxacillin Salt Agar Screen.

Oxacillin resistance mediated by mecA in Staphylococcus lugdunensis is emerging in some geographic areas. We evaluated cefoxitin disk diffusion (DD) and a new oxacillin agar (supplemented with 2 μg/ml oxacillin and 2% sodium chloride) screen for the detection of mecA-mediated resistance in S. lugdunensis. A total of 300 consecutive, non-duplicated clinical S. lugdunensis isolates from diverse sources in Hong Kong in 2019 were tested. The categorical agreement and errors obtained between cefoxitin DD test, oxacillin agar screen and mecA PCR were analyzed. Isolates with discordant results were further tested by MIC, penicillin binding protein 2a (PBP2a) assays, population analysis and molecular typing. PCR showed that 62 isolates were mecA-positive and 238 isolates were mecA-negative. For cefoxitin DD results interpreted using S. aureus/S. lugdunensis breakpoints, the categorical agreement (CA) for two brands of Muller-Hinton agars, MH-II (Becton Dickinson) and MH-E (bioMérieux) were both 96.0%; MEs were both 0%; and VMEs were 19.4 and 12.9%, respectively. The new oxacillin agar reliably differentiated mecA-positive and mecA-negative isolates (100% CA) without any ME or VME results. The 8 isolates with false susceptibility in the cefoxitin DD testing had cefoxitin and oxacillin MICs in the susceptible range. The isolates showed heterogeneous oxacillin resistance with resistant subpopulations at low frequencies. All had positive PBP2a results and were typed as sequence type 27/SCCmec V. The findings highlight the inability of cefoxitin DD and MIC tests for reliable detection of some mecA-positive S. lugdunensis isolates.

Antibacterial and synergistic activity of 6β-hydroxy-3-oxolup-20(29)-en-28-oic acid (6β-hydroxy betunolic acid) isolated from Schumacheria castaneifolia vahl

Multi- drug resistant microbial pathogens are a serious global health problem and thus new antibacterial agents, which are effective both alone and in combination with traditional antibiotics, are urgently needed. Hence, the objective of the present study was to investigate the antibacterial activity of 6β-hydroxy-3-oxolup-20(29)-en-28-oic acid (6β-hydroxy betunolic acid) isolated from the bark of Schumacheria castaneifolia and its effect when combined with oxacillin. Antibacterial potential of 6β-hydroxy betunolic acid alone was performed using broth micro dilution assay against sixteen bacterial strains which included eight standard strains [Staphylococcus aureus (ATCC 29213 and ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 35218 and ATCC 25922), carbapenemase producing Kebsiella pneumonia (ATCC BAA 1705), carbapenemase non-producing K. pneumonia (ATCC BAA 1706) and Pseudomonas aeruginosa (ATCC 27853)] and four strains each of clinically isolated meropenem resistant Acinetobactor sp. and methicillin resistant S. aureus (MRSA) which were included in the urgent threat list and serious threat list, respectively in 2019 by the Centers for Disease Control and Prevention in the United States. Its effect when combined with oxacillin was tested against S. aureus (ATCC 29213) and MRSAs using a checkerboard dilution method. The results indicated that 6β-hydroxy betunolic acid had antibacterial activity against the tested Gram positive organisms with MICs ranging from 16 to 32 mg L−1 (MIC of oxacillin and meropenem ranged from 0.25–16 and 0.03–128 mg L−1 respectively). The high MIC values (>1024 mg L−1) of 6β-hydroxy betunolic acid against Gram negative strains indicated a likely lack of activity. Further, 6β-hydroxy betunolic acid exhibited synergistic effect with oxacillin against Staphylococcus aureus (0.49) and showed an additive effect against all the tested MRSAs. The present study suggested that the antibacterial activity of the 6β-hydroxy betunolic acid is restricted to Staphylococcus isolates and possibly Enterococcus faecalis. Further testing on different types of Gram positives and identification of the exact mechanism of action would be of importance.

Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria.

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

Clonal Diversity, Low-Level and Heterogeneous Oxacillin Resistance of Oxacillin Sensitive MRSA.

PurposeThis study investigates the phenotypic and genotypic resistance features of OS-MRSA clinical isolates and their distinctive sensitivities to oxacillin.Methods1200 clinical isolates of Staphylococcus aureus were enrolled in this study. Automated antibiotics susceptibility tests on VITEK-2 and BD Phoenix-100TM, cefoxitin disc diffusion method, oxacillin broth microdilution method, mecA, and mecC gene detection were performed to identify OS-MRSA. MLST, PFGE, SCCmec, and spa typing methods were employed to determine genotypes of OS-MRSA isolates. Heterogeneous resistance of OS-MRSA isolates was detected using the population analysis profiling method, and PBP2a latex agglutination assay was used to detect the expression of PBP2a protein for 14 OS-MRSA isolates and their highly resistant subpopulations.ResultsA total of 14 OS-MRSA isolates (1.17%, 14/1200) were identified, and all of the isolates were confirmed to be positive with the mecA gene and negative with the mecC gene. All of the 14 OS-MRSA isolates were identified as MSSA by VITEK-2, BD Phonenix-100, and oxacillin broth microdilution methods, while 21.43% (3/14) isolates were determined to be MRSA by the cefoxitin disk diffusion method. Genotypes of the 14 OS-MRSA isolates were diverse, and no dominant clones were identified. The prevalence of pvl gene among 14 OS-MRSA isolates was high up to 64.29% (9/14). All of the isolates showed heterogeneous resistance to oxacillin, while frequencies of the oxacillin-resistant subpopulations ranged from 10−9 to 10−5 and differed significantly among different isolates.ConclusionThe overall prevalence of OS-MRSA was relatively lower, but lower oxacillin MICs, low testing sensitivity of routine antibiotics susceptibility testing methods and weak PBP2a protein expression were observed in this study. 14 OS-MRSA showed diverse genotypes and universal heterogeneous resistance, and inaccurate laboratory identification and improper antimicrobial usage may promote the induction of highly resistant subpopulations and lead to treatment failure.