USA300 Methicillin-resistant Staphylococcus Aureus Research Articles

Dissemination of meticillin-resistant Staphylococcus aureus sequence type 8 (USA300) in Taiwan

BackgroundIn Taiwan, sequence type (ST) 239 and ST59 were two major clones among methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates in the past two decades. USA300 (ST8) prevailed in Americas but not in outside areas. Recently USA300 (ST8) emerged and were increasingly identified in Taiwan; we thus conducted an islandwide study to explore the role of USA300 among MRSA isolates. Methods100 MRSA bloodstream isolates identified in 2020, respectively, from each of the six participating hospitals in Taiwan were collected and characterized. First ten ST8 isolates from each hospital were further analyzed by whole genome sequencing (WGS). ResultsOf the 590 confirmed MRSA isolates, a total of 22 pulsotypes and 21 STs were identified. The strain of pulsotype AI/ ST8 was the most common lineage identified, accounting for 187 isolates (31.7%) and dominating in 5 of 6 hospitals, followed by pulsotype A/ ST239 (14.7%), pulsotype C/ ST59 (13.9%) and pulsotype D/ ST59 (9.2%). Of the 187 pulsotype AI/ ST8 isolates, 184 isolates were characterized as USA300 and clustered in three major sub-pulsotypes, accounting for 78%. Ninety percent of the 60 ST8 isolates for WGS were clustered in three major clades. ConclusionsIn 2020, USA300 became the most common clone of MRSA in Taiwan, accounting for > 30% of MRSA bloodstream isolates islanwide. Most of USA300 isolates circulating in Taiwan might be imported at multiple occasions and evolved into at least 3 successful local clades. MRSA USA300 had successfully established its role in Taiwan, an area outside of Americas.

Discovery of N,N'-diarylurea molecules with activity against multidrug-resistant Staphylococcus aureus.

The emergence and global spread of methicillin-resistant Staphylococcus aureus (MRSA) pose a serious threat to public health, underscoring the urgent need for novel antibacterial interventions. Here, we screened 18 newly synthesized N,N'-diarylurea derivatives to identify compounds with activity against MRSA. Our investigations led to the discovery of a small molecule, SCB-24, which exhibited promising antimicrobial activity against MRSA USA300. Notably, SCB-24 demonstrated high activity even in the presence of 10% fetal bovine serum and showed excellent selectivity for bacterial over mammalian cells. SCB-24 also showed potent activity against various MRSA strains, including those resistant to second- and third-line antibiotics. Importantly, the efficacy of SCB-24 was inferior to that of vancomycin in MRSA-infected Galleria mellonella larvae. Overall, our findings suggest that SCB-24 has great potential as a new therapeutic for multidrug-resistant S. aureus infections.

Novel phenylthiazoles with a tert-butyl moiety: promising antimicrobial activity against multidrug-resistant pathogens with enhanced ADME properties.

The structure-activity relationship of a new tert-butylphenylthiazole series, with a pyrimidine linker, was investigated. We wished to expand knowledge of this novel class of antibiotics by generating 21 new derivatives bearing ≥2 heteroatoms in their side chains. Their activity was examined against isolates of methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, Escherichia coli, Neisseria gonorrhoeae, and Candida albicans. Two compounds with 1,2-diaminocyclohexane as a nitrogenous side chain showed promising activity against the highly infectious MRSA USA300 strain, with a minimum inhibitory concentration (MIC) of 4 μg mL-1. One of these two compounds demonstrated potent activity against C. difficile, with a MIC of 4 μg mL-1. Moderate activities against a C. difficile strain with a MIC of 8 μg mL-1 were noted. Some new compounds possessed antifungal activity against a wild fluconazole-resistant C. albicans strain, with MIC values of 4-16 μg mL-1. ADME and metabolism-simulation studies were performed for the most promising compound and compared with lead compounds. Our results revealed that one compound possessed greater penetration of bacterial membranes and metabolic resistance, which aided a longer duration of action against MRSA.

Unveiling Synergistic Potency: Exploring Butyrolactone I to Enhance Gentamicin Efficacy against Methicillin-Resistant Staphylococcus aureus (MRSA) Strain USA300.

Staphylococcus aureus, including MRSA strains, poses significant health risks, imposing a significant disease burden and mortality. We investigate butyrolactone I (BL-1), a marine-derived metabolite from Aspergillus terreus, enhancing aminoglycoside efficacy against MRSA. A promising synergy is observed with BL-1 and various aminoglycosides, marked by low fractional inhibitory concentration indexes (FICIs < 0.5). Comprehensive studies utilizing USA300 MRSA and gentamicin reveal a remarkable one-fourth reduction in minimum inhibitory concentration (MIC) with 20 μg/mL BL-1. A relative abundance assay indicates that BL-1 enhances gentamicin uptake while restraining extracellular presence, involving intricate transmembrane signaling and molecular interactions. RNA-Seq analysis yielded an unexpected revelation, unveiling a distinctive gene expression profile and distinguishing it from other treatment approaches. Furthermore, meticulous analyses validated the extensive perturbations induced by BL-1 exposure, affecting diverse biological functions, encompassing glycolysis, amino acid metabolisms, substance transmembrane transport, and virulence generation. These valuable insights inspired further confirmation of bacterial virulence and the modulation of membrane permeability resulting from BL-1 treatment. Phenotypic validations corroborated our observations, revealing reduced membrane permeability and hemolytic toxicity, albeit demanding a deeper comprehension of the intricate interplay underlying these actions. Our study contributes crucial mechanistic insights to the development of therapeutic strategies against this notorious pathogen and the judicious employment of aminoglycosides. Additionally, it elucidates marine-derived metabolites' ecological and functional roles, exemplified by fungal quorum sensing signals. These compounds could give producers a competitive edge, inhibiting microorganism proliferation and suggesting novel approaches for combating resistant pathogens.

Topical antimicrobial formulations using medicinal plant-derived essential oils targeting methicillin resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) causes a variety of healthcare-associated and community-acquired infections. Due to limited availability of effective antimicrobials for treating MRSA infections, there is a growing need to explore alternative therapeutic approaches. Here, the antimicrobial activities of 19 oils, popularly used for their medicinal properties, were tested against MRSA USA300. Oils obtained from cinnamon, clove, tangerine, and coriander showed the most promising activities, demonstrating bactericidal, anti-adhesive and anti-biofilm activities, and synergistic properties with common antibiotics. Given that clove and cinnamon oils showed the best activities, they were incorporated into topical formulations. Not only did the formulations with oils maintain antimicrobial and anti-adhesive activities, but their anti-biofilm property was potentiated. Tests on Galleria mellonella larvae suggested that the formulation is non-toxic. The formulations proposed here are a great alternative for the decolonisation of surfaces containing MRSA and can help circumventing antimicrobial resistance, a growing threat in the hospital environment.

Structural and Kinetic Characterization of the SpeG Spermidine/Spermine N-acetyltransferase from Methicillin-Resistant Staphylococcus aureus USA300.

Polyamines are simple yet critical molecules with diverse roles in numerous pathogenic and non-pathogenic organisms. Regulating polyamine concentrations affects the transcription and translation of genes and proteins important for cell growth, stress, and toxicity. One way polyamine concentrations are maintained within the cell is via spermidine/spermine N-acetyltransferases (SSATs) that acetylate intracellular polyamines so they can be exported. The bacterial SpeG enzyme is an SSAT that exhibits a unique dodecameric structure and allosteric site compared to other SSATs that have been previously characterized. While its overall 3D structure is conserved, its presence and role in different bacterial pathogens are inconsistent. For example, not all bacteria have speG encoded in their genomes; in some bacteria, the speG gene is present but has become silenced, and in other bacteria, it has been acquired on mobile genetic elements. The latter is the case for methicillin-resistant Staphylococcus aureus (MRSA) USA300, where it appears to aid pathogenesis. To gain a greater understanding of the structure/function relationship of SpeG from the MRSA USA300 strain (SaSpeG), we determined its X-ray crystal structure in the presence and absence of spermine. Additionally, we showed the oligomeric state of SaSpeG is dynamic, and its homogeneity is affected by polyamines and AcCoA. Enzyme kinetic assays showed that pre-incubation with polyamines significantly affected the positive cooperativity toward spermine and spermidine and the catalytic efficiency of the enzyme. Furthermore, we showed bacterial SpeG enzymes do not have equivalent capabilities to acetylate aminopropyl versus aminbutyl ends of spermidine. Overall, this study provides new insight that will assist in understanding the SpeG enzyme and its role in pathogenic and non-pathogenic bacteria at a molecular level.

Emergence of community-associated methicillin-resistant Staphylococcus aureus ΨUSA300 among Japanese people with HIV, resulted from stepwise mutations in 2010s

Although infection with the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 is extremely rare in Japan, the uniquely evolved clone ΨUSA300 has been reported in Japan. An outbreak of a distinct USA300 clone was recently reported in an HIV/AIDS referral hospital in Tokyo. The present study investigated the evolutionary origin and genetic diversity of USA300-related clones causing regional outbreaks among people living with HIV (PLWHIV) in Tokyo. MRSA isolates collected from PLWHIV in an HIV/AIDS referral center in Tokyo were subjected to whole-genome sequencing and their genetic features were compared with those of previously described USA300 MRSA genomes. Of the 28 MRSAs isolated in 2016–2019, 23 (82.1%) were identified as USA300, with 22 (95.6%) of the latter identified as ΨUSA300. Although the genomic structure of ΨUSA300 was identical to the structures of reference USA300 strains, one clade (cluster A) was found to have acquired 29 previously identified lineage-specific mutations in a stepwise manner. The estimated divergence dates of ΨUSA300 and Cluster A were 2009 and 2012, respectively. These findings suggested that the ΨUSA300 clone had spread among PLWHIVs in Tokyo in the early 2010s, with stepwise acquisition of lineage-specific nonsynonymous mutations.

Phylogenetic Analysis and Virulence Characteristics of Methicillin-Resistant Staphylococcus aureus ST45 in China: a Hyper-Virulent Clone Associated with Bloodstream Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 45 (ST45) was rarely found in China. This study was conducted to trace the transmission and evolution of emerging MRSA ST45 strains in mainland China and explore its virulence. A total of 27 ST45 isolates were included for whole-genome sequencing and genetic characteristic analysis. Epidemiological results showed that MRSA ST45 isolates were often obtained from blood, primarily originated in Guangzhou, and carried diverse virulence and drug resistance genes. Staphylococcal cassette chromosome mec type IV (SCCmec IV) dominated in MRSA ST45 (23/27, 85.2%). ST45-SCCmec V was located on a phylogenetic clade distinct from the SCCmec IV cluster. We selected two representative isolates, MR370 (ST45-SCCmec IV) and MR387 (ST45-SCCmec V), and performed hemolysin activity, a blood killing assay, a Galleria mellonella infection model, and a mouse bacteremia model, as well as real-time fluorescence quantitative PCR. MR370 was proved to have extreme virulence in the phenotypic assays and at the mRNA level compared with ST59, ST5, and USA300 MRSA strains. MR387 was comparable to USA300-LAC on the phenotype and was verified to have higher expression of scn, chp, sak, saeR, agrA, and RNAIII than USA300-LAC. The results emphasized the extraordinary performance of MR370 and the good potential of MR387 in virulence causing bloodstream infection. Meanwhile, we conclude that China MRSA ST45 showed two different clonotypes, which may be widespread in the future. The entire study is valuable as a timely reminder and reports virulence phenotypes of China MRSA ST45 for the first time. IMPORTANCE Methicillin-resistant Staphylococcus aureus ST45 is epidemic worldwide. This study contributed to the awareness of the Chinese hyper-virulent MRSA ST45 strains and served as a timely reminder of its wide dissemination of clonotypes. Further, we provide novel insights for prevention from the perspective of bloodstream infections. ST45-SCCmec V is a clonotype deserving special attention in China, and we performed genetic and phenotypic analyses for the first time on it.

Pre-epidemic evolution of the MRSA USA300 clade and a molecular key for classification.

USA300 has remained the dominant community and healthcare associated methicillin-resistant Staphylococcus aureus (MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American epidemic (SAE) clade and North American epidemic (NAE) clade. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Here, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome, to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage. Phylogenetic analysis with these genomes confirms that the NAE and SAE USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We provide evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South and North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

In Vitro and In Vivo Antibiofilm Activity of Red Onion Scales: An Agro-Food Waste

Red onion wastes (ROW) are valuable sources of bioactive metabolites with promising antimicrobial effects. Methicillin-resistant Staphylococcus aureus (MRSA) infections are a growing risk in hospitals and communities. This study aims to investigate the in vitro and in vivo antibiofilm activities of the acidified ethanolic extract of red onion scales (RO-T) and its fractions against an MRSA vaginal colonization model. The RO-T extract, as well as its anthocyanin-rich fraction (RO-P) and flavonoid-rich fraction (RO-S), recorded a promising antibacterial activity against highly virulent strains of bacteria (MRSA, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa). RO-S showed the highest antibacterial activity (MBC of 0.33 ± 0.11 mg/mL) against MRSA USA300 and significantly eradicated its biofilm formation with an IC50 of 0.003. Using a rat model, in vivo assessment on all samples, which were formulated as a hydrogel, revealed a significant reduction of MRSA bacterial load recovered from an infected vagina compared to that of the negative control group (NCG). RO-T extract and vancomycin groups recorded the highest antibacterial activity with a bacterial load 2.998 and 3.358 logs lower than the NCG, respectively. The histopathological investigation confirmed our findings. RO-T and RO-S were standardized for their quercetin content. Finally, ROW offers a new potent antibiofilm agent mostly due to its high quercetin content.

In silico identification of two peptides with antibacterial activity against multidrug-resistant Staphylococcus aureus

Here we report two antimicrobial peptides (AMPs), HG2 and HG4 identified from a rumen microbiome metagenomic dataset, with activity against multidrug-resistant (MDR) bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) strains, a major hospital and community-acquired pathogen. We employed the classifier model design to analyse, visualise, and interpret AMP activities. This approach allowed in silico discrimination of promising lead AMP candidates for experimental evaluation. The lead AMPs, HG2 and HG4, are fast-acting and show anti-biofilm and anti-inflammatory activities in vitro and demonstrated little toxicity to human primary cell lines. The peptides were effective in vivo within a Galleria mellonella model of MRSA USA300 infection. In terms of mechanism of action, HG2 and HG4 appear to interact with the cytoplasmic membrane of target cells and may inhibit other cellular processes, whilst preferentially binding to bacterial lipids over human cell lipids. Therefore, these AMPs may offer additional therapeutic templates for MDR bacterial infections.

The trend of environmental and clinical methicillin-resistant Staphylococcus aureus in a hospital in Taiwan: Impact of USA300.

The environment may facilitate transmission of health care-associated methicillin-resistant Staphylococcus aureus (MRSA) and the pathogen is frequently shed by patients. However, the molecular characteristics and genetic relatedness between clinical and environmental MRSA isolates remain largely unclear in the clinical setting. A total of 100 hospitalized patients with MRSA infection and 25 hospitalized patients without MRSA infection were enrolled in a medical center, Taiwan in 2019. Environmental and clinical MRSA isolates were characterized by antibiotic susceptibility testing and molecular methods. In the study, we detected 17 MRSA isolates in the environment that surrounded 15 MRSA-infected patients and one environmental MRSA isolate from one patient without MRSA infection. The molecular analysis revealed a high genetic diversity within either environmental or clinical MRSA isolates, while the USA300 clone (pulsotype AI, SCCmec IV, ST8, PVL-positive) accounts for 39% (7/18) of environmental and 33% (7/21) of clinical MRSA isolates. Moreover, 13 of the 15 MRSA-infected patients had identical paired clinical-environmental MRSA isolates, which exhibited indistinguishable genetic relatedness and highly similar antibiotic susceptibility phenotype, suggesting a possible transmission cycle of MRSA in the hospital. The environmental MRSA was closely linked to MRSA isolated from patients, suggesting that the environment may act as a reservoir of MRSA. Besides, the USA300 MRSA has become a major clone in the hospital setting. An effective and rigorous approach to environmental cleaning and decontamination is suggested to eradicate MRSA in the hospital.

Genomic Update of Phenotypic Prediction Rule for Methicillin-Resistant Staphylococcus aureus (MRSA) USA300 Discloses Jail Transmission Networks with Increased Resistance.

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of health care-associated (HA) and community-associated (CA) infections. USA300 strains are historically CA-MRSA, while USA100 strains are HA-MRSA. Here, we update an antibiotic prediction rule to distinguish these two genotypes based on antibiotic resistance phenotype using whole-genome sequencing (WGS), a more discriminatory methodology than pulsed-field gel electrophoresis (PFGE). MRSA clinical isolates collected from 2007 to 2017 underwent WGS; associated epidemiologic data were ascertained. In developing the rule, we examined MRSA isolates that included a population with a history of incarceration. Performance characteristics of antibiotic susceptibility for predicting USA300 compared to USA100, as defined by WGS, were examined. Phylogenetic analysis was performed to examine resistant USA300 clades. We identified 275 isolates (221 USA300, 54 USA100). Combination susceptibility to clindamycin or levofloxacin performed the best overall (sensitivity 80.7%, specificity 75.9%) to identify USA300. The average number of antibiotic classes with resistance was higher for USA100 (3 versus 2, P < 0.001). Resistance to ≤2 classes was predictive for USA300 (area under the curve (AUC) 0.84, 95% confidence interval 0.78 to 0.90). Phylogenetic analysis identified a cluster of USA300 strains characterized by increased resistance among incarcerated individuals. Using a combination of clindamycin or levofloxacin susceptibility, or resistance to ≤2 antibiotic classes, was predictive of USA300 as defined by WGS. Increased resistance was observed among individuals with incarceration exposure, suggesting circulation of a more resistant USA300 clade among at-risk community networks. Our phenotypic prediction rule could be used as an epidemiologic tool to describe community and nosocomial shifts in USA300 MRSA and quickly identify emergence of lineages with increased resistance.IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of health care-associated (HA) and community-associated (CA) infections, but the epidemiology of these strains (USA100 and USA300, respectively) now overlaps in health care settings. Although sequencing technology has become more available, many health care facilities still lack the capabilities to perform these analyses. In this study, we update a simple prediction rule based on antibiotic resistance phenotype with integration of whole-genome sequencing (WGS) to predict strain type based on antibiotic resistance profiles that can be used in settings without access to molecular strain typing methods. This prediction rule has many potential epidemiologic applications, such as analysis of retrospective data sets, regional monitoring, and ongoing surveillance of CA-MRSA infection trends. We demonstrate application of this rule to identify an emerging USA300 strain with increased antibiotic resistance among incarcerated individuals that deviates from the rule.

Bioguided Isolation of Antibiofilm and Antibacterial Pregnane Glycosides from Caralluma quadrangula: Disarming Multidrug-Resistant Pathogens.

Methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB) present a serious challenge because of their capability to cause biofilm resistance to commonly used antibiotics producing chronic infections and hindering the process of wound healing. In the current study, we investigated the antibacterial activity of Caralluma quadrangula extracts (MeOH, and its fractions CH2Cl2 and n-butanol) against multidrug-resistant MRSA USA300 and A. baumannii AB5057. In vitro, the MeOH extract and both fractions of C. quadrangula significantly inhibited biofilm formation and disrupted previously established biofilm by MRSA and MDRAB at all the tested concentrations (0.625, 0.313, and 0.156 mg/mL). In vivo, C. quadrangula extracts successfully decreased bacterial loads in MRSA-infected skin lesions in mice. Four pregnane glycosides and one flavone glycoside were isolated from the bioactive n-butanol fraction. The isolated compounds (Rus A–E) were tested for their biofilm inhibition and biofilm detachment activities. The results revealed that Rus C was the most active compound (IC50 = 0.139 mmole), while Rus E was the least active (IC50 = 0.818 mmole). These results support the potential use of C. quadrangula extracts or their isolated compounds for hindering the biofilm attachment and the virulence of MRSA and MDRAB and their application as a topical antimicrobial preparation for MRSA skin infections.

Antimicrobial effectiveness of wound matrices containing native extracellular matrix with polyhexamethylene biguanide.

A variety of wound matrix materials that are designed to help heal both acute and chronic wounds are currently available. Because wounds often encounter opportunistic microbes that can delay healing, the effectiveness of these materials is often suboptimal, resulting in delayed or compromised wound healing. The importance of reducing and controlling wound microbes is well recognised and there are several antimicrobial options available to address this unmet clinical need. This study compares the antimicrobial and wound healing capabilities, both in vivo and in vitro against methicillin‐resistant Staphylococcus aureus (MRSA) USA 300, for the following compounds: Collagen Wound Matrix‐Anti Microbial (CWM‐AM); Collagen Wound Matrix‐Anti Microbial XT (CWM‐AM XT); Antimicrobial Hydrofiber Wound Dressing (AHWD); Dermal Scaffold with Silver (DRSAg); Collagen Extracellular Matrix (CEM); Collagen Wound Matrix (CWM); Matrix Wound Dressing with Silver (MWDAg); Cadexomer Iodine Gel (CIG); Triple Antibiotic Ointment (TAO); and Antimicrobial Wound Gel (AWG). For the in vitro zone of inhibition assay, AWG and CIG had the largest diffused areas, followed by CWM‐AM and CWM‐AM XT. Furthermore, CWM‐AM, CWM‐AM XT, AWG, and CIG exhibited a persistent antimicrobial activity for up to 10 days after incubation. However, in the cytotoxicity studies performed using human fibroblasts, CWM‐AM and CWM‐AM XT had no detrimental effects in cell proliferation and viability, while AWG and CIG were cytotoxic and prohibitive for cell proliferation. Treatments were then assessed for microbiology and wound healing efficacy using an in vivo porcine deep reticular dermal wound model. CWM‐AM XT displayed the greatest in vivo antimicrobial activity against MRSA USA300 and expedited the reepithelialisation at a faster rate than other treatment groups. This study shows that a novel collagen matrix containing an antimicrobial agent can reduce the bacterial load and support healing.

A Bacteriophage-Based Electrochemical Biosensor for Detection of Methicillin-Resistant Staphylococcus aureus

Bacterial pathogens are important targets for detection and early diagnostics of infection in healthcare. Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that can cause severe complications such as pneumonia and septicemia from surgical site infections. In this work, a carbon nanotube (CNT)-based impedimetric biosensor using SATA-8505, a bacteriophage, as a recognition element was developed for the detection of MRSA USA300, a common strain of MRSA found in surgical infections. The biosensor allows for fast and selective detection of viable MRSA cells by measuring impedance changes on the electrode surface as a result of bacterial capture by the phage-functionalized CNT electrode. The immobilization of the bacteriophage was achieved by inducing an electric-field and using a charge-directed orientation strategy, and activity of the immobilized phage was confirmed by an infectivity study using disk diffusion methods. The biosensing platform showed high selectivity and sensitivity toward MRSA USA300 with a limit of detection of 1.23 × 102 CFU/ml in aqueous solution and 1.29 × 102 CFU/ml in blood plasma. The biosensing platform could potentially be integrated into a lab-on-a-chip platform for point of care use and for other pathogens such as Pseudomonas aeruginosa, Klebsiella and Listeria monocytogenes.

Genomic structure of ST8-t008 USA300 and USA300-LV MRSA in the Rhine-Neckar Region, Germany, 2012–2018

Objective: Methicillin-resistant Staphylococcus aureus (MRSA) ST8-t008 of the pulsotype USA300 and the Latin American variant (USA300-LV) are the predominant virulent MRSA clonal lineages on the American continent. In Europe, the occurrence of USA300 or USA300-LV has often been related to international travel or outbreaks in hospitals. The replacement of local epidemic MRSA clones by these hypervirulent clones has not yet been demonstrated in Europe. This study aimed to gain insight into the genetic relatedness of ST8-t008 MRSA encountered in previous studies in the Rhine-Neckar Region, Germany, and ST8-t008 MRSA from other geographic regions.Methods: Nineteen ST8-t008 MRSA isolated between 2012 and 2018 were compared with publicly available sequences of ST8-t008 MRSA from travellers returning from the tropics, and USA300 and USA300-LV that were previously encountered in Europe.Results: We identified 14 of 19 (73.7%) of the local ST8-t008 MRSA being related to USA300 and five of 19 (26.3%) belonging to the USA300-LV cluster. Four suspected transmission clusters were identified without any evidence of in-hospital transmission.Conclusion: The genetic relatedness of these local strains to publicly available sequences of ST8-t008 MRSA from other parts of Europe and to MRSA of travellers returning from the tropics pointed to multiple introductions into Germany. However, four suspected transmission clusters may be an indication of transmission within the community.

Evaluation of ebselen in resolving a methicillin-resistant Staphylococcus aureus infection of pressure ulcers in obese and diabetic mice.

Pressure ulcers (PUs) are a source of morbidity in individuals with restricted mobility including individuals that are obese or diabetic. Infection of PUs with pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), impairs ulcers from healing. The present study evaluated ebselen as a topical antibacterial to treat MRSA-infected PUs. Against two different S. aureus strains, including MRSA USA300, resistance to ebselen did not emerge after 14 consecutive passages. Resistance to mupirocin emerged after only five passages. Additionally, ebselen was found to exert a modest postantibiotic effect of five hours against two MRSA strains. Ebselen was subsequently evaluated in MRSA-infected PUs in two models using obese and diabetic mice. In obese mice, topical ebselen (89.2% reduction) and oral linezolid (84.5% reduction) similarly reduced the burden of MRSA in infected PUs. However, in diabetic mice, topical ebselen (45.8% reduction in MRSA burden) was less effective. Histopathological evaluation of ulcers in diabetic mice determined that ebselen treatment resulted in fewer bacterial colonies deep within the dermis and that the treatment exhibited evidence of epithelial regeneration. Topical mupirocin was superior to ebselen in reducing MRSA burden in infected PUs both in obese (98.7% reduction) and diabetic (99.3% reduction) mice. Ebselen’s antibacterial activity was negatively impacted as the bacterial inoculum was increased from 105 CFU/mL to 107 CFU/mL. These results suggest that a higher dose of ebselen, or a longer course of treatment, may be needed to achieve a similar effect as mupirocin in topically treating MRSA-infected pressure ulcers.

Clinical characteristics and molecular analysis of USA300 and ST 764 methicillin-resistant Staphylococcus aureus isolates from outpatients in Japan by PCR-Based open reading frame typing

IntroductionUSA300 is the most common community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain. Sequence type (ST) 764 MRSA is a new local variant of the ST 5 lineage. The objective of this study was to determine the clinical characteristics of USA300 and ST 764 infections among outpatients in Japan. MethodsWe obtained MRSA isolates from 132 outpatients who visited our hospital from January 2016 to December 2017 and compared USA300 infection group to ST 764 infection group. Molecular analysis, including that of various toxins and other virulence factors, of the MRSA isolates were performed. In particular, we investigated the relationships among PCR-based open reading frame typing (POT) scores, MRSA clones, and virulence factors. ResultsTwenty-seven USA300 isolates (20.5%) and 16 ST 764 isolates (12.1%) were identified. Although USA300 and ST 764 had lower rates of risk factors, their infection rates were higher. USA300-infected patients had higher rates of deep skin and soft tissue infections compared with the non-USA300 CA-MRSA-infected patients. Notably, the USA300 and ST 764 isolates had unique POT scores. ConclusionsOur results indicated that USA300 MRSA was spreading in an area 120 km west of Tokyo, Japan. We observed multiple cases of ST 764 MRSA infection, raising concerns about the antimicrobial resistance of ST 764, as it limits the choices of antibiotics to treat infection. The POT score can predict the presence of toxins and virulence factors, as well as the clone identity of MRSA with high accuracy.

Antimicrobial Nonsusceptibility Among Invasive MRSA USA300 Strains by Healthcare Exposure, Three Sites, 2005–2016

Background: Incidence of community-associated (CA) and healthcare-associated, community-onset (HACO) USA300 methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections has remained unchanged in recent years. Traditionally considered a CA strain, USA300 is increasingly associated with healthcare settings. We examined whether antimicrobial nonsusceptibility among USA300 strains could distinguish epidemiologic class (community vs hospital), and whether divergences in susceptibility were occurring over time. Methods: We used data on invasive MRSA infections from active, population, and laboratory-based surveillance during 2005–2016 from 11 counties in 3 states. Invasive cases were defined as MRSA isolation from a normally sterile site in a surveillance area resident. Cases were considered hospital-onset (HO) if the culture was obtained &gt;3 days after hospitalization and HACO if ≥1 of the following risk factors was present: hospitalization, surgery, dialysis, or residence in a long-term care facility in the past year; or central vascular catheter ≤2 days before culture. Otherwise, cases were considered CA. Sites submitted a convenience sample of clinical MRSA isolates for molecular typing and antimicrobial susceptibility testing. Molecular typing was performed by pulsed-field gel electrophoresis until 2008, when typing was inferred using a validated algorithm based on molecular characteristics. Reference broth microdilution was performed for 8 antimicrobials and interpreted based on CLSI interpretive criteria. We compared USA300 nonsusceptibility for HO and CA isolates. For antimicrobials with &gt;5% nonsusceptibility and for which HO isolates had greater nonsusceptibility than CA isolates, we compared nonsusceptibility for HACO and CA and analyzed annual trends in nonsusceptibility within each epidemiologic class (ie, CA, HACO, and HO) using linear regression. Results: Of 17,947 MRSA cases during 2005–2016, isolates were available for 6,685 (37%), and 2,120 were USA300 (34% CA, 52% HACO, 14% HO). HO isolates had more nonsusceptibility than CA isolates to gentamicin (2.2% vs 0.6%; P = .03), levofloxacin (47.8% vs 39.7%; P = .02), rifampin (3.7 vs 1.1%; P = .01), and trimethoprim-sulfamethoxazole (3.4% vs 0.6%; P = .04). HACO isolates also had more nonsusceptibility than CA isolates to levofloxacin (50.9% vs 39.7%; P &lt; .01). Levofloxacin nonsusceptibility increased during 2005–2016 for HACO and CA isolates (P &lt; .01), but not among HO isolates (P = .36) (Fig. 1). Conclusions: Overall, nonsusceptibility across drugs cannot distinguish USA300 isolates causing HO versus CA disease. Although HO isolates had higher levofloxacin nonsusceptibility than CA and HACO isolates early on, USA300 MRSA HACO isolates now have levofloxacin nonsusceptibility most similar to that of HO isolates. Further study could help to explore whether increases in fluoroquinolone nonsusceptibility among CA and HACO cases may be contributing to the persistence of USA300 strains.Disclosures: NoneFunding: None