Combined antimicrobial effect of two peptide nucleic acids against Staphylococcus aureus and S. pseudintermedius veterinary isolates

Background@#Staphylococcus aureus and S. pseudintermedius are the major etiological agents of staphylococcal infections in humans, livestock, and companion animals. The misuse of antimicrobial drugs has led to the emergence of antimicrobial-resistant Staphylococcus spp., including methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP). One novel therapeutic approach against MRSA and MRSP is a peptide nucleic acid (PNA) that can bind to the target nucleotide strands and block expression. Previously, two PNAs conjugated with cell-penetrating peptides (P-PNAs), antisense PNA (ASP)-cmk and ASP-deoD, targeting two essential genes in S. aureus, were constructed, and their antibacterial activities were analyzed. @*Objectives@#This study analyzed the combined antibacterial effects of P-PNAs on S. aureus and S. pseudintermedius clinical isolates. @*Methods@#S. aureus ATCC 29740 cells were treated simultaneously with serially diluted ASPcmk and ASP-deoD, and the minimal inhibitory concentrations (MICs) were measured. The combined P-PNA mixture was then treated with S. aureus and S. pseudintermedius veterinary isolates at the determined MIC, and the antibacterial effect was examined. @*Results@#The combined treatment of two P-PNAs showed higher antibacterial activity than the individual treatments. The MICs of two individual P-PNAs were 20 and 25 μM, whereas that of the combined treatment was 10 μM. The application of a combined treatment to clinical Staphylococcus spp. revealed S. aureus isolates to be resistant to P-PNAs and S.pseudintermedius isolates to be susceptible. @*Conclusions@#These observations highlight the complexity of designing ASPs with high efficacy for potential applications in treating staphylococcal infections in humans and animals.

Pathogenic and phylogenetic characteristics of non-O157 Shiga toxin-producing Escherichia coli isolates from retail meats in South Korea

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx₁, stx(2c), stx(2e), or stx₁ + stx(2b)) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.

Proteomic Analysis of the Serum from Chicken Infected by Avian Influenza Virus

Avian influenza (AI) is an infectious, low pathogenic virus that is endemic all over the world and poses a potential threat to the poultry industry. Vaccination is a widely used effective method to prevent avian influenza virus. Here we employed a comparative proteomics approach [two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF)] to characterize proteome in the sera from the specific pathogen free (SPF) chickens, the vaccinated chickens, and the naturally infected chickens. We identified total 58 proteins that were differentially expressed in the sera of three groups. Among them ovotransferrin and vitamin D-binding protein were more expressed in the sera of naturally infected chickens compare with other groups. Our results suggested that the level of these two proteins in the serum may help to discriminate the naturally infected chicken from the vaccinated chicken.

Detection of the Avian Influenza Viruses Nonstructural Protein 1 for Distinction between Vaccinated and Infected Chickens Using Synthetic Peptide-Based ELISA

Avian influenza (AI) virus infects both animal and human. Low pathogenic AI virus infections (some H7 and H9 subtypes) have been reported all over the world and pose a potential threat to the poultry industry. Vaccination is the most effective way to prevent virus infection. However, vaccination makes it difficult to differentiate between vaccinated chickens and infected chickens. In order to differentiate vaccinated chickens from naturally infected chickens, we adopted synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) using the peptide sequences from nonstructural protein 1 (NS1) of H9N2. Five synthetic peptides were designed using Protein Variability Sever (http://imed.med.ucm.es/PVS/) and synthesized. NS1-1 ~ NS1-4 peptides failed to detect serum antibodies from both vaccinated and naturally infected chickens. NS1-5 peptide from the C-terminal NS1 protein detected serum antibody from naturally infected chickens but not vaccinated chickens. These results imply that NS1-5 peptide may be a useful tool to differentiate naturally infected chicken from vaccinated chicken as being used in the synthetic peptide-based ELISA.