Comparative Study of Two-Dimensional (2D) vs. Three-Dimensional (3D) Organotypic Kertatinocyte-Fibroblast Skin Models for Staphylococcus aureus (MRSA) Infection

The invasion of skin tissue by Staphylococcus aureus is mediated by mechanisms that involve sequential breaching of the different stratified layers of the epidermis. Induction of cell death in keratinocytes is a measure of virulence and plays a crucial role in the infection progression. We established a 3D-organotypic keratinocyte-fibroblast co-culture model to evaluate whether a 3D-skin model is more effective in elucidating the differences in the induction of cell death by Methicillin-resistant Staphylococcus aureus (MRSA) than in comparison to 2D-HaCaT monolayers. We investigated the difference in adhesion, internalization, and the apoptotic index in HaCaT monolayers and our 3D-skin model using six strains of MRSA representing different clonal types, namely, ST8, ST30, ST59, ST22, ST45 and ST239. All the six strains exhibited internalization in HaCaT cells. Due to cell detachment, the invasion study was limited up to two and a half hours. TUNEL assay showed no significant difference in the cell death induced by the six MRSA strains in the HaCaT cells. Our 3D-skin model provided a better insight into the interactions between the MRSA strains and the human skin during the infection establishment as we could study the infection of MRSA in our skin model up to 48 h. Immunohistochemical staining together with TUNEL assay in the 3D-skin model showed co-localization of the bacteria with the apoptotic cells demonstrating the induction of apoptosis by the bacteria and revealed the variation in bacterial transmigration among the MRSA strains. The strain representing ST59 showed maximum internalization in HaCaT cells and the maximum cell death as measured by Apoptotic index in the 3D-skin model. Our results show that 3D-skin model might be more likely to imitate the physiological response of skin to MRSA infection than 2D-HaCaT monolayer keratinocyte cultures and will enhance our understanding of the difference in pathogenesis among different MRSA strains.

VraSR Regulatory System Contributes to the Virulence of Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) in a 3D-Skin Model and Skin Infection of Humanized Mouse Model

The vancomycin-resistance associated sensor/regulator, VraSR two-component regulatory-system (VraSR), regulates virulence and the response of Staphylococcus aureus (SA) to environmental stress. To investigate the role of VraSR in SA skin and soft tissue infections (SSTI), we inactivated the VraSR of a clinical CA-MRSA ST30 strain by insertional mutation in vraR gene using the TargeTron-Gene Knockout System. We constructed an organotypic keratinocyte fibroblast co-culture (3D-skin model) and a humanized mouse as SSTI infection models. In the 3D-skin model, inactivation of VraSR in the strains ST30 and USA300 showed 1-log reduction in adhesion and internalization (p < 0.001) compared to the respective wildtype. The mutant strains of ST30 (p < 0.05) and USA300-LAC (p < 0.001) also exhibited reduced apoptosis. The wildtype ST30 infection in the humanized mouse model demonstrated increased skin lesion size and bacterial burden compared to BALB/c mice (p < 0.01). The response of the humanized mouse towards the MRSA infection exhibited human similarity indicating that the humanized mouse SSTI model is more suitable for evaluating the role of virulence determinants. Inactivation of VraSR in ST30 strain resulted in decreased skin lesion size in the humanized mouse SSTI model (p < 0.05) and reduction in apoptotic index (p < 0.01) when compared with the wildtype. Our results reveal that inactivating the VraSR system may be a potent anti-virulence approach to control MRSA infection.

Staphylococcus aureus in poultry, with special emphasis on methicillin-resistant strain infection: A comprehensive review from one health perspective

Staphylococcus aureus is a Gram-positive coccus normally present on the skin and internal organs of animals, birds, and humans. Under certain conditions, S. aureus could produce septicemia and affection of the skin, joints, and heart, as well as sepsis and death. The pathogenicity of S. aureus is associated with the presence of some virulent surface proteins and the production of some virulent toxins and enzymes. This pathogen is considered one of the most important and worldwide foodborne causes as it is incriminated in most cases of food poisoning. The hazardous use of antibiotics in the veterinary field leads to the development of multidrug-resistant S. aureus strains that can be transmitted to humans. The incidence of methicillin-resistant S. aureus (MRSA) strains has increased globally. These resistant strains have been detected in live animals, poultry, and humans. In addition, retail animal products, especially those of avian origin, are considered the main source of MRSA strains that can be easily transmitted to humans. MRSA infection is regarded as nosocomial or occupational. Humans get infected with MRSA strains through improper handling or preparation of contaminated animals or poultry carcasses or improper cooking with contaminated meat. Live birds also can transmit MRSA to close-contact workers in poultry farms. Transmission of MRSA infection in hospitals is from an infected individual to a healthy one. Prevention and control of MRSA are based on the application of hygienic measures in farms as well as proper processing, handling, and cooking of retail poultry products. The cooperation between veterinary and human practitioners is a must to avoid the possibility of zoonotic transmission. Accordingly, this review focused on the sources and transmission of MRSA infection, virulence and resistance factors, incidence and prevalence in poultry and different products, antibiotic resistance, and prevention and control strategies.

Nemonoxacin enhances antibacterial activity and anti-resistant mutation ability of vancomycin against methicillin-resistant Staphylococcus aureus in an in vitro dynamic pharmacokinetic/pharmacodynamic model

Reduced susceptibility and emergence of resistance to vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) have led to the development of various vancomycin based combinations. Nemonoxacin is a novel nonfluorinated quinolone with antibacterial activity against MRSA. The present study aimed to investigate the effects of nemonoxacin on antibacterial activity and the anti-resistant mutation ability of vancomycin for MRSA and explore whether quinolone resistance genes are associated with a reduction in the vancomycin minimal inhibitory concentration (MIC) and mutant prevention concentration (MPC) when combined with nemonoxacin. Four isolates, all with a vancomycin MIC of 2 μg/mL, were used in a modified in vitro dynamic pharmacokinetic/pharmacodynamic model to investigate the effects of nemonoxacin on antibacterial activity (M04, M23 and M24) and anti-resistant mutation ability (M04, M23 and M25, all with MPC ≥19.2 μg/mL) of vancomycin. The mutation sites of gyrA , gyrB , parC , and parE of 55 clinical MRSA isolates were sequenced. We observed that in M04 and M23, the combination of vancomycin (1g q12h) and nemonoxacin (0.5g qd) showed a synergistic bactericidal activity and resistance enrichment suppression. All clinical isolates resistant to nemonoxacin harbored gyrA (S84→L) mutation; gyrA (S84→L) and parC (E84→K) mutations were the two independent risk factors for the unchanged vancomycin MPC in combination. Nemonoxacin enhances the bactericidal activity and suppresses resistance enrichment ability of vancomycin against MRSA with a MIC of 2 μg/mL. Our in vitro data support the combination of nemonoxacin and vancomycin for the treatment of MRSA infection with a high MIC.

The Effectivity of Thymus vulgaris Extract on IL-1 Level and Bacterial Count in The Heart of Balb-c Mice Infected with MRSA

Background: The increasing phenomenon resistance of bacteria to various types of antibiotics, for example MRSA (Methicillin Resistant Staphylococcus aureus) became a serious problem. Over the last few decades S. aureus has become the dominant cause of endocarditis. MRSA data in Indonesia are still rarely reported. This has led to the importance of extracting natural substances that have anti-bacterial effects or that modulate immune response such as Thymus vulgaris (TV). It was hypothesized that TV can overcome MRSA infection by increasing IL-1, which is the initial cytokine in a natural immune response stimulated by the presence of S. aureus. Objective: This study was to investigate the effect of Thymus vulgaris extract on IL-1 and bacterial counts in the heart of balb-c mice infected with MRSA. Methods: There were 30 male Balb-c mice randomly divided into six groups. Group K (mice infected with MRSA without treatment),P1(mice treated with TV extract 7 days later infected with MRSA), P2(mice infected and treated with vancomycin), P3(mice infected and treated with TV+vancomycin extract), P4(mice infected and treated with amoxicillin), and P5 (mice infected and treated with TV extract+amoxicillin). IL-1 and bacterial count was assessed by ELISA and microbiological culture. Results: The result showed that there was a significant increase in IL-1 between groups (p=0.001).The median decrease in the bacterial count in the heart was 0 in almost treatment group except for P4 group which its median was 26.000 (p=0.161). The correlation between IL-1 levels and MRSA count in the heart had a weak negative correlation (r=-0.182). Conclusion: Thymus vulgaris extract was effective to increase IL-1 levels significantly but was not proven to decrease MRSA count in the heart of balb-c mice.

Systematic Review: A Comparison between Vancomycin and Daptomycin for Sepsis Infection Antibiotic Therapy

BACKGROUND: Sepsis is a dangerous condition that threatens life because of immune system dysregulation caused by an infection resulting in organ failure. One of the most common resistant strain bacteria that can cause sepsis is Methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin is the first-line therapy for treating sepsis infection caused by MRSA, but recently there have been some MRSA strains that are resistant to vancomycin therapy. AIM: This study aimed to review comparison between vancomycin and daptomycin for sepsis infection antibiotics therapy. MATERIALS AND METHODS: This research was a systematic review using three databases such as PubMed, ProQuest, and ScienceDirect. The journal articles included in this study were about randomized controlled trial (RCT) studies published from 2011 to 2020. RESULTS: This research included seven RCT studies, but none of them discuss the usage of daptomycin for sepsis treatment caused by MRSA. They discuss more the effect of dose, method of administration, and side effects of vancomycin therapy in relation to the outcome of the patient. CONCLUSIONS: Because of the lack of RCT articles that conducted experiments of daptomycin usage for sepsis treatment caused by MRSA infection, this research could not compare the effectiveness between vancomycin and daptomycin. However, from some case reports included in this research, there was evidence that the usage of daptomycin base after vancomycin treatment failure will cause another treatment failure.

Necrotizing pneumonia caused by methicillin-resistant Staphylococcus aureus

We report a fatal case of methicillin-resistant Staphylococcus aureus (MRSA)-induced necrotizing pneumonia that was refractory to adequate vancomycin treatment (trough value, 13.1 µg/mL), drainage of a hydropneumothorax, and veno-arterial extracorporeal membrane oxygenation. MRSA infection can cause rapidly progressive disease with a high case fatality rate, even with appropriate treatment.