Methicillin-resistant Staphylococcus aureus (MRSA) is a predominant nosocomial infection-causing bacteria. The aim of this study was to develop a novel single-bacteria multiplex digital PCR assays (SMD-PCR), which is capable of simultaneously detecting and discriminating Methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. This protocol employed TaqMan probes to detect SAOUHSC_00106 and mecA genes, with the latter being linked to methicillin resistance. A total of 72 samples from various specimen types were evaluated. The accuracy rates for the sputum samples, pus samples, swab samples, ear secretion samples, and catheter samples were 94.44%, 100%, 92%, 100%, and 100%, respectively. Our results showed that the clinical practicability of SMD-PCR has applicability to the rapid detection of MRSA without DNA extraction or bacterial culture, and can be utilized for the rapid detection of Staphylococcus aureus and the timely identification of MRSA in clinical samples, thereby providing an advanced platform for the rapid diagnosis of clinical MRSA infection.

UNASSIGNED: Over 75% of clinical microbiological infections are caused by bacterial biofilms that grow on wounds or implantable medical devices. This work describes the development of a new poly(diallyldimethylammonium chloride) (PDADMAC)/alginate-coated gold nanorod (GNR/Alg/PDADMAC) that effectively disintegrates the biofilms of Staphylococcus aureus (S. aureus), a prominent pathogen responsible for hospital-acquired infections.
UNASSIGNED: GNR was synthesised via seed-mediated growth method, and the resulting nanoparticles were coated first with Alg and then PDADMAC. FTIR, zeta potential, transmission electron microscopy, and UV-Vis spectrophotometry analysis were performed to characterise the nanoparticles. The efficacy and speed of the non-coated GNR and GNR/Alg/PDADMAC in disintegrating S. aureus-preformed biofilms, as well as their in vitro biocompatibility (L929 murine fibroblast) were then studied.
UNASSIGNED: The synthesised GNR/Alg/PDADMAC (mean length: 55.71 ± 1.15 nm, mean width: 23.70 ± 1.13 nm, aspect ratio: 2.35) was biocompatible and potent in eradicating preformed biofilms of methicillin-resistant (MRSA) and methicillin-susceptible S. aureus (MSSA) when compared to triclosan, an antiseptic used for disinfecting S. aureus colonisation on abiotic surfaces in the hospital. The minimum biofilm eradication concentrations of GNR/Alg/PDADMAC (MBEC50 for MRSA biofilm = 0.029 nM; MBEC50 for MSSA biofilm = 0.032 nM) were significantly lower than those of triclosan (MBEC50 for MRSA biofilm = 10,784 nM; MBEC50 for MRSA biofilm 5967 nM). Moreover, GNR/Alg/PDADMAC was effective in eradicating 50% of MRSA and MSSA biofilms within 17 min when used at a low concentration (0.15 nM), similar to triclosan at a much higher concentration (50 µM). Disintegration of MRSA and MSSA biofilms was confirmed by field emission scanning electron microscopy and confocal laser scanning microscopy.
UNASSIGNED: These findings support the potential application of GNR/Alg/PDADMAC as an alternative agent to conventional antiseptics and antibiotics for the eradication of medically important MRSA and MSSA biofilms.

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

Staphylococcus aureus (S. aureus) is a common opportunistic and zoonotic pathogen in the world and could easily cause human infections and food contaminations. This study investigated the sequence typing and resistance profiles of S. aureus isolates from patient and food samples in Shijiazhuang, China. A total of 101 S. aureus isolates were distributed into six clonal complexes (CCs) and 16 singletons. A total of 86 patient isolates were distributed into six clonal CCs and 12 singletons, including a new ST. CC59, CC5, CC22, and CC398 were the predominant CCs of patient isolates. A total of 15 foodborne S. aureus isolates were distributed into 3 CCs and 4 STs, and CC1 was the most prevalent CC. Moreover, 101 S. aureus isolates had high resistance to penicillin and low resistance to chloramphenicol and rifampicin. A total of 39 strains of methicillin-resistant Staphylococcus aureus (MRSA) were detected in this study, including thirty-eight strains of patient isolates (44.2%, 38/86) and one strain of food isolates (6.7%, 1/15). MRSA-ST5, MRSA-ST59, and MRSA-ST239 were the predominant MRSA isolates in hospitals. The present study explained the relationship between S. aureus isolated from patient and food samples and indicated the risks of S. aureus in infectious diseases.

Methicillin-susceptible Staphylococcus aureus (MSSA) is a more prevalent neonatal intensive care unit (NICU) pathogen than methicillin-resistant S. aureus (MRSA). However, the introduction and spread of MSSA, the role of systematic decolonization, and optimal infection prevention and control strategies remain incompletely understood. We previously screened infants hospitalized in a university-affiliated level III to IV NICU twice monthly over 18 months for S. aureus colonization and identified several prevalent staphylococcal protein A (spa) types. Here, we performed whole-genome sequencing (WGS) and phylogenetic comparisons of 140 isolates from predominant spa types t279, t1451, and t571 to examine possible transmission routes and identify genomic and epidemiologic features associated with the spread of dominant clones. We identified two major MSSA clones: sequence type 398 (ST398), common in the local community, and ST1898, not previously encountered in the region. ST398 NICU isolates formed distinct clusters with closely related community isolates from previously published data sets, suggesting multiple sources of acquisition, such as family members or staff, including residents of the local community. In contrast, ST1898 isolates were nearly identical, pointing to clonal expansion within the NICU. Almost all ST1898 isolates harbored plasmids encoding mupirocin resistance (mupA), suggesting an association between the proliferation of this clone and decolonization efforts with mupirocin. Comparative genomics indicated genotype-specific pathways of introduction and spread of MSSA via community-associated (ST398) or health care-associated (ST1898) sources and the potential role of mupirocin resistance in dissemination of ST1898. Future surveillance efforts could benefit from routine genotyping to inform clone-specific infection prevention strategies. IMPORTANCE Methicillin-susceptible Staphylococcus aureus (MSSA) is a significant pathogen in neonates. However, surveillance efforts in neonatal intensive care units (NICUs) have focused primarily on methicillin-resistant S. aureus (MRSA), limiting our understanding of colonizing and infectious MSSA clones which are prevalent in the NICU. Here, we identify two dominant colonizing MSSA clones during an 18-month surveillance effort in a level III to IV NICU, ST398 and ST1898. Using genomic surveillance and phylogenetic analysis, coupled with epidemiological investigation, we found that these two sequence types had distinct modes of spread, namely the suggested exchange with community reservoirs for ST398 and the contribution of antibiotic resistance to dissemination of ST1898 in the health care setting. This study highlights the additional benefits of whole-genome surveillance for colonizing pathogens, beyond routine species identification and genotyping, to inform targeted infection prevention strategies.

BACKGROUND: Methicillin resistance, inducible clindamycin resistance (ICR), biofilm production, and increased minimum inhibitory concentration (MIC) of vancomycin in Staphylococcus aureus are major causes of antibiotic treatment failure and increased morbidity and mortality. The surveillance of such isolates and the study of their antimicrobial pattern are essential in managing the infections caused by these isolates. This study aimed to determine methicillin resistance, biofilm production, and ICR in S. aureus isolates from a tertiary care hospital in Kathmandu, Nepal.
METHODS: A total of 217 S. aureus isolated from different samples were processed following standard laboratory procedures. Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion technique. Methicillin-resistant S. aureus (MRSA) were identified by the cefoxitin disk diffusion test, and biofilm producers were examined using the microtiter plate technique. D-test and E-test were performed to determine inducible clindamycin resistance and minimum inhibitory concentration of vancomycin, respectively.
RESULTS: Among the 217 S. aureus isolates, 78.3% were multidrug-resistant (MDR), 47.0% were MRSA, 62.2% were biofilm producers, and 50.7% showed ICR. All MRSA isolates exhibited MIC levels of vancomycin within the susceptible range. Biofilm producers and MRSA isolates showed elevated antimicrobial resistance. MRSA was significantly associated with MDR. Biofilm-producing and multidrug-resistant MRSA isolates showed significantly higher MIC levels of vancomycin (p = 0.0013 and < 0.0001, respectively), while ICR was significantly higher in MDR (p = 0.0001) isolates.
CONCLUSIONS: High multidrug resistance, MRSA, and ICR in this study call for routine evaluation of antibiotic susceptibility patterns of S. aureus. Vancomycin can be used to treat serious staphylococcal infections. Clindamycin should be prescribed only after performing the D-test. Drugs like teicoplanin, chloramphenicol, doxycycline, amikacin, and levofloxacin can treat MRSA infections.

Staphylococcus aureus (S. aureus) is one of the main pathogens causing mastitis in dairy cows. The current work mainly focuses on the pathway of apoptosis induction in MAC-T cells caused by S. aureus infection or other factors. However, the physiological characteristics of S. aureus infected MAC-T cells and the resulting mRNA expression profile remain unknown particularly in the case of diverse drug resistant strains. Methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains were used to infect MAC-T cells to investigate this issue. The adhesion, invasion and apoptosis ability of MRSA-infected group and MSSA-infected group was assessed over time (2, 4, 6, 8, and 12 h). After 8 h, the RNA sequencing was conducted on the MRSA-infected and the MSSA-infected with uninfected MAC-T cells as controls. The results showed that the adhesion and invasion ability of MRSA-infected and MSSA-infected to MAC-T cells increased and then decreased with infection time, peaking at 8 h. The adhesion and invasion rates of the MSSA-infected were substantially lower than those of the MRSA-infected, and the invasion rate of the MSSA-infected group was nearly non-existent. Then the apoptosis rate of MAC-T cells increased as the infection time increased. The transcriptome analysis revealed 549 differentially expressed mRNAs and 390 differentially expressed mRNAs in MRSA-infected and MSSA-infected MAC-T cells, respectively, compared to the uninfected MAC-T cells. According to GO analysis, these differentially expressed genes were involved in immune response, inflammation, apoptosis, and other processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to adhesion, invasion inflammation and apoptosis, including AMPK, FOXO, HIF-1, IL-17, JAK-STAT, MAPK, mTOR, NF-κB, p53, PI3K-Akt, TNF, Toll-like receptor, Rap1, RAS, prion disease, the bacterial invasion of epithelial cells pathway. We found 86 DEGs from 41 KEGG-enriched pathways associated with adhesion, invasion, apoptosis, and inflammation, all of which were implicated in MAC-T cells resistance to MRSA and MSSA infection. This study offers helpful data toward understanding the effect of different drug-resistant S. aureus on dairy cow mammary epithelial cells and aid in the prevention of mastitis in the dairy industry.

Staphylococcus aureus (S. aureus) sequence type 398 (ST398) has aroused great concern for its spread throughout the world. ST398 community-acquired MRSA (CA-MRSA) has been given greater emphasis because of its high virulence and high probability of treatment failure. Herein, A 22-year-old male was admitted to our hospital with a history of fever, chest pain and dyspnea for 2 days. A chest CT scan showed infiltrative and nodular shadows. The sequence type of the isolates from blood culture was ST398, the virulence genes detected was PVL gene (lukS-PV and lukF-PV). Despite resuscitation efforts, he died of multiple organ failure on admission 3rd day. This is the first described case of severe pneumonia and sepsis due to hematogenous spread of scalp furuncles caused by Panton-Valentine leukocidin (PVL) positive community-acquired methicillin-susceptible S. aureus (CA-MSSA) ST398 strains in an immunocompentent adult in mainland China. This report highlight the emergence CA-PVL-MSSA ST398 infection and its association with life-threatening infections. Early decolonization and identification of ST398 is critical. Severe skin and soft tissue infections should be suspected for ST398 PVL-MSSA.

BACKGROUND: Staphylococcus aureus (S. aureus), especially methicillin-resistant Staphylococcus aureus (MRSA), is considered a common zoonotic pathogen, causing severe infections. The objective of this study was to investigate the antimicrobial susceptibility, resistance genes and molecular epidemiology among MRSA and methicillin-susceptible Staphylococcus aureus (MSSA) isolated from food animals in Sichuan Province, China.
METHODS: This study was conducted on 236 S. aureus isolates. All isolates were subjected to antimicrobial susceptibility testing by using a standard microbroth dilution method. The Polymerase Chain Reaction (PCR) was performed to identify genes encoding the β-lactams resistance (blaZ, mecA), macrolides (ermA, ermB, ermC) and aminoglycosides (aacA-aphD). The molecular structures and genomic relatedness of MRSA isolates were determined by staphylococcal chromosome cassette mec (SCCmec) typing and pulsed-field gel electrophoresis (PFGE), respectively.
RESULTS: Among 236 isolates, 24 (10.17 %) were recognized as MRSA. MRSA isolates showed different resistance rates to 11 antimicrobials ranging from 33.33 to 100 %, while for MSSA isolates the rates varied from 8.02 to 91.51 %. Multi-drug resistance phenotype was found in all MRSA isolates. The ermC gene encoding macrolides-lincosamides-streptogramin B was the most prevalent gene detected in 87.29 % of the S. aureus isolates, followed by ermB (83.05 %), blaZ (63.98 %), aacA-aphD (44.07 %), ermA (11.44 %) and mecA (11.02 %) genes. The prevalence of resistance genes in MRSA isolates was significantly higher than that of MSSA. Regarding the molecular morphology, SCCmec III (12/24, 50 %) was the most common SCCmec type. Furthermore, the PFGE typing showed that 24 MRSA were divided into 15 cluster groups (A to O), the major pulsotype J encompassed 25 % of MRSA isolates.
CONCLUSIONS: The S. aureus isolates from food animals in Sichuan province of China have severe antimicrobials resistance with various resistance genes, especially MRSA isolates. Additionally, the genetic pool of MRSA isolates is diverse and complex, and further investigation is necessary.

Staphylococcus aureus is an opportunistic pathogen leading to food poisoning as well as human infections. The present study examined the prevalence and characterization of antimicrobial-resistant S. aureus in sushi from 42 outlets and in pork products from eight outlets in Beijing, China. The total bacterial counts were between 3.0 and 8.9 log CFU/g (mean 5.5 ± 1.5 log CFU/g) in sushi products and 4.8 to 7.4 log CFU/g (mean 5.6 ± 0.8 log CFU/g) in pork products. The mean counts of coliforms were 2.7 and 2.9 log CFU/g in sushi and pork, respectively. Staphylococcus aureus was isolated from seven sushi outlets (13 isolates) and two pork outlets (2 isolates) with average counts below 2 log CFU/g in all cases. A total of 15 S. aureus isolates were further characterized. Six lineages of S. aureus were present, including ST398 (n = 5), ST25 (n = 4), ST15 (n = 2), ST59 (n = 2), ST8 (n = 1) and ST2631 (n = 1). Thirteen isolates contained the scn virulence marker, whereas four and eight isolates contained the virulence marker edinB and enterotoxin genes, respectively. Characterization of antimicrobial resistance profiles documented resistances to ampicillin (n = 15), penicillin (n = 14), ceftazidime (n = 6), erythromycin (n = 4), tetracycline (n = 3), clindamycin (n = 3), and gentamicin (n = 1). Three MRSA isolates were obtained, one from pork (ST398) and two from one sushi outlet (ST59). They were all resistant to at least three classes of antimicrobials and two of them contained the scn gene and enterotoxin genes. Twelve sushi isolates and one of the pork isolates contained the scn gene, indicating that they were of human origin. This emphasizes the potential importance of transmission through foods of antimicrobial-resistant S. aureus including MRSA. We also showed that S. aureus exhibited geographical variation with regards to ST profiles, antimicrobial-resistance and virulence genes when comparing isolates from sushi products sold in Beijing and Copenhagen, Denmark. Whereas food safety is not compromised by the presence of low amounts of S. aureus in sushi, this study shows that with regards to public health such foods may serve as vehicles for transmission of multidrug-resistant S. aureus and MRSA lineages.