Antibacterial Activity of Novel Cationic Peptides against Clinical Isolates of Multi-Drug Resistant Staphylococcus pseudintermedius from Infected Dogs

Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent antistaphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 ‘‘D isoform’’ demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammaliancells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and ciprofloxacin increased 32 and 8 fold, respectively; under similar conditions. Taken together, these results support designing of peptide-based therapeutics for combating MRSP infections, particularly for topical application

Antibacterial Characterization of Novel Synthetic Thiazole Compounds against Methicillin-Resistant Staphylococcus pseudintermedius

Staphylococcus pseudintermedius is a commensal organism of companion animals that is a significant source of opportunistic infections in dogs. With the emergence of clinical isolates of S. pseudintermedius (chiefly methicillin-resistant S. pseudintermedius (MRSP)) exhibiting increased resistance to nearly all antibiotic classes, new antimicrobials and therapeutic strategies are urgently needed. Thiazole compounds have been previously shown to possess potent antibacterial activity against multidrug-resistant strains of Staphylococcus aureus of human and animal concern. Given the genetic similarity between S. aureus and S. pseudintermedius, this study explores the potential use of thiazole compounds as novel antibacterial agents against methicillin-sensitive S. pseudintermedius (MSSP) and MRSP. A broth microdilution assay confirmed these compounds exhibit potent bactericidal activity (at sub-microgram/mL concentrations) against both MSSA and MRSP clinical isolates while the MTS assay confirmed three compounds (at 10 μg/mL) were not toxic to mammalian cells. A time-kill assay revealed two derivatives rapidly kill MRSP within two hours. However, this rapid bactericidal activity was not due to disruption of the bacterial cell membrane indicating an alternative mechanism of action for these compounds against MRSP. A multistep resistance selection analysis revealed compounds 4 and 5 exhibited a modest (twofold) shift in activity over ten passages. Furthermore, all six compounds (at a subinihibitory concentration) demonstrated the ability to re-sensitize MRSP to oxacillin, indicating these compounds have potential use for extending the therapeutic utility of β-lactam antibiotics against MRSP. Metabolic stability analysis with dog liver microsomes revealed compound 3 exhibited an improved physicochemical profile compared to the lead compound. In addition to this, all six thiazole compounds possessed a long post-antibiotic effect (at least 8 hours) against MRSP. Collectively the present study demonstrates these synthetic thiazole compounds possess potent antibacterial activity against both MSSP and MRSP and warrant further investigation into their use as novel antimicrobial agents

Permeabilization of the cytoplasmic membrane of SP3 as a function of peptide concentration at 5X MIC (A) and 10 X MIC (B), indicated by percent of propidium iodide fluorescence.

<p>Each experiment was done in triplicate, and the values represent means ± standard deviations.</p

Transmission electron microscopy (TEM) micrographs of untreated and peptide treated <i>S. pseudintermedius</i> (SP3).

<p>Control bacteria either dividing cells (A) or single celled (B), the cells are round and intact, with a well-defined cell membrane. The intracellular DNA region exhibits a highly inhomogeneous electron density. C&D: 1X MIC RRIKA; E&F: 10X MIC RRIKA, G: 10X MIC RR; H: 10X MIC (KFF)₃K; I: 10X MIC IK8 “D isoform”; K: 10X MIC WR-12; L: 1X MIC penetratin; M: 10X MIC penetratin.</p

Drug resistance development profiles of <i>S. pseudintermedius</i> (SP6) exposed to ½ X MIC concentrations of peptides (IK8 “D isoform” and RRIKA) and antibiotics (amikacin and ciprofloxacin) for 10 serial passages.

<p>Drug resistance development profiles of <i>S. pseudintermedius</i> (SP6) exposed to ½ X MIC concentrations of peptides (IK8 “D isoform” and RRIKA) and antibiotics (amikacin and ciprofloxacin) for 10 serial passages.</p

Global transcriptional analysis reveals surface remodeling of Anaplasma marginale in the tick vector

Pathogens dependent upon vectors for transmission to new hosts undergo environment specific changes in gene transcription dependent on whether they are replicating in the vector or the mammalian host. Differential gene transcription, especially of potential vaccine candidates, is of interest in Anaplasma marginale, the tick-borne causative agent of bovine anaplasmosis. RNA-seq technology allowed a comprehensive analysis of the transcriptional status of A. marginale genes in two conditions: bovine host blood and tick derived cell culture, a model for the tick vector. Quantitative PCR was used to assess transcription of a set of genes in A. marginale infected tick midguts and salivary glands at two time points during the transmission cycle. Genes belonging to fourteen pathways or component groups were found to be differentially transcribed in A. marginale in the bovine host versus the tick vector. One of the most significantly altered groups was composed of surface proteins. Of the 56 genes included in the surface protein group, eight were up regulated and 26 were down regulated. The down regulated surface protein encoding genes include several that are well studied due to their immunogenicity and function. Quantitative PCR of a set of genes demonstrated that transcription in tick cell culture most closely approximates transcription in salivary glands of recently infected ticks. The ISE6 tick cell culture line is an acceptable model for early infection in tick salivary glands, and reveals disproportionate down regulation of surface protein genes in the tick. Transcriptional profiling in other cell lines may help us simulate additional microenvironments. Understanding vector-specific alteration of gene transcription, especially of surface protein encoding genes, may aid in the development of vaccines or transmission blocking therapies

The values of FIC index for the combination of peptides and antimicrobial compounds against <i>Staphylococcus pseudintermedius</i> (SP3).

a<p>The FIC index was determined in the presence of a constant amount of peptide, equal to one -quarter of the peptide MIC.</p><p>The values of FIC index for the combination of peptides and antimicrobial compounds against <i>Staphylococcus pseudintermedius</i> (SP3).</p

Cytotoxicity assay showing the percent mean absorbance at 490 nm after incubating macrophage cell line (J774A.1) (A &B) and human keratinocyte (HaCat) (C&D) with peptides at different concentrations.

<p>Melittin and DMSO served as positive control in J774A.1and HaCat cells, respectively. Water (peptide diluent) served as negative control. Cell viability was measured by MTS assay. Results are expressed as means from three measurements ± standard deviation. Data were analyzed using one-way ANOVA, with post hoc Tukey's multiple comparisons test to determine statistical significance. Two asterisks (**) means statistical different (p<0.01) from the negative control while (ns) means there was no statistical significance from the negative control.</p

Minimum inhibitory concentration (MIC) of peptides against methicillin resistant <i>Staphylococcus pseudintermedius</i> (MRSP) strains.

<p>Abbreviation:OXA: oxacillin, AMK: amikacin, CEF: cefpodxime, CLIN: clindamycin, GEN: gentamycin, CHL: chloramphenicol, ENR: enrofloxacin, MARB: marbofloxacin, ERM: erythromycin, BAC: bacitracin, NEO: neomycin, TOB: tobramycin, CIP: ciprofloxacin, OXY: Oxytetracyclin, TMP-SMX: trimethoprime sulphamethoxazole, TIC: ticarcillin, IMI: imipenem.</p><p>Minimum inhibitory concentration (MIC) of peptides against methicillin resistant <i>Staphylococcus pseudintermedius</i> (MRSP) strains.</p

Bacterial killing kinetics of peptides (RRIKA, RR, WR-12, (KFF)₃K, IK8 “D isoform” and penetratin) and antibiotics amikacin at 5X MIC against SP 3 in MHB (Mueller Hinton broth).

<p>Samples treated with peptide diluent (sterile water plus 0.2% acetic acid) were used as a control. The killing curves were identical (overlapping in the figure) for RRIKA, RR and (KFF)₃K. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen).</p