scholarly journals Expression of type II toxin-antitoxin systems and ClpP protease of methicillin-resistant Staphylococcus aureus under thermal and oxidative stress conditions

2021 ◽  
Author(s):  
Samira Karimaei ◽  
Behrooz Sadeghi Kalani ◽  
Nader Shahrokhi ◽  
Rahil Mashhadi ◽  
Mohammad Reza Pourmand
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Stress Responses ◽  
Stress Conditions ◽  
Bacterial Survival ◽  
Methicillin Resistant ◽  
Persistent Infections ◽  
Qrt Pcr ◽  
Oxidative Stresses ◽  
Antibacterial Target

Background and Objectives: Staphylococcus aureus is a main human pathogen that causes a variety of chronic to persistent infections. Across the diverse factors of pathogenesis in bacteria, Toxin-Antitoxin (TA) systems can be considered as an antibacterial target due to their involvement in cellular physiology counting stress responses. Here, the expression of TA system genes and ClpP protease was investigated under the thermal and oxidative conditions in S. aureus strains. Materials and Methods: The colony-forming unit (CFU) was used to determine the effects of thermal and oxidative stresses on bacterial survival. Moreover, the expressions of TA system genes in S. aureus strains were evaluated 30 min and 1 h after thermal and oxidative stresses, respectively, by quantitative reverse transcriptase real-time PCR (qRT-PCR). Results: The cell viability was constant across thermal stress while oxidative stress induction showed a significantly decrease in the growth of Methicillin-Resistant S. aureus (MRSA) strain. Based on the qRT-PCR results, the expression of mazF gene increased under both thermal and oxidative stresses in the MRSA strain. Conclusion: A putative TA system (namely immA/irrA) most likely has a role under the stress condition of S. aureus. The MRSA strain responds to stress by shifting the expression level of TA genes that has diverse effects on the survival of the pathogen due to the stress conditions. The TA systems may be introduced as potential targets for antibacterial treatment.

Expression of Type II Toxin/Antitoxin systems and ClpP protease of Methicillin-Resistant Staphylococcus aureus under stress condition

2020 ◽  
Author(s):  
Samira Karimaei ◽  
Behrooz Sadeghi Kalani ◽  
Nader Shahrokhi ◽  
Rahil Mashhadi ◽  
Mohammad Reza Pourmand
Keyword(s):  
Staphylococcus Aureus ◽  
Stress Responses ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Stress Condition ◽  
Cell Physiology ◽  
Type Ii ◽  
Methicillin Resistant ◽  
Persistent Infections ◽  
Oxidative Stresses ◽  
Antibacterial Target

Abstract BackgroundStaphylococcus aureus is a major human pathogen causing chronic to persistent infections. Amongst diverse factors of pathogenesis in bacteria, toxin-antitoxin (TA) systems have a potential to be presented as an antibacterial target due to their participation in cell physiology including stress responses. This study was conducted to determine the effects of thermal and oxidative stresses on expression of type II Toxin/Antitoxin systems and ClpP protease in Methicillin-Resistant Staphylococcus aureus (MRSA).Materials/methodsExpression of type II TA genes (mazF, relE1, relE2 and immA) and clpP gene in MRSA strain were evaluated following thermal and oxidative stresses by qRT-PCR techniques.ResultsThe cell viability was constant across thermal stress, whereas oxidative stress induction resulted in a significant reduction in the growth of MRSA strain. The result of RT-qPCR revealed that TA genes were expressed in stress conditions and expression of mazF gene increased under both thermal and oxidative stresses in MRSA strain.ConclusionsBased on our results, the MRSA strain responded to stress by altering the expression level of TA genes. In overall, TA system could be an antibacterial target in S. aureus that can revitalize the research on TA systems in this pathogen.

scholarly journals Stress Survival Islet 2, Predominantly Present in Listeria monocytogenes Strains of Sequence Type 121, Is Involved in the Alkaline and Oxidative Stress Responses

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Eva Harter ◽  
Eva Maria Wagner ◽  
Andreas Zaiser ◽  
Sabrina Halecker ◽  
Martin Wagner ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Listeria Monocytogenes ◽  
Stress Responses ◽  
Food Processing ◽  
Stress Conditions ◽  
Content Type ◽  
Oxidative Stresses ◽  
Stress Survival ◽  
Oxidative Stress Responses ◽  
And Oxidative Stress

ABSTRACT The foodborne pathogen Listeria monocytogenes is able to survive a variety of stress conditions leading to the colonization of different niches like the food processing environment. This study focuses on the hypervariable genetic hot spot lmo0443 to lmo0449 haboring three inserts: the stress survival islet 1 (SSI-1), the single-gene insert LMOf2365_0481, and two homologous genes of the nonpathogenic species Listeria innocua: lin0464, coding for a putative transcriptional regulator, and lin0465, encoding an intracellular PfpI protease. Our prevalence study revealed a different distribution of the inserts between human and food-associated isolates. The lin0464-lin0465 insert was predominantly found in food-associated strains of sequence type 121 (ST121). Functional characterization of this insert showed that the putative PfpI protease Lin0465 is involved in alkaline and oxidative stress responses but not in acidic, gastric, heat, cold, osmotic, and antibiotic stresses. In parallel, deletion of lin0464 decreased survival under alkaline and oxidative stresses. The expression of both genes increased significantly under oxidative stress conditions independently of the alternative sigma factor σB. Furthermore, we showed that the expression of the protease gene lin0465 is regulated by the transcription factor lin0464 under stress conditions, suggesting that lin0464 and lin0465 form a functional unit. In conclusion, we identified a novel stress survival islet 2 (SSI-2), predominantly present in L. monocytogenes ST121 strains, beneficial for survival under alkaline and oxidative stresses, potentially supporting adaptation and persistence of L. monocytogenes in food processing environments. IMPORTANCE Listeria monocytogenes strains of ST121 are known to persist for months and even years in food processing environments, thereby increasing the risk of food contamination and listeriosis. However, the molecular mechanism underlying this remarkable niche-specific adaptation is still unknown. Here, we demonstrate that the genomic islet SSI-2, predominantly present in L. monocytogenes ST121 strains, is beneficial for survival under alkaline and oxidative stress conditions, which are routinely encountered in food processing environments. Our findings suggest that SSI-2 is part of a diverse set of molecular determinants contributing to niche-specific adaptation and persistence of L. monocytogenes ST121 strains in food processing environments.

scholarly journals Modulation of Fibronectin Adhesins and Other Virulence Factors in a Teicoplanin-Resistant Derivative of Methicillin-Resistant Staphylococcus aureus

2004 ◽  
Vol 48 (8) ◽  
pp. 2958-2965 ◽  
Author(s):  
Adriana Renzoni ◽  
Patrice Francois ◽  
Dongmei Li ◽  
William L. Kelley ◽  
Daniel P. Lew ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Steady State ◽  
Resistant Strain ◽  
Mrna Levels ◽  
Glycopeptide Resistance ◽  
Methicillin Resistant ◽  
Qrt Pcr ◽  
Sigma B ◽  
Steady State Levels ◽  
The Impact

ABSTRACT The impact of glycopeptide resistance on the molecular regulation of Staphylococcus aureus virulence and attachment to host tissues is poorly documented. We compared stable teicoplanin-resistant methicillin-resistant S. aureus (MRSA) strain 14-4 with its teicoplanin-susceptible MRSA parent, strain MRGR3, which exhibits a high degree of virulence in a rat model of chronic foreign body MRSA infection. The levels of fibronectin-mediated adhesion and surface display of fibronectin-binding proteins were higher in teicoplanin-resistant strain 14-4 than in its teicoplanin-susceptible parent or a teicoplanin-susceptible revertant (strain 14-4rev) that spontaneously emerged during tissue cage infection. Quantitative reverse transcription-PCR (qRT-PCR) showed four- and twofold higher steady-state levels of fnbA and fnbB transcripts, respectively, in strain 14-4 than in its teicoplanin-susceptible counterparts. Analysis of global regulatory activities by qRT-PCR revealed a strong reduction in the steady-state levels of RNAIII and RNAII in the teicoplanin-resistant strain compared to in its teicoplanin-susceptible counterparts. In contrast, sarA mRNA levels were more than fivefold higher in strain 14-4 than in MRGR3 and 14-4rev. Furthermore, the alternative transcription factor sigma B had a higher level of functional activity in the teicoplanin-resistant strain than in its teicoplanin-susceptible counterparts, as evidenced by significant increases in both the sigma B-dependent asp23 mRNA levels and the sarA P3 promoter-derived transcript levels, as assayed by qRT-PCR and Northern blotting, respectively. These data provide further evidence that the emergence of glycopeptide resistance is linked by still poorly understood molecular pathways with significant pleiotropic changes in the expression and regulation of some major virulence genes. These molecular and phenotypic changes may have a profound impact on the bacterial adhesion and colonization properties of such multiresistant organisms.

scholarly journals ComparativeIn VitroActivities of Oritavancin, Dalbavancin, and Vancomycin against Methicillin-Resistant Staphylococcus aureus Isolates in a Nondividing State

2016 ◽  
Vol 60 (7) ◽  
pp. 4342-4345 ◽  
Author(s):  
Adam Belley ◽  
David Lalonde Seguin ◽  
Francis Arhin ◽  
Greg Moeck
Keyword(s):  
Staphylococcus Aureus ◽  
Bactericidal Activity ◽  
Antibacterial Agents ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Methicillin Resistant ◽  
Content Type ◽  
Skin Structure ◽  
Persistent Infections ◽  
Time Kill

ABSTRACTAntibacterial agents that kill nondividing bacteria may be of utility in treating persistent infections. Oritavancin and dalbavancin are bactericidal lipoglycopeptides that are approved for acute bacterial skin and skin structure infections in adults caused by susceptible Gram-positive pathogens. Using time-kill methodology, we demonstrate that oritavancin exerts bactericidal activity against methicillin-resistantStaphylococcus aureus(MRSA) isolates that are maintained in a nondividing statein vitro, whereas dalbavancin and the glycopeptide vancomycin do not.

scholarly journals A Novel Aza-Derivative Inhibits agr Quorum Sensing Signaling and Synergizes Methicillin-Resistant Staphylococcus aureus to Clindamycin

2021 ◽  
Vol 12 ◽  
Author(s):  
Giulia Bernabè ◽  
Matteo Dal Pra ◽  
Vittoria Ronca ◽  
Anthony Pauletto ◽  
Giovanni Marzaro ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Response Regulator ◽  
Accessory Gene ◽  
Mobility Shift ◽  
Signaling Mechanism ◽  
Methicillin Resistant ◽  
Qrt Pcr ◽  
Mobility Shift Assay

Increasing antibiotic resistance and diminishing pharmaceutical industry investments have increased the need for molecules that can treat infections caused by dangerous pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Quorum Sensing (QS) is a signaling mechanism that regulates bacterial virulence in pathogens. A report demonstrating that the anti-inflammatory drug Diflunisal reduces MRSA virulence factors’ expression prompted us to design, synthesize and test 16 aza-analogs as inhibitors of S. aureus virulence factors controlled by the accessory gene regulator (agr) QS system. At first, we evaluated by qRT-PCR the activity of compounds on rnaIII expression, a QS related gene. Azan-7 was the most active molecule tested and it did not show cytotoxic activity in human cell lines. Moreover, we demonstrated that it did not affect bacterial proliferation. Regulation of MRSA virulence genes by Azan-7 was investigated using qRT-PCR and RNAseq. Azan-7 significantly reduced hla, psmα, hysA, agrA, cap1A, and cap1C gene expression. In silico docking demonstrated that Azan-7 binds the response regulator AgrA. This data was confirmed by electrophoretic mobility shift assay (EMSA) reporting that Azan-7 binding to AgrA protein strongly reduced the AgrA-DNA complex formation at the P3 promoter region involved in the regulation of rnaIII transcription. Azan-7 inhibited MRSA-mediated haemolysis, reduced survival of the pathogen at low pH levels, and increased macrophage killing. In addition, Azan-7 enhanced MRSA susceptibility to clindamycin both in planktonic growth and biofilm. Azan-7 did not induce resistance over 10 days in culture. It was equally active against all the AgrA MRSA subtypes encountered among clinical isolates, but it was not active against Staphylococcus epidermidis, although the AgrA proteins show an approximate 80% homology. These results demonstrate that Azan-7 inhibits the expression of MRSA virulence factors by interfering in the QS and synergizes MRSA biofilm with clindamycin, indicating the compound as a promising candidate for the treatment of MRSA infections.

scholarly journals Withania somnifera promotes stress resistant activity in Drosophila melanogaster

2018 ◽  
Vol 8 (6-s) ◽  
pp. 83-88
Author(s):  
B.P. Renuka Prasad ◽  
J.S. Ashadevi
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Drosophila Melanogaster ◽  
Stress Tolerance ◽  
Withania Somnifera ◽  
Stress Conditions ◽  
High Dose ◽  
Oxidative Stresses ◽  
Paraquat Toxicity ◽  
Emotional Strain

Stress is a state of mental or emotional strain of an individual.  In recent years nutritional antioxidants study have gain more attention in minimizing the stress like oxidative stress. The stress resistant ability in an organism can be increased by the supplementation of herbal resources. However, few plant extracts are known to have stress resistant ability and increases the tolerance capacity. Plants containing high antioxidant and other bioactive compounds promote tolerance capacity. An antioxidant rich plant has been proved to decreases the   lipid peroxidation. Here, we investigated the potential protective effect of ethanolic extract of Withania somnifera (WS), against Paraquat toxicity on stress tolerance capacity using Drosophila melanogaster. Wild-type fruit flies of Oregon-K strain were fed with standard food media with 1mg/ml and 10 mg/ml of WS.  The oxidative stress was induced by exposing the extract supplemented flies to Paraquat (20 mM).  The stress tolerance capacity of flies was measured by subjecting to desiccation and oxidative stresses. Further, locomotor activity, lipid peroxidation were also studied along with the quantification of triglycerides, glycogen in WS fed flies under stress conditions. Our result reveals that PQ induced WS fed flies showed greater survivability, better locomotor ability when compared to PQ induced flies.WS fed flies increases about 73.55% of resistance ability under oxidative conditions and increased by 59.15% under desiccation than PQ induced flies. WS was more effective in protecting against Paraquat toxicity. The flies fed with high dose of WS (10mg/ml) showed greater improvement of the tolerance ability when subjected to desiccation and oxidative stresses. Further, the data on biochemical analysis reveals that lipid peroxidation activities were found to be significantly low and the triglyceride as well as glycogen quantities were found to be significantly high in WS fed flies compare to –ve control under both desiccation and oxidative stress conditions.  Together, these findings suggest that WS promotes stress resistant ability by modulating metabolism and reducing oxidative damage. Keywords: Drosophila melanogaster, Withania somnifera, Oxidative stress assay, Desiccation Assay, Negative Geotaxis, 

scholarly journals (456) Abiotic Stress Responses in Cucumber (Cucumis sativus L.) Plants Expressing the Arabidopsis thaliana Transcriptional Activators-CBF1and CBF3

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1036E-1037
Author(s):  
Mohamed Tawfik ◽  
Alejandra Ferenczi ◽  
Daniel Enter ◽  
Rebecca Grumet
Keyword(s):  
Oxidative Stress ◽  
Abiotic Stress ◽  
Drought Stress ◽  
Stress Responses ◽  
Stress Resistance ◽  
Photochemical Efficiency ◽  
Leaf Damage ◽  
Cucumis Sativus L ◽  
Oxidative Stresses ◽  
Major Factors

Abiotic stresses (e.g., salinity, drought, cold, oxidative stress) can be major factors limiting plant productivity worldwide. We sought to increase abiotic stress resistance in cucumber by expressing the A. thaliana transcription factors CBF1and CBF3, which regulate genes responsible for enhanced dehydration-stress resistance in Arabidopsis. Our previous studies in the greenhouse and field demonstrated increased salinity tolerance in CBF-expressing cucumber lines. In the current studies, we tested response of CBF-cucumber plants to drought, chilling, and oxidative stresses. Transgenic cucumber plants subjected to drought stress in the greenhouse showed elevated levels of the stress-inducible compatible solute, proline, compared to the nontransgenic controls. Preliminary results also indicate greater photochemical efficiency in CBF-expressing plants under drought stress conditions compared to the nontransgenic controls. Under nonstressed conditions, there were no significant differences in growth between the transgenic and the nontransgenic cucumber plants; however, after a cycle of drought stress, CBF-cucumber lines had less growth reduction compared to the nontransgenic counterparts. The advantage in growth was less pronounced after a second cycle of drought. We also evaluated the transgenic cucumber plants under chilling conditions (i.e., low, nonfreezing temperatures within the 0 to 12 °C range). Based on plant height and cotyledon and leaf damage measurements, transgenic cucumber seedlings did not show chilling tolerance compared to the wild-type control. The response of transgenic CBF-cucumber plants to oxidative stress using methyl viologen is also being evaluated.

scholarly journals Genotoxic, Metabolic, and Oxidative Stresses Regulate the RNA Repair Operon ofSalmonella entericaSerovar Typhimurium

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Jennifer E. Kurasz ◽  
Christine E. Hartman ◽  
David J. Samuels ◽  
Bijoy K. Mohanty ◽  
Anquilla Deleveaux ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Stress Responses ◽  
Nitrogen Limitation ◽  
Selective Advantage ◽  
Stress Conditions ◽  
Rna Repair ◽  
Content Type ◽  
E Coli ◽  
Oxidative Stresses

ABSTRACTThe σ54regulon inSalmonella entericaserovar Typhimurium includes a predicted RNA repair operon encoding homologs of the metazoan Ro60 protein (Rsr), Y RNAs (YrlBA), RNA ligase (RtcB), and RNA 3′-phosphate cyclase (RtcA). Transcription from σ54-dependent promoters requires that a cognate bacterial enhancer binding protein (bEBP) be activated by a specific environmental or cellular signal; the cognate bEBP for the σ54-dependent promoter of thersr-yrlBA-rtcBAoperon is RtcR. To identify conditions that generate the signal for RtcR activation inS. Typhimurium, transcription of the RNA repair operon was assayed under multiple stress conditions that result in nucleic acid damage. RtcR-dependent transcription was highly induced by the nucleic acid cross-linking agents mitomycin C (MMC) and cisplatin, and this activation was dependent on RecA. Deletion ofrtcRorrtcBresulted in decreased cell viability relative to that of the wild type following treatment with MMC. Oxidative stress from peroxide exposure also induced RtcR-dependent transcription of the operon. Nitrogen limitation resulted in RtcR-independent increased expression of the operon; the effect of nitrogen limitation required NtrC. The adjacent toxin-antitoxin module,dinJ-yafQ, was cotranscribed with the RNA repair operon but was not required for RtcR activation, although YafQ endoribonuclease activated RtcR-dependent transcription. Stress conditions shown to induce expression the RNA repair operon ofEscherichia coli(rtcBA) did not stimulate expression of theS. Typhimurium RNA repair operon. Similarly, MMC did not induce expression of theE. colirtcBAoperon, although when expressed inS. Typhimurium,E. coliRtcR responds effectively to the unknown signal(s) generated there by MMC exposure.IMPORTANCEHomologs of the metazoan RNA repair enzymes RtcB and RtcA occur widely in eubacteria, suggesting a selective advantage. Although the enzymatic activities of the eubacterial RtcB and RtcA have been well characterized, the physiological roles remain largely unresolved. Here we report stress responses that activate expression of the σ54-dependent RNA repair operon (rsr-yrlBA-rtcBA) ofS. Typhimurium and demonstrate that expression of the operon impacts cell survival under MMC-induced stress. Characterization of the requirements for activation of this tightly regulated operon provides clues to the possible functions of operon componentsin vivo, enhancing our understanding of how this human pathogen copes with environmental stressors.

scholarly journals Inducible expression of (pp)pGpp synthetases in Staphylococcus aureus is associated with activation of stress response genes

2020 ◽  
Author(s):  
Petra Horvatek ◽  
Andrew Magdy Fekry Hanna ◽  
Fabio Lino Gratani ◽  
Daniela Keinhörster ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Stress Response ◽  
Stringent Response ◽  
Trna Synthetase ◽  
Oxidative Stress Response ◽  
Stress Conditions ◽  
Additional Stress ◽  
Iron Storage ◽  
Transcriptional Induction

AbstractThe stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog (RSHSau) upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ, upon cell wall stress. We used RNA-Seq to compare the global effects in response to transcriptional induction of the synthetase domain of RSH (RSH-Syn), RelP or RelQ without the need to apply additional stress conditions. Enzyme expression resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than RSH-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. Genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., katA, sodA) and the the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenole soluble modulins (PSMs) were highly upregulated upon (pp)pGpp synthesis. Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.SignificanceMost bacteria make use of the second messenger (pp)pGpp to reprogram bacterial metabolism under nutrient-limiting conditions. In the human pathogen Staphylococcus aureus, (pp)pGpp plays an important role in virulence, phagosomal escape and antibiotic tolerance. Here, we analyzed the immediate consequences of (pp)pGpp synthesis upon transcriptional induction of the (pp)pGpp-producing enzymes RSH, RelP or RelQ. (pp)pGpp synthesis provokes immediate changes in the nucleotide pool and severely impacts the expression of hundreds of genes. A main consequence of (pp)pGpp synthesis in S. aureus is the induction of ROS-inducing toxic phenol-soluble modulins (PSMs) and simultaneous expression of the detoxifying system to protect the producer. This mechanism is likely of special advantage for the pathogen after phagocytosis.

scholarly journals An Enzybiotic Regimen for the Treatment of Methicillin-Resistant Staphylococcus aureus Orthopaedic Device-Related Infection

Antibiotics ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1186
Author(s):  
Eric T. Sumrall ◽  
Marloes I. Hofstee ◽  
Daniel Arens ◽  
Christian Röhrig ◽  
Susanne Baertl ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Trauma Surgery ◽  
In Vitro Models ◽  
Fracture Repair ◽  
Combination Regimen ◽  
Bacterial Survival ◽  
Methicillin Resistant ◽  
Orthopaedic Device

Orthopaedic device-related infection (ODRI) presents a significant challenge to the field of orthopaedic and trauma surgery. Despite extensive treatment involving surgical debridement and prolonged antibiotic therapy, outcomes remain poor. This is largely due to the unique abilities of Staphylococcus aureus, the most common causative agent of ODRI, to establish and protect itself within the host by forming biofilms on implanted devices and staphylococcal abscess communities (SACs). There is a need for novel antimicrobials that can readily target such features. Enzybiotics are a class of antimicrobial enzymes derived from bacteria and bacteriophages, which function by enzymatically degrading bacterial polymers essential to bacterial survival or biofilm formation. Here, we apply an enzybiotic-based combination regimen to a set of in vitro models as well as in a murine ODRI model to evaluate their usefulness in eradicating established S. aureus infection, compared to classical antibiotics. We show that two chimeric endolysins previously selected for their functional efficacy in human serum in combination with a polysaccharide depolymerase reduce bacterial CFU numbers 10,000-fold in a peg model and in an implant model of biofilm. The enzyme combination also completely eradicates S. aureus in a SAC in vitro model where classical antibiotics are ineffective. In an in vivo ODRI model in mice, the antibiofilm effects of this enzyme regimen are further enhanced when combined with a classical gentamicin/vancomycin treatment. In a mouse model of methicillin-resistant S. aureus (MRSA) ODRI following a fracture repair, a combined local enzybiotic/antibiotic treatment regimen showed a significant CFU reduction in the device and the surrounding soft tissue, as well as significant prevention of weight loss. These outcomes were superior to treatment with antibiotics alone. Overall, this study demonstrates that the addition of enzybiotics, which are distinguished by their extremely rapid killing efficacy and antibiofilm activities, can enhance the treatment of severe MRSA ODRI.

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