The Immediate and Delayed Maximal Nonirritating Skin Testing Concentrations of ß-Lactam Antibiotics.

BACKGROUND: Maximal skin testing (ST) nonirritant concentrations (NICs) are consistent for penicillin and aminopenicillin among guidelines. However, there is variability among guidelines for maximal ST NICs of cephalosporins. OBJECTIVE: To determine maximal immediate and delayed ST NICs of 15 ß-lactams in ß-lactam-tolerant and ß-lactam-naïve participants. METHODS: We performed a single-center, nonrandomized prospective study between September 2019 and January 2022 in adult participants. Participants received skin prick testing (SPT) and intradermal test (IDT) injections at 6 increasing concentrations of 1 or more ß-lactams. A concentration was considered irritant when more than 5% of participants had a positive test. A positive test was defined as a wheal ≥3 mm compared with negative control accompanied by a ≥5 mm flare for SPT/IDT and induration ≥5 mm with associated erythema at 48 hours for delayed readings (dIDT). Sensitivity analyses using 3 alternative IDT positive criteria were conducted. RESULTS: A total of 747 participants with a median age of 64 (interquartile range: 54-72) years (52% male, 85% White, and 92% non-Hispanic) underwent 20,858 skin tests. All undiluted SPT concentrations were nonirritant. We found the following maximal IDT/dIDT NICs (mg/mL): ampicillin (41.6/125), ampicillin-sulbactam (93.8/187.5), aztreonam (6.3/25), cefazolin (55/165), cefepime (35/140), cefoxitin (45/90), ceftaroline (7.5/15), ceftriaxone (58.3/175), cefuroxime (55/110), ertapenem (16.6/50), imipenem-cilastin (6.3/25), meropenem (8.3/25), nafcillin (31.3/62.5), oxacillin (20.9/83.5), and piperacillin-tazobactam (112.5/225). dIDTs were almost all completely nonirritant close to or at undiluted concentrations. There were no differences when we applied 3 IDT positivity criteria to our raw data. CONCLUSIONS: Our results suggest that SPTs with undiluted stock ß-lactam antibiotic concentrations are nonirritant. Compared with previously published nonirritant concentrations, we propose a 2- to 50-fold increase to the maximal IDT and dIDT NICs of 15 ß-lactam antibiotics. When performing dIDTs, a higher concentration should be used rather than the same IDT concentration.

A rare biofilm dispersion strategy demonstrated by Staphylococcus aureus under oxacillin stress.

Staphylococcus aureus (S. aureus), a versatile Gram-positive bacterium, is implicated in a spectrum of infections, and its resilience is often attributed to biofilm formation. This study investigates the effect of sub-inhibitory doses of oxacillin on biofilm formation by methicillin-resistant S. aureus (MRSA). Specifically, it examines how these doses influence biofilms' development, maturation, and dispersal. The biofilm's zenith reached 48 h of incubation, followed by a noteworthy decline at 96 h and a distinctive clearance zone around biofilm-positive cells exposed to oxacillin. Scanning electron micrographs unveiled an intriguing active biofilm dispersal mechanism, a rarity in this species. Among 180 isolates, only three carrying the elusive icaD gene exhibited this phenomenon. icaD gene was absent in their counterparts. Notably, the icaD gene emerges as a distinctive marker, crucial in regulating biofilm dispersion and setting these isolates apart. The captivating interplay of oxacillin, biofilm dynamics, and genetic signatures disintegrate novel dimensions in understanding MRSA's adaptive strategies and underscores the importance of the icaD gene in engineering biofilm resilience.

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

BACKGROUND: Multi-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. METHODS: In 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. RESULTS: Oxacillin 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. CONCLUSION: The 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.

Performance evaluation of cefoxitin screen test on two different automated antimicrobial susceptibility test systems: a comparative study.

Reliable detection of mecA and mecC-mediated beta-lactam resistance using automated antimicrobial susceptibility test systems is critical for patient care. The aim of this study was to compare the performance of the new cefoxitin screen test (oxsf02n) on the Vitek 2 card (Vitek 2) and BD Phoenix PMC-100 Gram-Positive AST Panel (Phoenix) against the reference method for the detection of mecA (and mecC)-mediated beta-lactam resistance. Two hundred fifty clinical fresh and stock Staphylococcus spp. isolates were included. There were 120 mecA-positive, 10 mecC-positive, and 120 mecA and mecC-negative isolates. Cefoxitin screen and oxacillin tests were performed on Vitek 2 and Phoenix and by their respective reference methods (disk diffusion for the cefoxitin screen test and broth microdilution for oxacillin) for all isolates. PCR testing was also performed to confirm the presence of mecA and/or mecC genes. Results from each system were compared to the reference methods. Statistical hypotheses were evaluated both for Vitek 2 compared to the reference methods and Vitek 2 compared to the Phoenix. Compared to the reference method, the Vitek 2 cefoxitin screen test had 100% sensitivity/98% specificity and the Phoenix cefoxitin screen test had 84% sensitivity/100% specificity for the detection of mecA (and mecC)-mediated beta-lactam resistance. When the oxacillin test was combined with the cefoxitin screen for Vitek 2, the sensitivity and specificity were unchanged. However, when the oxacillin and cefoxitin screen tests were combined for the Phoenix, the sensitivity increased to 100% and the specificity remained unchanged (100%). When considering cefoxitin alone, the Vitek 2 screen test showed a higher sensitivity than the Phoenix for the detection of mecA and mecC-mediated beta-lactam resistance. However, currently, both systems use a combination of the cefoxitin and oxacillin tests to interpret the final result, and both reached a high level of performance when cefoxitin and oxacillin results were combined.IMPORTANCEThis research marks the inaugural evaluation of the revamped cefoxitin screen test version in Vitek 2, juxtaposing it against reference methods and a primary competitor BD Phoenix.

Metabolic remodeling by RNA polymerase gene mutations is associated with reduced ß-lactam susceptibility in oxacillin-susceptible MRSA.

The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to ß-lactam antibiotics, these strains can easily acquire reduced ß-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced ß-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. IMPORTANCE: The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to ß-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced ß-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Dhvar5-chitosan nanogels and their potential to improve antibiotics activity.

Infection is one of the main causes of orthopedic implants failure, with antibiotic-resistant bacteria playing a crucial role in this outcome. In this work, antimicrobial nanogels were developed to be applied in situ as implant coating to prevent orthopedic-device-related infections. To that regard, a broad-spectrum antimicrobial peptide, Dhvar5, was grafted onto chitosan via thiol-norbornene "photoclick" chemistry. Dhvar5-chitosan nanogels (Dhvar5-NG) were then produced using a microfluidic system. Dhvar5-NG (1010 nanogels (NG)/mL) with a Dhvar5 concentration of 6 µg/mL reduced the burden of the most critical bacteria in orthopedic infections - methicillin-resistant Staphylococcus aureus (MRSA) - after 24 h in medium supplemented with human plasma proteins. Transmission electron microscopy showed that Dhvar5-NG killed bacteria by membrane disruption and cytoplasm release. No signs of cytotoxicity against a pre-osteoblast cell line were verified upon incubation with Dhvar5-NG. To further explore therapeutic alternatives, the potential synergistic effect of Dhvar5-NG with antibiotics was evaluated against MRSA. Dhvar5-NG at a sub-minimal inhibitory concentration (109 NG/mL) demonstrated synergistic effect with oxacillin (4-fold reduction: from 2 to 0.5 µg/mL) and piperacillin (2-fold reduction: from 2 to 1 µg/mL). This work supports the use of Dhvar5-NG as adjuvant of antibiotics to the prevention of orthopedic devices-related infections.

Staphylococcus aureus nasal colonization and susceptibility profile to antimicrobials in hemodialysis patients using a protocol of seven collections.

BACKGROUND: Patients colonized with Staphylococcus aureus in their nasal passages have a higher risk of acquiring infection, especially if they are immunocompromised or have comorbidities such as chronic renal failure undergoing hemodialysis (HD). OBJECTIVE: This study aimed to report the prevalence of nasal carriage of S. aureus among HD patients utilizing a seven-week sampling protocol and to assess the susceptibility of these isolates to various antimicrobial agents. METHODS: Over seven consecutive weeks, nasal swab samples were collected from 47 HD patients, resulting in a total of 329 samples. The microorganisms were identified using biochemical methods and subjected to antimicrobial susceptibility testing via disk diffusion and microdilution techniques. RESULTS: Out of all the patients analyzed, 25 individuals (53.19%) were found to be colonized by S. aureus, with 21 of them displaying intermittent colonization. Additionally, 38% showed positive results for S. aureus in only the 6th or 7th week of sampling. Within the 58 isolates, 17.2% (n=10) exhibited methicillin (oxacillin)-resistance and 25.86% (n=15) displayed elevated vancomycin MIC values (2 µg/ml). Based on the results, daptomycin and gentamicin were found to be effective treatment options. However, 31% of the isolates (n=18) exhibited a MIC of 1 µg/ml for daptomycin. CONCLUSION: Over half of the patients were colonized by S. aureus, but mostly on an intermittent basis. The identification of oxacillin resistance and high vancomycin and daptomycin MICs serve as warnings for possible future complications in managing bacteremia caused by S. aureus in these patients.

Comparative Analysis of Two Commercial Automated Systems with Agar Dilution for Oxacillin Susceptibility and Their Association with Genotypes of Invasive Staphylococcus aureus Isolates (2011-2021).

Background: Determining oxacillin susceptibility using reference methods and automated systems is crucial for treating invasive infections caused by Staphylococcus aureus. This study compares the oxacillin susceptibility results from the two automated systems with agar dilution and correlates them with genotypes of invasive S. aureus. Methods: Non-duplicate S. aureus invasive isolates were collected over an 11-year period. The oxacillin susceptibility was determined with Phoenix 100 (Jan 2011 to Aug 2018) or Vitek 2 (Sep 2018 to Dec 2021), and susceptibility for oxacillin and cefoxitin was determined with agar dilution. Methicillin-resistant S. aureus (MRSA) was confirmed with mecA existence, and the genotype was determined using SCCmec. The association between genotype and antibiotic susceptibility using two automated systems and agar dilution was evaluated. Results: A total of 842 invasive S. aureus, including 443 mecA+ MRSA and 399 mecA- MSSA, were collected. The susceptibility rates of oxacillin determined by two automated systems and agar dilution were 68.8% (76.8% for Phoenix 100 and 57.6% for Vitek 2) and 54.0%, respectively. When compared with the oxacillin susceptibility using agar dilution, the categorical agreement for Phoenix 100 and Vitek 2 were 0.46% and 0.88%, respectively (p < 0.001). One hundred and forty-three isolates were misinterpreted as oxacillin-susceptible S. aureus (OSSA) using automated systems while comparing with agar dilution, among which molecularly community-associated MRSA (CA-MRSA) outnumbered healthcare-associated MRSA (HA-MRSA) (99 vs 34, p < 0.001). There were 70 mecA+ OSSA (OS-MRSA) using agar dilution, among which 42 harbored SCCmec types were predominantly categorized as CA-MRSA (38, p < 0.001). Conclusion: The categorical agreement of Vitek 2 in determining oxacillin susceptibility and predicting mecA existence is comparable with agar dilution, whereas Phoenix 100 is not. Most of those ORSA determined by agar dilution but misinterpreted as OSSA by automated systems and OS-MRSA are categorized as CA-MRSA.

Inhibition of the blaOXA-48 gene expression in Klebsiella pneumoniae by a plasmid carrying CRISPRi-Cas9 system.

Antibiotic resistance is an increasing concern that threatens the effectiveness of treating bacterial infections. The spread of carbapenem resistant Klebsiella pneumoniae poses a significant threat to global public health. To combat this issue, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system is being developed. This system includes a single guide RNA (sgRNA) and a nuclease dead Cas9 (dCas9), which work together to downregulate gene expression. Our project involved the use of the CRISPRi system to reduce gene expression of the beta-lactamase oxacillin-48 (blaOXA-48) gene in K. pneumoniae. We designed a sgRNA and cloned it into pJMP1363 plasmid harboring the CRISPRi system. The pJMP1363-sgRNA construct was transformed in K. pneumoniae harboring the blaOXA-48 gene. The MIC test was used to evaluate the antimicrobial resistance, and quantitative real-time RT-PCR was used to confirm the inhibition of the OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA construct expression. The Galleria mellonella larvae model was also utilized for in vivo assay. Following the transformation, the MIC test indicated a 4-fold reduction in meropenem resistance, and qRT-PCR analysis revealed a 60-fold decrease in the mRNA OXA-48 harboring the pJMP1363-sgRNA construct expression. Additionally, G. mellonella larvae infected with OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA showed higher survival rates. Based on the findings, it can be concluded that the CRISPR interference technique has successfully reduced antibiotic resistance and virulence in the K. pneumoniae harboring the blaOXA-48 gene.

Staphylococcus aureus AbcA transporter enhances persister formation under ß-lactam exposure.

We evaluated the role of Staphylococcus aureus AbcA transporter in bacterial persistence and survival following exposure to the bactericidal agents nafcillin and oxacillin at both the population and single-cell levels. We show that AbcA overexpression resulted in resistance to nafcillin but not oxacillin. Using distinct fluorescent reporters of cell viability and AbcA expression, we found that over 6-14 hours of persistence formation, the proportion of AbcA reporter-expressing cells assessed by confocal microscopy increased sixfold as cell viability reporters decreased. Similarly, single-cell analysis in a high-throughput microfluidic system found a strong correspondence between antibiotic exposure and AbcA reporter expression. Persister cells grown in the absence of antibiotics showed neither an increase in nafcillin MIC nor in abcA transcript levels, indicating that survival was not associated with stable mutational resistance or abcA overexpression. Furthermore, persister cell levels on exposure to 1×MIC and 25×MIC of nafcillin decreased in an abcA knockout mutant. Survivors of nafcillin and oxacillin treatment overexpressed transporter AbcA, contributing to an enrichment of the number of persisters during treatment with pump-substrate nafcillin but not with pump-non-substrate oxacillin, indicating that efflux pump expression can contribute selectively to the survival of a persister population.

Um raro caso de vasculite leucocitoclástica induzida por oxacilina / A rare case of oxacillin-induced leukocytoclastic vasculitis

A vasculite leucocitoclástica é uma patologia cujos mecanismos estão associados ao processo de inflamação vascular. Estima-se que até 24% dos casos de vasculite estão relacionados ao uso de fármacos, sendo os antimicrobianos beta-lactâmicos um dos grupos farmacológicos comumente associados a este desfecho adverso. A oxacilina, uma penicilina semissintética, possui um anel beta-lactâmico que confere atividade biológica e está associada com maior frequência a relatos de vasculite leucocitoclástica. No entanto, casos semelhantes relacionados a esse antimicrobiano são raros, sendo identificados apenas três casos na literatura. Diante desse contexto, relatamos um quarto caso de vasculite leucocitoclástica em um homem de 56 anos, em tratamento com oxacilina, que desenvolveu a vasculite no 3º dia de uso do antimicrobiano. Além da suspensão da oxacilina, ele foi tratado com 125 mg/dia de metilprednisolona endovenosa por sete dias, seguido de 20 mg/dia de prednisona oral por quatro dias, resultan-do em remissão satisfatória das lesões cutâneas e ausência de novos desfechos adversos. Este caso corrobora a possível relação causal entre o uso de oxacilina e o desenvolvimento da vasculite leucocitoclástica, apesar de sua ocorrência ser rara. A resposta favorável às intervenções terapêuticas, incluindo a suspensão da oxacilina e o uso de corticosteroides, destaca a eficácia dessas abordagens no tratamento dessa complicação (AU).

Leukocytoclastic vasculitis is a pathology whose mechanisms are associated with the process of vascular inflammation. It is estimated that up to 24% of the cases of vasculitis are drug-related, with beta-lactam antimicrobials be-ing one of the pharmacological groups commonly associated with this adverse outcome. Oxacillin, a semisynthetic penicillin, has a beta-lactam ring that confers biological activity and is most frequently associated with reports of leukocytoclastic vasculitis. However, similar cases related to this antimicrobial are rare, with only three cases identified in the literature. Against this background, we report a fourth case of leukocytoclastic vasculitis in a 56-year-old man, on oxacillin treatment, who developed the vasculitis on the 3rd day of antimicrobial use. In addition to oxacillin suspension, he was treated with 125 mg/day of intravenous methylprednisolone for seven days, followed by 20 mg/day of oral prednisone for four days, resulting in satisfactory remission of the skin lesions and no new adverse outcomes. This case provides further evidence supporting the potential causal relationship between the use of oxacillin and the development of leukocytoclastic vasculitis, albeit a rare occurrence. The positive response to therapeutic interventions, such as oxacillin suspension and corticosteroid treatment, underscores the effectiveness of these approaches in addressing this complication (AU),

A hope for ineffective antibiotics to return to treatment: investigating the anti-biofilm potential of melittin alone and in combination with penicillin and oxacillin against multidrug resistant-MRSA and -VRSA.

Background: The emergence and rapid spread of multi-drug resistant (MDR) bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), have posed a significant challenge to the medical community due to their ability to form biofilm and develop resistance to common antibiotics. Traditional antibiotics that were once effective in treating bacterial infections are now becoming increasingly ineffective, leading to severe consequences for patient outcomes. This concerning situation has called for urgent research to explore alternative treatment strategies. Recent studies have shown that antimicrobial peptides (AMPs) hold promise as effective agents against biofilm-associated drug-resistant infections as well as to enhance the efficacy of conventional antibiotics. Accordingly, we aimed to investigate the antimicrobial and antibiofilm effects of melittin AMP, both alone and in combination with penicillin and oxacillin, against biofilm-forming MDR-MRSA and -VRSA. Methods: In this study, we investigated the kinetics of biofilm formation and assessed various parameters related to the antimicrobial and antibiofilm efficacy of melittin and antibiotics, both alone and in combination, against MDR-MRSA and -VRSA. The antimicrobial parameters included the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Fractional Inhibitory Concentration Index (FICi), Fractional Bactericidal Concentration Index (FBCi), and the antibiofilm activity of melittin and antibiotics indicated by the Minimum Biofilm Inhibitory Concentration (MBIC), Minimal Biofilm Eradication Concentration (MBEC), Fractional Biofilm Inhibitory Concentration Index (FBICi), and Fractional Biofilm Eradication Concentration Index (FBECi). Results: The MIC results showed that all S. aureus isolates were resistant to penicillin (≥0.25 µg/mL), and 66% of isolates were resistant to oxacillin. The geometric means of the MIC values for penicillin, oxacillin, and melittin were 19.02, 16, and 1.62 µg/ml, respectively, and the geometric means of the MBC values for penicillin, oxacillin, and melittin were 107.63, 49.35, and 5.45 µg/ml, respectively. The study revealed that the combination indexes of melittin-penicillin and melittin-oxacillin, as determined by FIC values against all isolates, were 0.37 and 0.03, respectively. Additionally, melittin-penicillin and melittin-oxacillin exhibited combination indexes based on FBC values against all isolates at 1.145 and 0.711, respectively. Besides, melittin inhibited the biofilm formation of all S. aureus isolates, with MBIC values ranging from 10 to 1.25 µg/mL, and MBEC values ranging from 40 to 10 µg/mL. Generally, the combination indexes of melittin-penicillin and melittin-oxacillin, determined using FBIC values against all isolates, were 0.23 and 0.177, respectively. Moreover, melittin-penicillin and melittin-oxacillin typically had combination indexes based on FBEC values against all isolates at 5 and 2.97, respectively. Conclusion: In conclusion, our study provides evidence that melittin is effective against both planktonik and biofilm forms of MRSA and VRSA and exhibits significant synergistic effects when combined with antibiotics. These results suggest that melittin and antibiotics could be a potential candidate for further investigation for in vivo infections caused by MDR S. aureus. Furthermore, melittin has the potential to restore the efficacy of penicillin and oxacillin antibiotics in the treatment of MDR infections. Applying AMPs, like melittin, to revive beta-lactam antibiotics against MRSA and VRSA is an innovative approach against antibiotic-resistant bacteria. Further research is needed to optimize dosage and understand melittin mechanism and interactions with beta-lactam antibiotics for successful clinical applications.

Phenotypic and genotypic characterization of oxacillin-susceptible and mecA positive Staphylococcus aureus strains isolated in Uruguay / Caracterización fenotípica y genotípica de cepas de Staphylococcus aureus sensibles a oxacilina y mecA positivas aisladas en Uruguay

Abstract The aim of this study was to characterize phenotypically and genotypically 27 mecApositive Staphylococcus aureus strains with oxacillin MICs of ≤2 g/ml by Vitek 2, isolated indifferent regions of Uruguay. Susceptibility to oxacillin and cefoxitin was studied by gradient dif-fusion, 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 isolateswere susceptible to oxacillin; one strain was susceptible to cefoxitin by disk diffusion and 3strains by gradient diffusion. Phoenix and MicroScan panels detected methicillin resistance in25 and 27 strains, respectively. Twenty-six strains tested positive for PBP2a. Twenty-six strainscarried SCCmec V and 24 belonged to pulsotype A. One strain carried SCCmec IV and did notbelong to pulsotype A. Cefoxitin disk diffusion test and PBP2a detection correctly identified 26of these 27 strains as MRSA. PFGE results suggest the dissemination of a cluster of MRSA carryingSCCmec V.

Resumen El objetivo de este estudio fue caracterizar fenotípicamente y genotípicamente 27 cepas de Staphylococcus aureus positivas para mecA y con CIM de oxacilina <2 pg/ml según Vitek 2, obtenidas en diferentes regiones del país. La sensibilidad frente a la oxacilina y la cefoxitina se estudió por difusión en gradiente, por disco-difusión (cefoxitina) y por los sistemas Phoenix y MicroScan. Se analizó la portación de PBP2a, se realizó la tipificación de SCCmec y las cepas se compararon mediante PFGE. Resultaron sensibles a oxacilina por difusión en gradiente 26 cepas; una fue sensible a cefoxitina por disco-difusión y 3 lo fueron por difusión en gradiente. Los sistemas Phoenix y MicroScan detectaron resistencia a meticilina en 25 y 27 cepas, respectivamente. Asimismo, 26 cepas portaban PBP2a y 26 cepas mostraron presencia de SCCmec V, 24 correspondieron al pulsotipo A. Una portaba SCCmec IV y no perteneció al pulsotipo A. La prueba de disco-difusión con cefoxitina y la detección de PBP2a identificaron 26 de 27 cepas como MRSA. La PFGE sugiere la diseminación de un grupo MRSA con SCCmec V. © 2022 Asociación Argentina de Microbiología. Publicado por Elsevier Espana, S.L.U. Este es un artículo Open Access bajo la licencia CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Tercer caso de vasculitis leucocitoclástica cutánea secundaria al uso de oxacilina: reporte de caso / Third case in the literature of cutaneous leukocytoclastic vasculitis secondary to oxacilin use

Resumen La vasculitis leucocitoclastica es una patologìa que compromete los vasos pequeños y cuya causa predominantemente se ha descrito como idiopatica. Se presenta el caso de una mujer de 78 años hipertensa, diabética y con enfermedad renal crónica en estadio 5, que presentó lesiones limitadas a la piel posterior a la administración de oxacilina para manejo de bacteremia por SAMS. La presentación clínica se basó en purpuras palpables predominantemente en miembros inferiores y lesiones dolorosas coalescentes que formaban ampollas de contenido hemorrágico. Estas lesiones resolvieron gradualmente después del cambio de la terapia mencionada anteriormente. La biopsia fue compatible con vasculitis leucocitoclástica, con paraclínicos que descartaron causas infecciosas y autoinmunes.

Abstract Leukocytoclastic vasculitis is a pathology that involves small vessels and whose cause has been predominantly described as idiopathic. The clinical case of a 78-year-old woman with hypertension, diabetic and chronic stage 5 kidney disease, who presented limited skin lesions after administration of oxacillin for management of bacteremia by MSSA. The clinical presentation consisted on palpable purpura predominantly in the lower limbs and painful coalescent lesions that formed blisters of hemorrhagic content. Lesions gradually resolved after the change of the therapy mentioned above. The biopsy was compatible with leukocytocastic vasculitis, with paraclinics who ruled out infectious and autoimmune causes.

Talinum paniculatum leaves with in vitro antimicrobial activity against reference and clinical strains of Staphylococcus aureus interfere with oxacillin action / Las hojas de Talinum paniculatum con actividad antimicrobiana in vitro contra cepas de referencia y clínicas de Staphylococcus aureus interfieren con la acción de la oxacilina

SUMMARY Propose: We evaluated the antibacterial potential of the crude leaf extract (CLE) and fractions hexane (HX) and ethyl acetate (EtOAc) from Talinum paniculatum alone and in association with oxacillin (OXA) against OXA-resistant Staphylococcus aureus (ORSA, environment isolates) and OXA-sensitive S. aureus (OSSA, ATCC 25923). Furthermore, toxicity tests were performed. Methods: The antibacterial activity was evaluated through checkerboard assay (broth microdilution) to establish the minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC). Toxicity test in mice was assessed. Results: The MIC values for the CLE and its fractions against ORSA and OSSA were in the order of HX (500 μg ml-1) = EtOAc < CLE (4000 μg ml-1). EtOAc and HX presented outstanding antibacterial activities against ORSA, and these fractions were bactericidal toward OSSA. Conversely, the associations between plant product (CLE, EtOAc, or HX) and OXA exhibited no synergistic effects. During these associations, there was an increase in OXA MICs anywhere from 2- to 4092-fold. The CLE presented absence of toxicity at a dose of 5 g kg-1 (in vivo). Conclusion: Although T. paniculatum be a good source of bioactive compounds with antistaphylococcal potential, the researchers should be cautious, since its edible leaf may interfere with OXA therapy (mitigating OXA-induced growth inhibition or killing of S. aureus and enhancing S. aureus resistance).

RESUMEN Propósito: evaluamos el potencial antibacteriano del extracto de hoja en bruto (EHB) y las fracciones hexano (HX) y acetato de etilo (AcOEt) de Talinum paniculatum solo y en asociación con oxacilina (OXA) contra Staphylococcus aureus resistente a OXA (ORSA, ambientales) y S. aureus sensible a OXA (OSSA, ATCC 25923). Además, se realizaron pruebas de toxicidad. Métodos: la actividad antibacteriana se evaluó mediante microdilución en caldo para establecer las concentraciones inhibitorias mínimas (CIM) y bactericidas mínimas (CBM). Se evaluó la toxicidad en ratones. Resultados: los valores de CIM para el EHB y sus fracciones contra ORSA y OSSA fueron del orden de HX (500 μg ml-1) = AcOEt < EHB (4000 μg ml-1). AcOEt y HX presentaron actividades antibacterianas sobresalientes contra ORSA, y estas fracciones fueron bactericidas hacia OSSA. Por el contrario, las asociaciones entre el producto vegetal (EHB, AcOEt o HX) y OXA no mostraron efectos sinérgicos. Durante estas asociaciones, hubo un aumento en las CIM de OXA de 2 a 4092 veces. EHB no mostró toxicidad a una dosis de 5 g kg-1. Conclusión: aunque T. paniculatum es una buena fuente de compuestos bioactivos con potencial antiestofilocócico, los investigadores deben ser cautelosos, ya que su hoja comestible puede interferir con la terapia con OXA (mitigando la inhibición del crecimiento inducida por OXA o la muerte de S. aureus y promoviendo resistencia bacteriana).

Chemical composition and antimicrobial activity of two extract of propolis against isolates of Staphylococcus spp. and multiresistant bacterials / Composição química e atividade antimicrobiana de dois extratos de própolis contra isolados de Staphylococcus spp. e bactérias multirresistentes

There is a growing need to discover and develop alternative therapies for the treatment of mastitis caused by Staphylococcus spp. and multidrug-resistant bacterial infections. This study examined the chemical composition and antimicrobial potential of two propolis extracts (EPA and EPB) against seventy-seven isolates of Staphylococcus spp. obtained from subclinical bovine mastitis; three clinical strains of MRSA and two from clinical strains of S. aureus ATCC, identified as S. aureus ATCC 6538 and S. aureus ATCC 25923. The total phenolic content was determined by the Folin-Ciocalteau method, the total flavonoid content by the Dowd method and the phenolic profile was quantified by HPLC-DAD. The MBC values of the extracts were evaluated by broth microdilution method. The amount of total phenolic and flavonoid compounds was higher in EPA than EPB. Both extracts revealed the presence of caffeic, coumaric, cinnamic, ferulic and 3,4-dihydroxybenzoic acids, with higher concentrations of coumaric and cinnamic acids. Staphylococcus spp. isolates were susceptible to EPA (90.9%), EPB (83.1%) and oxacillin (80.5%). The oxacillin susceptible isolates were also susceptible to EPA (70.1%) and EPB (80.6%), whereas those oxacillin-resistant strains were also susceptible to EPA (40.0%) and to EPB (26.7%). MBC ranged from 34.3 to 68.7µm/mL for EPA and from 68.7 to 137.5µg/mL for EPB. Both extracts inhibited significantly (100%) the clinical strains of MRSA, S. aureus ATCC 6538 and S. aureus ATCC 25923 at the concentration of 68.7µg/mL. It is concluded that both extracts of propolis, whose main constituents are coumaric and cinnamic acids, have high antimicrobial activity against the microorganisms studied, and EPA also against oxacillin-resistant strains. These findings reinforce its potential use for the treatment of bovine mastitis.(AU)

É cada vez mais oportuna a necessidade de descobrir e desenvolver terapias alternativas para tratamento da mastite causada por Staphylococcus spp. e de infecções bacterianas multirresistentes. Este estudo examinou a composição química e o potencial antimicrobiano de dois extratos etanólicos de própolis (EPA e EPB) contra setenta e sete isolados de Staphylococcus spp. obtidos a partir de mastite bovina subclínica; três estirpes clínicas de MRSA e duas de linhagens clínicas de S. aureus ATCC, identificadas como, S. aureus ATCC 6538 e S. aureus ATCC 25923, ambas metacilina resistentes. O teor total de fenólicos foi determinado pelo método de Folin-Ciocalteau, o teor de flavonoides totais pelo método Dowd e o perfil fenólico foi quantificado por HPLC-DAD. CBM dos extratos foi avaliada pelo método de microdiluição em caldo. A quantidade total de compostos fenólicos e flavonoides foi maior no EPA do que no EPB. Ambos os extratos revelaram a presença dos ácidos cafeico, cumárico, cinâmico, ferúlico e 3,4-di-hidroxibenzóico, com maiores concentrações de ácidos cumárico e cinâmico. Os isolados de Staphylococcus spp. foram sensíveis a EPA (90,9%), EPB (83,1%) e oxacilina (80,5%). Os isolados suscetíveis à oxacilina também foram suscetíveis ao EPA (70,1%) e ao EPB (80,6%), enquanto os do resistente à oxacilina foram suscetíveis ao EPA (40,0%) e ao EPB (26,7%). MBC variou de 34,3 a 68,7µm/mL para EPA e de 68,7 a 137,5µg/mL para EPB. Ambos os extratos inibiram significativamente (100%) as linhagens clínicas de MRSA, S. aureus ATCC 6538 e S. aureus ATCC 25923 na concentração de 68,7µg/mL. Conclui-se que os extratos etanólicos da própolis, cujos principais constituintes são os ácidos cumário e cinâmico, possuem atividade antimicrobiana contra os micro-organismos estudados, e o EPA também contra as cepas resistentes à oxacilina. Estes achados reforçam seu potencial uso para o tratamento da mastite bovina.(AU)

Ceftaroline resistance in Staphylococcus pseudintermedius gene mecA carriers / Resistência à ceftarolina em Staphylococcus pseudintermedius portadores do gene mecA

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) being a constant concern, ceftaroline fosamil has been recently approved as a new cephalosporin, active against MRSA, for use in humans; only rare cases of resistance have been reported till date. There is no report of resistance to ceftaroline in Staphylococcus pseudintermedius, which is the main bacterium causing dermatitis and otitis in dogs. To evaluate staphylococcal resistance to ceftaroline, 35 isolates of methicillin-resistant S. pseudintermedius (MRSP), carrying the mecA gene, from 26 dogs with folliculitis and nine dogs with external otitis, underwent disk diffusion test with cefoxitin, oxacillin, and ceftaroline. Tests with cefoxitin and oxacillin showed > 90% sensitivity in methicillin resistance detection. In the disk diffusion test, 97.14% (34/35) were resistant to cefoxitin, 94.29% (33/35) to oxacillin, and 31.43% (11/35) to ceftaroline. Of the ceftaroline-resistant strains, 27.27% (3/11) were obtained from the ears of dogs while the rest (8/11) were from the skin. The current report is the first description of MRSP resistance to ceftaroline.(AU)

Infecções causadas por Staphylococcus aureus resistente à meticilina (MRSA) são uma preocupação médica constante. A ceftarolina fosamila é uma nova cefalosporina ativa contra Staphylococcus aureus resistente à meticilina recentemente aprovada para uso em humanos e raros casos de resistência relatados até agora. Não há relatos de resistência à ceftarolina em Staphylococcus pseudintermedius, principal bactéria causadora de dermatite e otite em cães. Com o objetivo de avaliar a resistência estafilocócica à ceftarolina, 35 amostras de S. pseudintermedius resistentes à meticilina (MRSP), portadoras do gene mecA, provenientes de 26 cães com foliculite e 9 com otite externa foram submetidos ao teste de disco-difusão com cefoxitina, oxacilina e ceftarolina. Os testes realizados com cefoxitina e oxacilina mostraram mais de 90% de sensibilidade na detecção da resistência à meticilina em ambas. No teste da disco-difusão, 97,14% (1/35) foram resistentes à cefoxitina, 94,29% (3/35) à oxacilina e 31,43% (11/35) à ceftarolina. Das cepas resistentes às ceftarolina, 27,27 (3/11) foram provenientes de ouvido de cães e as demais (8/11), provenientes da pele, sendo essa primeira descrição de resistência de MRSP à ceftarolina na literatura atual.(AU)

Isoenzyme genotyping and phylogenetic analysis of oxacillin-resistance Staphylococcus aureus isolates

Aim: The propagation of S. aureus in hospital and dental environments is considered an important public health problem since resistant strains can cause serious infections in humans. The genetic variability of 99 oxacillin-resistant S. aureus isolates (ORSA) from the dental patients (oral cavity) and environments (air) was studied by isoenzyme genotyping. Methods: S. aureus isolates were studied using isoenzyme markers (alcohol dehydrogenase, sorbitol dehydrogenase, mannitol-1-phosphate dehydrogenase, malate dehydrogenase, glucose dehydrogenase, D-galactose dehydrogenase, glucose-6-phosphate dehydrogenase, catalase and α/ß-esterase) and genetic (Nei's statistics) and cluster analysis (UPGMA algorithm). Results: A highly frequent polyclonal pattern was observed in this population of ORSA isolates, suggesting various sources of contamination or microbial dispersion. Genetic relationship analysis showed a high degree of polymorphism between the strains, and it revealed three taxa (A, B and C) distantly genetically related (0.653≤dij≤1.432) and fifteen clusters (I to XV) moderately related (0.282≤dij<0.653). These clusters harbored two or more highly related strains (0≤dij<0.282), and the existence of microevolutionary processes in the population of ORSA. Conclusion: This research reinforces the hypothesis of the existence of several sources of contamination and/or dispersal of ORSA of clinical and epidemiologically importance, which could be associated with carriers (patients) and dental environmental (air) (AU)

Sphygmomanometers and thermometers as potential fomites of Staphylococcus haemolyticus: biofilm formation in the presence of antibiotics

BACKGROUND The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.

Resistencia a oxacilina, eritromicina y gentamicina en cepas de Staphylococcus coagulasa negativa aisladas de hemocultivos / Resistance to oxacillin, erythromycin and gentamycin in coagulase negative Staphylococcus strains isolated from blood cultures

La resistencia a los antimicrobianos en bacterias Gram positivas como Staphylococcus coagulasa negativa constituye una amenaza mundial emergente. El propósito de la presente investigación fue identificar los genes de resistencia a oxacilina (mecA), eritromicina (erm y msrA), y gentamicina aac(6´)/aph(2´´), en cepas de Staphylococcus coagulasa negativa aisladas de hemocultivos de pacientes atendidos en el Hospital Universitario de Maracaibo. La detección fenotípica se realizó mediante métodos automatizados. Se utilizó la reacción en cadena de la polimerasa para detectar la presencia de genes de resistencia a los antimicrobianos. Se estudiaron 34 cepas cuya distribución por especie fue: S. haemolyticus (38,23%), S. epidermidis (29,42%), S. hominis (26,47%), S. xylosus y S. capitis (5,88% cada uno). Todas las cepas fueron resistentes a oxacilina. La resistencia a gentamicina varió entre 38,46% y 100%; mientras que la resistencia a eritromicina osciló entre 77,78% y 100%. Los análisis mostraron la presencia de los genes mecA (100%), ermA (35,2%), ermC (41,17%), msrA (17,64%), y aac(6´)/aph(2´´) (61,76%). En conclusión, se encontró una alta frecuencia de genes de resistencia a estos antibióticos y la Unidad de Cuidados Intensivos fue el servicio médico donde se aisló el mayor porcentaje de cepas portadoras de estos genes.

Resistance to antimicrobials in Gram-positive bacteria such as coagulase negative Staphylococcus is an emerging global threat. The purpose of this research was to identify the genes for resistance to oxacillin (mecA), erythromycin (erm and msrA), and gentamicin aac(6´)/aph(2´´), in Staphylococcus coagulase negative strains isolated from blood cultures from patients attended at the University Hospital in Maracaibo. Phenotypic detection was performed using automated methods. Polymerase chain reaction was used for the detection of antimicrobial resistance genes. Be studied 34 strains whose distribution by species was: S. haemolyticus (38.23%), S. epidermidis (29.42%), S. hominis (26.47%), S. xylosus and S. capitis (5.88% each one). All strains were resistant to oxacillin. Gentamicin resistance varied between 38.46% and 100%; while the erythromycin resistance ranged between 77.78% and 100%. The analyses showed the presence of genes mecA (100%), ermA (35.2%), ermC (41.17%), msrA (17.64%), and aac(6´)/aph (2´´) (61,76%). In conclusion, is found a high frequency of genes for resistance to these antibiotics and the intensive care unit was the health service where the highest percentage of isolated strains carriers of these genes.