Molecular characterisation of Campylobacter concisus : a potential etiological agent of gastroenteritis in children

Campylobacter concisus is a human oral cavity bacterium, which has been linked to oral cavity infections such as gingivitis and periodontitis for the last twenty years. C. concisus has also been isolated from non-oral sources since 1989 and was considered as a potential aetiological agent of enteritis in children since 1994. Some patient subgroups, mainly children under 24 months of age, the elderly, and immunocompromised patients are known to be most susceptible to C. concisus enteric infections. However, C. concisus was isolated from saliva and stool of healthy individuals and was recently detected in the synovial fluid of patients with Campylobacter-associated reactive arthritis. Yet, little is known about this bacterium regarding its mode of transmission, reservoir, and its true potential as a pathogen, due to the lack of any established typing procedure and other virulence related studies. C. concisus clinical strains isolated from children suffering from gastroenteritis at the Royal Children's Hospital (Melbourne), were grouped and characterized in this study using molecular methods to clarify the identity of these isolates and to detect any possible virulence factors that could be related to the pathogenicity of this bacterium. The clinical isolates were grouped into two molecular groups (genomospecies) using PCR amplification of the 23S rDNA of C. concisus with the majority of the isolates (>70%) belonging to genomospecies A. However, the protein profiles for these isolates were divergent even within the same molecular group, but they were different from the protein profiles of other closely related Campylobacter spp. isolates, which indicate the complex nature of this species. Both secreted and cell-bound hemolytic activities were detected in C. concisus clinical isolates by different hemolysis assays using mammalian and non-mammalian erythrocytes. Variable levels of hemolysis were detected for all tested strains on different types of blood. The species specificity of red blood cell hemolysis and the difference in the hemolytic activity levels have been previously reported for Campylobacter spp.' and other pathogenic bacteria. The fraction containing the membrane-bound hemolytic activity found in C. concisus clinical isolates was extracted and characterised using different physical and chemical tests. This hemolysin(s) is a stable calcium-dependent and iron regulated protein with phospholipase A2 enzymatic activity that prefers phosphatidylcholine as a substrate when compared to other lipid components of mammalian cells. Similar cell-associated hemolytic activities produced by C. coli and C. jejuni strains were previously related to pathogenicity in these bacteria. The phospholipase A hemolytic extract of C. concisus caused stable damaging effects on eRO tissue culture cell lines. Vacuolation and cytolytic effects were noticed on eRO cells after 18 hours of incubation with C. concisus diluted and concentrated hemolytic extracts respectively. Remolysins and hemolytic phospholipases of several pathogens such as Serratia marcescens, Haemophilus ducreyi and in Legionella spp. have been reported to cause a damaging effect on human epithelial cells. Moreover, the phospholipases AI, A2 and e of H. pylori were linked to the degradation of the phospholipid components of the mucosal barrier and phospholipase A of H. pylori is thought to have a role in colonisation. Therefore, further studies are required to determine whether this hemolysin isolated from C. concisus produces a vacuolation effect on human epithelial cells similar to that produced by VacA toxin of Helicobacter pylori or VcVac from Vibrio cholerae. The sequencing and analysis of the C. concisus pldA gene in this study indicated that the nucleotide sequence of this gene in clinical isolates of C. concisus resembles the pldA gene sequence encoding outer-membrane phospholipase A in other pathogenic campylobacters such as C. coli and C. jejuni. The pldA gene of C. concisus encodes for a 35 kDa protein with 97% similarity to the 35 kDa phospholipase A protein found in C. coli. When the nucleotide sequences of the flanking regions of the pldA gene were analysed in this study, they revealed that the pldA gene in C. concisus is located upstream of the' ceuB and ceuC genes, similar to the position of the pldA gene in the C. coli and the C. jejuni genome. Therefore, the presence of this gene in C. concisus clinical isolates indicates the possible pathogenic role for this gene and its encoded PLA protein in gastroenteritis cases caused by C. concisus. However further sequencing studies are needed to analyse both the ceuB and ceuC genes in different C. concisus strains belonging to both genomospecies and also from different geographic origins to detect any similarity between these genes and other pathogenic campylobacters. C. concisus clinical strains from different clinical sources should be screened for the presence of other virulence-related genes such as those encoding for cytotoxins, lipopolysaccharides (LPS) and flagellin to clarify the pathogenic role of this opportunistic pathogen. It is recommended that C. concisus gastroenteritis cases in children under the age of primary dentition should be further investigated and highlighted leading to more awareness in the community. The detection of possible virulence factors in C. concisus gastroenteritis-related strains in this study, in addition to the recent findings for its possible relation to other infections such as in Campylobacter-associated reactive arthritis indicates the importance of this opportunistic pathogen in patients with low or impaired immunity such as young children, the elderly and immunocompromised patients

A bioactive peptide analogue for myxoma virus protein with a targeted cytotoxicity for human skin cancer in vitro

Background - Cancer is an international health problem, and the search for effective treatments is still in progress. Peptide therapy is focused on the development of short peptides with strong tumoricidal activity and low toxicity. In this study, we investigated the efficacy of a myxoma virus peptide analogue (RRM-MV) as a candidate for skin cancer therapy. RRM-MV was designed using the Resonant Recognition Model (RRM) and its effect was examined on human skin cancer and normal human skin cells in vitro

Key Role of Mfd in the Development of Fluoroquinolone Resistance in Campylobacter jejuni

Campylobacter jejuni is a major food-borne pathogen and a common causative agent of human enterocolitis. Fluoroquinolones are a key class of antibiotics prescribed for clinical treatment of enteric infections including campylobacteriosis, but fluoroquinolone-resistant Campylobacter readily emerges under the antibiotic selection pressure. To understand the mechanisms involved in the development of fluoroquinolone-resistant Campylobacter, we compared the gene expression profiles of C. jejuni in the presence and absence of ciprofloxacin using DNA microarray. Our analysis revealed that multiple genes showed significant changes in expression in the presence of a suprainhibitory concentration of ciprofloxacin. Most importantly, ciprofloxacin induced the expression of mfd, which encodes a transcription-repair coupling factor involved in strand-specific DNA repair. Mutation of the mfd gene resulted in an approximately 100-fold reduction in the rate of spontaneous mutation to ciprofloxacin resistance, while overexpression of mfd elevated the mutation frequency. In addition, loss of mfd in C. jejuni significantly reduced the development of fluoroquinolone-resistant Campylobacter in culture media or chickens treated with fluoroquinolones. These findings indicate that Mfd is important for the development of fluoroquinolone resistance in Campylobacter, reveal a previously unrecognized function of Mfd in promoting mutation frequencies, and identify a potential molecular target for reducing the emergence of fluoroquinolone-resistant Campylobacter

Phospholipase A in gram-negative bacteria and its role in pathogenesis

Phospholipase A (PLA) is one of the few enzymes present in the outer membrane of Gram-negative bacteria, and is likely to be involved in the membrane disruption processes that occur during host cell invasion. Both secreted and membrane-bound phospholipase A(2) activities have been described in bacteria, fungi and protozoa. Recently there have been increasing reports on the involvement of PLA in bacterial invasion and pathogenesis. This review highlights the latest findings on PLA as a virulence factor in Gram-negative bacteria

Toward development of novel peptide-based cancer therapeutics:computational design and experimental evaluation

Drug research and discovery are of critical importance in human health care. Computational approaches for drug lead discovery and optimization have proven successful in many recent research programs. These methods have grown in their effectiveness not only because of improved understanding of the basic science, the biological events and molecular interactions that de fi ne a target for therapeutic intervention, but also because of advances in algorithms, representations, and mathematical procedures for studying such processes. Advances in genomics and proteomics and development of new bioinformatics methods contribute greatly to the process of rational drug design, which can be a cost-effective solution to drug discovery. Peptides are emerging as a novel class of drugs for cancer therapy, and many efforts have been made to develop peptide-based pharmacologically active compounds. In this study, we present and discuss three novel bioactive peptide analogues, designed using the Resonant Recognition Model (RRM), and discuss their biological effects on normal and tumor cells from mouse and human origin

The effect of extremely low frequency (ELF) pulsed electromagnetic field (PEMF) on bacteria staphylococcus aureus

Current interest in the application of pulsed electromagnetic fields (PEMF) as alternative therapy for different medical conditions has proven to be successful. There are studies that demonstrate the effectiveness of low frequency PEMF in facilitating wound healing. There are many factors that can affect wound healing and cause improper or impaired tissue repair. Of particular interest are the infected wounds where bacteria or other microorganisms have colonized that cause either a delay in wound healing or deterioration of the wound. In most cases, wounds are typically contaminated by bacteria. In this study, we investigate the application of the applied PEMF on the selected bacterial cultures. In our previous study [1], we presented and discussed the design and development of an extremely low frequency (ELF) PEMF system that produces time varying magnetic field in the range of 0.5 mT to 2.5 mT for a frequency range of 2–500 Hz. Here, we report simulation results of the induced magnetic field distribution and the region of uniformity produced by the system of two pairs of air core Helmholtz coils. We also present the experimental evaluation of varying parametric changes in the applied ELF PEMF on the bacteria Staphylococcus aureus. The selection of this gram-positive bacterium for this study is attributed to the versatile role of these bacteria in infecting wounded tissues. These bacteria are also within easy reach and can be bred at a temperature of 37◦C. Here, the Staphylococcus aureus bacteria in broth were exposed to the ELF PEMF of magnetic flux density, B = 0.5–2.5 mT, frequency (f) range of 2–500 Hz and time of exposure, t = 90 min. This is done in order to compare studied bacterial cultures viability (number of colony-forming units (CFU)). The number of colony-forming units aids in quantifying the experimental result. Fresh bacterial culture is used throughout the experiment. Findings from our investigation have direct implication in determining the optimal characteristics of the applied ELF PEMF for possible treatment of infected tissue and thus, wound healing promotion

Development and evaluation of a multiplex PCR for the detection of campylobacter concisus and other campylobacter spp. from gastroenteritis cases

We developed and evaluated a multiplex PCR (m-PCR) for application in routine diagnostic labor- atories to detect Campylobacter spp. in stool samples including C. concisus, C. jejuni, and C. coli. When this m-PCR was applied on spiked faecal samples, C. concisus, C. jejuni, and C. coli were spe- cifically identified at 105 cells/gm of faeces. To compare the sensitivity of the m-PCR with conven- tional culture techniques, the same spiked stool samples were cultured on an antibiotic free Co- lumbia blood agar using the filtration technique. The detection limit of conventional culture me- thod was 105 cells/gm of stool for C. concisus and 106 cells/gm of stool for C. jejuni and C. coli. The m-PCR was applied to test 127 faecal samples from children with gastroenteritis and the results were compared with the conventional bacterial cultures data. By this m-PCR technique, C. jejuni was detected in 7 samples, C. coli in 2 samples, and C. concisus in 7 samples. However, the conven- tional culture results for these samples were 6 for C. jejuni, 2 for C. coli and only one sample was positive for C. concisus. In total, 19 samples were positive for Campylobacter spp. by m-PCR while only 9 samples were positive for Campylobacter spp. by culture. In conclusion, m-PCR is more sen- sitive than the culture technique to detect C. concisus and other fastidious campylobacters in faeces

Advances in methods for therapeutic peptide discovery, design and development

Drug discovery and development are intense, lengthy and interdisciplinary processes. Traditionally, drugs were discovered by synthesizing compounds in time-consuming multi-step experimental investigations followed by in vitro and in vivo biological screening. Promising candidates were then further studied for their pharmacokinetic properties, metabolism and potential toxicity. Today, the process of drug discovery has been revolutionized due to the advances in genomics, proteomics, and bioinformatics. Efficient technologies such as combinatorial chemistry, high throughput screening (HTS), virtual screening, de novo design and structure-based drug design contribute greatly to drug discovery. Peptides are emerging as a novel class of drugs for cancer therapy, and many efforts have been made to develop peptide-based pharmacologically active compounds. This paper presents a review of current advances and novel approaches in experimental and computational drug discovery and design. We also present a novel bioactive peptide analogue, designed using the Resonant Recognition Model (RRM), and discuss its potential use for cancer therapeutics

Experimental evaluation of antimicrobial effects of the synthetic peptide on pathogenic bacteria

Antibiotics are common anti-infection drugs used these days. However, their excessive applications have led to the rising antibiotic resistance, a serious phenomenon in modern medicine that is regarded as one of the pre-eminent public health concerns of the 21st century. As a result, there is a growing need to find alternative drugs to eradicate the microbial resistance. Antimicrobial peptides (AMPs) are natural defence molecules found in virtually all life forms. They are evolutionary conserved components of the innate immune defence. AMPs utilize different mechanisms of action for killing bacteria, which can vary depending on a type of particular bacterium. Generally, AMPs are broad-spectrum antibiotics that act not only against bacteria but also certain viruses and fungi. A number of studies suggested a possibility of using AMPs as an alternative therapy for treatment of microbial infections

The Akt signaling pathway in skin cancer cell lines treated with RRM-MV, a short peptide analogue of the MT5 protein of myxoma virus

In silico methods can help in identifying drug targets via bioinformatics tools. In our previous studies, a 2.34 kDa short linear peptide analogue to myxoma virus M-T5 protein was computationally designed using the resonant recognition model (RRM). Quantitative and qualitative cell cytotoxicity assays showed that the peptide analogue can induce a dose and time dependent tumoricidal effect on skin cancer cells but not on normal cells. It has been reported that the viral protein (M-T5) binds with p-Akt to regulate Akt signaling in some human cancer types. Hence in this study we investigated the effects induced by the peptide analogue RRM- MV on the Akt pathway in skin cancer and normal cells. Akt expression levels in cells treated with RRM-MV, in the presence or absence of PI3K/Akt inhibitor, were detected in mammalian skin cancer and normal cells using immunoblotting. The results revealed that different endogenous levels of p-Akt were expressed in the human melanoma and carcinoma cells following treatment with RRM-MV. Yet, it did not appear to affect the p-Akt expression in the mouse melanoma cells. Furthermore, RRM-MV treatment did not seem to affect the level of total Akt in any type of cancer cells under the same experimental conditions. Hence, the bioactive peptide RRM-MV appears to be targeting the Akt pathway in some types of skin cancer cells. Elucidating the mechanism of RRM-MV effects on other cellular apoptosis pathways requires further investigation