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Antibiotics
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Novel Strategies to Control Antimicrobial Resistance
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Novel Strategies to Control Antimicrobial Resistance

A special issue of Antibiotics (ISSN 2079-6382). This special issue belongs to the section "Antibiotics Use and Antimicrobial Stewardship".

Deadline for manuscript submissions: closed (31 May 2020) | Viewed by 67100

Special Issue Editor

Dr. Raffaela Pero

E-Mail Website
Guest Editor
Department of Molecular Medicine and Biotechnology, University of Naples, Naples, Italy
Interests: microbiology; human defensins; Helicobacter pylori; microbiome; genomics; epigenetics; cell biology; cancer biology; gene regulation; antimicrobial peptides
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Antimicrobial resistance is today one of the most important public health problems and is destined to become one of the main health challenges of the coming decades.
The increasing incidence of infections with multiresistant micro-organisms (multi-drug-resistant organisms, MDRO) dramatically reduces the therapeutic equipment available to the doctor, leading to an increase in the complexity of treatments with an increase in morbidity, mortality, and costs associated with healthcare and generates a social alarm that is difficult to manage.

The advancement of effective means of control and rapid detection is essential for the treatment and prevention of multiple bacterial infections resistant to antibiotics.
Many control systems, including combination therapy, phages, and systems based on the antibiotic resistance mechanism (efflux pump and beta-lactamase inhibitors) have been designed to improve antibacterial activity against multiple antibiotic-resistant pathogens. Different approaches have been developed for rapid, selective, and sensitive detection for multiple antibiotic-resistant pathogens, including nucleic acid probes, molecular-based techniques, chemical methods, and physical methods.

In this Special Issue of Antibiotics, we invite you to send contributions concerning any aspects related, but not limited to prevention, new therapeutic agents, drug discovery, diagnostics, and drug resistance.

Furthermore, studies based on antibiotic mechanisms and any biological activity related to the therapeutic potential of antimicrobial peptides, including direct and indirect modes of action, are welcome for this Special Issue.

Dr. Raffaela Pero
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Antibiotics is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Antimicrobial resistance
  • Prevention
  • Antibiotic mechanisms
  • Antibiotic activity
  • Combination therapy
  • Phages therapy
  • Multi-drug-resistant organisms (MDRO)
  • Drug discovery
  • Antimicrobial peptides
  • Diagnostics resistance

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Published Papers (11 papers)

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Research

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20 pages, 1236 KiB  
Article
Artificial Intelligence to Get Insights of Multi-Drug Resistance Risk Factors during the First 48 Hours from ICU Admission
by Inmaculada Mora-Jiménez, Jorge Tarancón-Rey, Joaquín Álvarez-Rodríguez and Cristina Soguero-Ruiz
Antibiotics 2021, 10(3), 239; https://doi.org/10.3390/antibiotics10030239 - 27 Feb 2021
Cited by 15 | Viewed by 3278
Abstract
Multi-drug resistance (MDR) is one of the most current and greatest threats to the global health system nowadays. This situation is especially relevant in Intensive Care Units (ICUs), where the critical health status of these patients makes them more vulnerable. Since MDR confirmation [...] Read more.
Multi-drug resistance (MDR) is one of the most current and greatest threats to the global health system nowadays. This situation is especially relevant in Intensive Care Units (ICUs), where the critical health status of these patients makes them more vulnerable. Since MDR confirmation by the microbiology laboratory usually takes 48 h, we propose several artificial intelligence approaches to get insights of MDR risk factors during the first 48 h from the ICU admission. We considered clinical and demographic features, mechanical ventilation and the antibiotics taken by the patients during this time interval. Three feature selection strategies were applied to identify statistically significant differences between MDR and non-MDR patient episodes, ending up in 24 selected features. Among them, SAPS III and Apache II scores, the age and the department of origin were identified. Considering these features, we analyzed the potential of machine learning methods for predicting whether a patient will develop a MDR germ during the first 48 h from the ICU admission. Though the results presented here are just a first incursion into this problem, artificial intelligence approaches have a great impact in this scenario, especially when enriching the set of features from the electronic health records. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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14 pages, 1214 KiB  
Article
N-Nonyloxypentyl-l-Deoxynojirimycin Inhibits Growth, Biofilm Formation and Virulence Factors Expression of Staphylococcus aureus
by Eliana De Gregorio, Anna Esposito, Adriana Vollaro, Maria De Fenza, Daniele D’Alonzo, Antonella Migliaccio, Vita Dora Iula, Raffaele Zarrilli and Annalisa Guaragna
Antibiotics 2020, 9(6), 362; https://doi.org/10.3390/antibiotics9060362 - 26 Jun 2020
Cited by 15 | Viewed by 3535
Abstract
Staphylococcus aureus is one of the major causes of hospital- and community-associated bacterial infections throughout the world, which are difficult to treat due to the rising number of drug-resistant strains. New molecules displaying potent activity against this bacterium are urgently needed. In this [...] Read more.
Staphylococcus aureus is one of the major causes of hospital- and community-associated bacterial infections throughout the world, which are difficult to treat due to the rising number of drug-resistant strains. New molecules displaying potent activity against this bacterium are urgently needed. In this study, d- and l-deoxynojirimycin (DNJ) and a small library of their N-alkyl derivatives were screened against S. aureus ATCC 29213, with the aim to identify novel candidates with inhibitory potential. Among them, N-nonyloxypentyl-l-DNJ (l-NPDNJ) proved to be the most active compound against S. aureus ATCC 29213 and its clinical isolates, with the minimum inhibitory concentration (MIC) value of 128 μg/mL. l-NPDNJ also displayed an additive effect with gentamicin and oxacillin against the gentamicin- and methicillin-resistant S. aureus isolate 00717. Sub-MIC values of l-NPDNJ affected S. aureus biofilm development in a dose-dependent manner, inducing a strong reduction in biofilm biomass. Moreover, real-time reverse transcriptase PCR analysis revealed that l-NPDNJ effectively inhibited at sub-MIC values the transcription of the spa, hla, hlb and sea virulence genes, as well as the agrA and saeR response regulator genes. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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13 pages, 1184 KiB  
Article
HNP-1 and HBD-1 as Biomarkers for the Immune Systems of Elite Basketball Athletes
by Raffaela Pero, Mariarita Brancaccio, Cristina Mennitti, Luca Gentile, Adelaide Franco, Sonia Laneri, Margherita G. De Biasi, Chiara Pagliuca, Roberta Colicchio, Paola Salvatore, Giovanni D’Alicandro, Daniela Terracciano, Michele Cennamo, Evelina La Civita, Antonietta Liotti, Cristina Mazzaccara, Giulia Frisso, Barbara Lombardo and Olga Scudiero
Antibiotics 2020, 9(6), 306; https://doi.org/10.3390/antibiotics9060306 - 7 Jun 2020
Cited by 19 | Viewed by 4032
Abstract
Acute or strenuous exercise is sometimes related to upper respiratory tract infections in athletes. Practicing intense and regular exercise can lead to incorrect activation of the immune system, causing athletes to be excluded from training programs and competitions. Defensins are small antimicrobial peptides [...] Read more.
Acute or strenuous exercise is sometimes related to upper respiratory tract infections in athletes. Practicing intense and regular exercise can lead to incorrect activation of the immune system, causing athletes to be excluded from training programs and competitions. Defensins are small antimicrobial peptides that are part of the innate immune system and dynamically involved in several biological activities. In this study, we highlight the role of human defensins in competitive basketball athletes. In particular, we consider the behavior of alpha- and beta-defensins together with white blood cells in a cohort of players. Moreover, we focus our attention on cortisol, a physiological indicator of stress, and testosterone, both of which are human hormones involved in muscle metabolism. The free-testosterone/cortisol ratio is considered to be an indicator of overtraining among athletes. This paper provides an up-to-date information of the role of human defensins as self-defense molecules during a continuous stressor such as long-term exercise, and it recognizes them as potential markers of infection. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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11 pages, 1356 KiB  
Article
PYED-1 Inhibits Biofilm Formation and Disrupts the Preformed Biofilm of Staphylococcus aureus
by Adriana Vollaro, Anna Esposito, Eliana Pia Esposito, Raffaele Zarrilli, Annalisa Guaragna and Eliana De Gregorio
Antibiotics 2020, 9(5), 240; https://doi.org/10.3390/antibiotics9050240 - 8 May 2020
Cited by 14 | Viewed by 3701
Abstract
Pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1 (PYED-1), a heterocyclic corticosteroid derivative of deflazacort, exhibits broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria. Here, we investigated the effect of PYED-1 on the biofilms of Staphylococcus aureus, an etiological agent of biofilm-based chronic infections such as osteomyelitis, indwelling medical [...] Read more.
Pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1 (PYED-1), a heterocyclic corticosteroid derivative of deflazacort, exhibits broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria. Here, we investigated the effect of PYED-1 on the biofilms of Staphylococcus aureus, an etiological agent of biofilm-based chronic infections such as osteomyelitis, indwelling medical device infections, periodontitis, chronic wound infections, and endocarditis. PYED-1 caused a strong reduction in biofilm formation in a concentration dependent manner. Furthermore, it was also able to completely remove the preformed biofilm. Transcriptional analysis performed on the established biofilm revealed that PYED-1 downregulates the expression of genes related to quorum sensing (agrA, RNAIII, hld, psm, and sarA), surface proteins (clfB and fnbB), secreted toxins (hla, hlb, and lukD), and capsular polysaccharides (capC). The expression of genes that encode two main global regulators, sigB and saeR, was also significantly inhibited after treatment with PYED-1. In conclusion, PYED-1 not only effectively inhibited biofilm formation, but also eradicated preformed biofilms of S. aureus, modulating the expression of genes related to quorum sensing, surface and secreted proteins, and capsular polysaccharides. These results indicated that PYED-1 may have great potential as an effective antibiofilm agent to prevent S. aureus biofilm-associated infections. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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7 pages, 396 KiB  
Communication
Urinary Pharmacokinetic and Pharmacodynamic Profiles of Fosfomycin against Extended-Spectrum β-Lactamase-Producing Escherichia coli with Canine Ex Vivo Modeling: A Pilot Study
by Kazuki Harada, Takae Shimizu, Koji Kawaguchi, Takeshi Furuhashi and Genki Ishihara
Antibiotics 2020, 9(5), 230; https://doi.org/10.3390/antibiotics9050230 - 5 May 2020
Cited by 2 | Viewed by 5278
Abstract
Fosfomycin is a candidate drug for extended-spectrum β-lactamase (ESBL)-producing bacteria, but its efficacy is yet to be investigated in dogs. This study investigated the urinary pharmacokinetic/pharmacodynamic (PK/PD) profile of fosfomycin orally administered at 80 mg/kg to six healthy dogs to assess its efficacy [...] Read more.
Fosfomycin is a candidate drug for extended-spectrum β-lactamase (ESBL)-producing bacteria, but its efficacy is yet to be investigated in dogs. This study investigated the urinary pharmacokinetic/pharmacodynamic (PK/PD) profile of fosfomycin orally administered at 80 mg/kg to six healthy dogs to assess its efficacy for canine urinary tract infections (UTIs) caused by ESBL-producing bacteria. Four strains of ESBL-producing Escherichia coli (ESBL-EC) characterized by fosfomycin minimum inhibitory concentrations (MICs) of 0.5, 1, 2, and 32 µg/mL were used. Urine samples for the measurement of urinary drug concentrations and urinary bactericidal titers (UBTs) were obtained after drug administration. The urinary concentrations (µg/mL, mean ± SE) were 1348.2 ± 163.5, 1191.6 ± 260.2, and 661.1 ± 190.4 at 0–4, 4–8, and 8–12 h, respectively, after drug administration. The mean urinary area under the curve during the test period (AUC0–12) of fosfomycin was estimated to be 12,803.8 µg·h/mL. The UBTs for all tested strains fluctuated closely with urine concentration during the test period (r = 0.944–1.000), and the area under the UBT-versus-time curve correlated with the urinary AUC/MIC of each strain (r = 0.991). According to the optimal urinary PK/PD target value, fosfomycin at 80 mg/kg twice daily may be suitable for the treatment of canine UTIs caused by ESBL-EC presenting MIC ≤ 128 µg/mL. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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12 pages, 1106 KiB  
Article
Antibacterial and Antivirulence Activity of Glucocorticoid PYED-1 against Stenotrophomonas maltophilia
by Anna Esposito, Adriana Vollaro, Eliana Pia Esposito, Daniele D’Alonzo, Annalisa Guaragna, Raffaele Zarrilli and Eliana De Gregorio
Antibiotics 2020, 9(3), 105; https://doi.org/10.3390/antibiotics9030105 - 2 Mar 2020
Cited by 11 | Viewed by 3905
Abstract
Stenotrophomonas maltophilia, an environmental Gram-negative bacterium, is an emerging nosocomial opportunistic pathogen that causes life-threatening infections in immunocompromised patients and chronic pulmonary infections in cystic fibrosis patients. Due to increasing resistance to multiple classes of antibiotics, S. maltophilia infections are difficult to [...] Read more.
Stenotrophomonas maltophilia, an environmental Gram-negative bacterium, is an emerging nosocomial opportunistic pathogen that causes life-threatening infections in immunocompromised patients and chronic pulmonary infections in cystic fibrosis patients. Due to increasing resistance to multiple classes of antibiotics, S. maltophilia infections are difficult to treat successfully. This makes the search for new antimicrobial strategies mandatory. In this study, the antibacterial activity of the heterocyclic corticosteroid deflazacort and several of its synthetic precursors was tested against S. maltophilia. All compounds were not active against standard strain S. maltophilia K279a. The compound PYED-1 (pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1) showed a weak effect against some S. maltophilia clinical isolates, but exhibited a synergistic effect with aminoglycosides. PYED-1 at sub-inhibitory concentrations decreased S. maltophilia biofilm formation. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis demonstrated that the expression of biofilm- and virulence- associated genes (StmPr1, StmPr3, sphB, smeZ, bfmA, fsnR) was significantly suppressed after PYED-1 treatment. Interestingly, PYED-1 also repressed the expression of the genes aph (3′)-IIc, aac (6′)-Iz, and smeZ, involved in the resistance to aminoglycosides. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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Review

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21 pages, 355 KiB  
Review
Phage Therapy in Veterinary Medicine
by Rosa Loponte, Ugo Pagnini, Giuseppe Iovane and Giuseppe Pisanelli
Antibiotics 2021, 10(4), 421; https://doi.org/10.3390/antibiotics10040421 - 11 Apr 2021
Cited by 41 | Viewed by 6411
Abstract
To overcome the obstacle of antimicrobial resistance, researchers are investigating the use of phage therapy as an alternative and/or supplementation to antibiotics to treat and prevent infections both in humans and in animals. In the first part of this review, we describe the [...] Read more.
To overcome the obstacle of antimicrobial resistance, researchers are investigating the use of phage therapy as an alternative and/or supplementation to antibiotics to treat and prevent infections both in humans and in animals. In the first part of this review, we describe the unique biological characteristics of bacteriophages and the crucial aspects influencing the success of phage therapy. However, despite their efficacy and safety, there is still no specific legislation that regulates their use. In the second part of this review, we describe the comprehensive research done in the past and recent years to address the use of phage therapy for the treatment and prevention of bacterial disease affecting domestic animals as an alternative to antibiotic treatments. While in farm animals, phage therapy efficacy perspectives have been widely studied in vitro and in vivo, especially for zoonoses and diseases linked to economic losses (such as mastitis), in pets, studies are still few and rather recent. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
20 pages, 291 KiB  
Review
Some Suggestions from PK/PD Principles to Contain Resistance in the Clinical Setting—Focus on ICU Patients and Gram-Negative Strains
by Chiara Adembri, Andrea Novelli and Stefania Nobili
Antibiotics 2020, 9(10), 676; https://doi.org/10.3390/antibiotics9100676 - 6 Oct 2020
Cited by 20 | Viewed by 4023
Abstract
The containment of the phenomenon of resistance towards antimicrobials is a priority, especially in preserving molecules acting against Gram-negative pathogens, which represent the isolates more frequently found in the fragile population of patients admitted to Intensive Care Units. Antimicrobial therapy aims to prevent [...] Read more.
The containment of the phenomenon of resistance towards antimicrobials is a priority, especially in preserving molecules acting against Gram-negative pathogens, which represent the isolates more frequently found in the fragile population of patients admitted to Intensive Care Units. Antimicrobial therapy aims to prevent resistance through several actions, which are collectively known as “antimicrobial stewardship”, to be taken together, including the application of pharmacokinetic/pharmacodynamic (PK/PD) principles. PK/PD application has been shown to prevent the emergence of resistance in numerous experimental studies, although a straight translation to the clinical setting is not possible. Individualized antibiotic dosing and duration should be pursued in all patients, and even more especially when treating intensive care unit (ICU) septic patients in whom optimal exposure is both difficult to achieve and necessary. In this review, we report on the available data that support the application of PK/PD parameters to contain the development of resistance and we give some practical suggestions that can help to translate the benefit of PK/PD application to the bedside. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
15 pages, 3266 KiB  
Review
Methicillin-Resistant Staphylococcus aureus: Risk for General Infection and Endocarditis Among Athletes
by Mariarita Brancaccio, Cristina Mennitti, Sonia Laneri, Adelaide Franco, Margherita G. De Biasi, Arturo Cesaro, Fabio Fimiani, Elisabetta Moscarella, Felice Gragnano, Cristina Mazzaccara, Giuseppe Limongelli, Giulia Frisso, Barbara Lombardo, Chiara Pagliuca, Roberta Colicchio, Paola Salvatore, Paolo Calabrò, Raffaela Pero and Olga Scudiero
Antibiotics 2020, 9(6), 332; https://doi.org/10.3390/antibiotics9060332 - 18 Jun 2020
Cited by 12 | Viewed by 6170
Abstract
The first studies on Staphylococcus aureus (SA) infections in athletes were conducted in the 1980s, and examined athletes that perform in close physical contact, with particular attention to damaged or infected skin. Recent studies have used molecular epidemiology to shed light on the [...] Read more.
The first studies on Staphylococcus aureus (SA) infections in athletes were conducted in the 1980s, and examined athletes that perform in close physical contact, with particular attention to damaged or infected skin. Recent studies have used molecular epidemiology to shed light on the transmission of SA in professional athletes. These studies have shown that contact between athletes is prolonged and constant, and that these factors influence the appearance of infections caused by SA. These results support the need to use sanitary measures designed to prevent the appearance of SA infections. The factors triggering the establishment of SA within professional sports groups are the nasal colonization of SA, contact between athletes and sweating. Hence, there is a need to use the most modern molecular typing methods to evaluate the appearance of cutaneous SA disease. This review aims to summarize both the current SA infections known in athletes and the diagnostic methods employed for recognition, pointing to possible preventive strategies and the factors that can act as a springboard for the appearance of SA and subsequent transmission between athletes. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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23 pages, 4132 KiB  
Review
Multimodal Role of Amino Acids in Microbial Control and Drug Development
by Muhammad Idrees, Afzal R. Mohammad, Nazira Karodia and Ayesha Rahman
Antibiotics 2020, 9(6), 330; https://doi.org/10.3390/antibiotics9060330 - 17 Jun 2020
Cited by 50 | Viewed by 15780
Abstract
Amino acids are ubiquitous vital biomolecules found in all kinds of living organisms including those in the microbial world. They are utilised as nutrients and control many biological functions in microorganisms such as cell division, cell wall formation, cell growth and metabolism, intermicrobial [...] Read more.
Amino acids are ubiquitous vital biomolecules found in all kinds of living organisms including those in the microbial world. They are utilised as nutrients and control many biological functions in microorganisms such as cell division, cell wall formation, cell growth and metabolism, intermicrobial communication (quorum sensing), and microbial-host interactions. Amino acids in the form of enzymes also play a key role in enabling microbes to resist antimicrobial drugs. Antimicrobial resistance (AMR) and microbial biofilms are posing a great threat to the world’s human and animal population and are of prime concern to scientists and medical professionals. Although amino acids play an important role in the development of microbial resistance, they also offer a solution to the very same problem i.e., amino acids have been used to develop antimicrobial peptides as they are highly effective and less prone to microbial resistance. Other important applications of amino acids include their role as anti-biofilm agents, drug excipients, drug solubility enhancers, and drug adjuvants. This review aims to explore the emerging paradigm of amino acids as potential therapeutic moieties. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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16 pages, 2150 KiB  
Review
Human Defensins: A Novel Approach in the Fight against Skin Colonizing Staphylococcus aureus
by Olga Scudiero, Mariarita Brancaccio, Cristina Mennitti, Sonia Laneri, Barbara Lombardo, Margherita G. De Biasi, Eliana De Gregorio, Chiara Pagliuca, Roberta Colicchio, Paola Salvatore and Raffaela Pero
Antibiotics 2020, 9(4), 198; https://doi.org/10.3390/antibiotics9040198 - 21 Apr 2020
Cited by 44 | Viewed by 9082
Abstract
Staphylococcus aureus is a microorganism capable of causing numerous diseases of the human skin. The incidence of S. aureus skin infections reflects the conflict between the host skin′s immune defenses and the S. aureus’ virulence elements. Antimicrobial peptides (AMPs) are small protein molecules [...] Read more.
Staphylococcus aureus is a microorganism capable of causing numerous diseases of the human skin. The incidence of S. aureus skin infections reflects the conflict between the host skin′s immune defenses and the S. aureus’ virulence elements. Antimicrobial peptides (AMPs) are small protein molecules involved in numerous biological activities, playing a very important role in the innate immunity. They constitute the defense of the host′s skin, which prevents harmful microorganisms from entering the epithelial barrier, including S. aureus. However, S. aureus uses ambiguous mechanisms against host defenses by promoting colonization and skin infections. Our review aims to provide a reference collection on host-pathogen interactions in skin disorders, including S. aureus infections and its resistance to methicillin (MRSA). In addition to these, we discuss the involvement of defensins and other innate immunity mediators (i.e., toll receptors, interleukin-1, and interleukin-17), involved in the defense of the host against the skin disorders caused by S. aureus, and then focus on the evasion mechanisms developed by the pathogenic microorganism under analysis. This review provides the “state of the art” on molecular mechanisms underlying S. aureus skin infection and the pharmacological potential of AMPs as a new therapeutic strategy, in order to define alternative directions in the fight against cutaneous disease. Full article
(This article belongs to the Special Issue Novel Strategies to Control Antimicrobial Resistance)
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