Pathogens, Volume 6, Issue 3 (September 2017) – 18 articles

Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat for effective treatment of several difficult-to-treat infections in humans. To identify potential new treatment options for MRSA infections, we screened a clinical compound library consisting of 1524 compounds using a growth inhibition assay in 96-well plates. We identified 34 agents which are either bacteriostatic or bactericidal against log-phase clinical MRSA strain USA300. Among them, 9 candidates (thonzonium, cetylpyridinium, trilocarban, benzododecinium, bithionol, brilliant green, chlorquinaldol, methylbenzethonium and green violet) are known antiseptics, 11 candidates are known antibiotics currently recommended for the treatment of MRSA. We identified 9 new drug candidates, 5 of which (thiostrepton, carbomycin, spiramycin, clofazimine and chloroxine) are antibiotics used for treating other infections than S. aureus infections; 4 of which (quinaldine blue, closantel, dithiazanine iodide and pyrvinium pamoate) are drugs used for treating parasitic diseases or cancer. We ranked these new drug candidates according to their MICs against the MRSA strain USA300. Our findings may have implications for more effective treatment of MRSA infections. Full article

Heparan sulfate (HS) is ubiquitously expressed on mammalian cells. It is a polysaccharide that binds growth factors, cytokines, and chemokines, and thereby controls several important physiological functions. Ironically, many human pathogens including viruses interact with it for adherence to host cells. HS functions can be regulated by selective modifications and/or selective cleavage of the sugar chains from the cell surface. In mammals, heparanase (HPSE) is the only known enzyme capable of regulating HS functions via a selective endoglycosidase activity that cleaves polymeric HS chains at internal sites. During homeostasis, HPSE expression and its endoglycosidase activity are tightly regulated; however, under stress conditions, including infection, its expression may be upregulated, which could contribute directly to the onset of several disease pathologies. Here we focus on viral infections exemplified by herpes simplex virus, dengue virus, human papillomavirus, respiratory syncytial virus, adenovirus, hepatitis C virus, and porcine respiratory and reproductive syncytial virus to summarize recent advances in understanding the highly significant, but emerging roles, of the enzyme HPSE in viral infection, spread and pathogenesis. Full article

Both Mycobacterium spp. and Methylobacterium spp. are opportunistic premise plumbing pathogens that are found on pipe surfaces in households. However, examination of data published in prior microbiological surveys indicates that Methylobacterium spp. and Mycobacterium spp. tend not to coexist in the same household plumbing biofilms. That evidence led us to test the hypothesis that Methylobacterium spp. in biofilms could inhibit the adherence of Mycobacterium avium. Measurements of adherence of M. avium cells to stainless steel coupons using both culture and PCR-based methods showed that the presence of Methylobacterium spp. biofilms substantially reduced M. avium adherence and vice versa. That inhibition of M. avium adherence was not reduced by UV-irradiation, cyanide/azide exposure, or autoclaving of the Methylobacterium spp. biofilms. Further, there was no evidence of the production of anti-mycobacterial compounds by biofilm-grown Methylobacterium spp. cells. The results add to understanding of the role of microbial interactions in biofilms as a driving force in the proliferation or inhibition of opportunistic pathogens in premise plumbing, and provide a potential new avenue by which M. avium exposures may be reduced for at-risk individuals. Full article

The foodborne pathogen Listeria monocytogenes is a concern in food safety because of its ability to form biofilm and to persist in food industry. In this mini-review, the issue represented by this pathogen and some of the latest efforts performed in order to investigate the composition of biofilms formed by L. monocytogenes are summarized. Full article

Human papillomavirus (HPV) is suggested to infect trophoblasts in the placenta, and HPV infections are reported to be more prevalent in pregnancies with adverse outcomes. Results are however controversial, and studies investigating the molecular consequences of placental HPV infections are lacking. We studied HPV DNA localization in the placenta in cases of spontaneous abortion/spontaneous preterm delivery as well as in elective abortion/normal full-term delivery. Using in vitro assays, we investigated downstream effects of HPV16 E6 and E7 expression in trophoblast cells at the gene expression level in order to gain increased biological insight into the interaction between HPV and the cellular host. Fluorescent in situ hybridization (FISH), combined with fluorescent immunohistochemistry (FIHC) to target the trophoblast marker CK7 clearly showed, that HPV DNA resides within syncytiotrophoblast cells in the placenta. In vitro HPV16 E6 and E7-transfected trophoblasts were analyzed by RNA sequencing, and results were validated by reverse transcription real time polymerase chain reaction (RT-qPCR) for selected genes in cell lines, as well as in patient material. We show that HPV16 E6 and E7 upregulate interferon-induced antiviral response genes ISG15 and IFIT1 in a human trophoblast cell line two-days post-transfection. This is a response that is not observed when assessing the gene expression levels of the same genes in HPV16-positive placenta samples. Investigations on viral activity find that HPV16 E6 and E7 are not transcribed in patients, possibly suggesting that HPV16 syncytiotrophoblast infection may be latent. We conclude that HPV localizes to syncytiotrophoblast cells of the placenta, and that active expression of HPV16 E6 and E7 induce an immediate interferon-induced antiviral response in trophoblast cells, which is not present in HPV-positive placenta samples, suggesting latent infection. Full article

Lipid rafts are liquid-ordered membrane microdomains that form by preferential association of 3-β-hydroxysterols, sphingolipids and raft-associated proteins often having acyl modifications. We isolated lipid rafts of the protozoan parasite Trypanosoma brucei and determined the protein composition of lipid rafts in the cell. This analysis revealed a striking enrichment of flagellar proteins and several putative signaling proteins in the lipid raft proteome. Calpains and intraflagellar transport proteins, in particular, were found to be abundant in the lipid raft proteome. These findings provide additional evidence supporting the notion that the eukaryotic cilium/flagellum is a lipid raft-enriched specialized structure with high concentrations of sterols, sphingolipids and palmitoylated proteins involved in environmental sensing and cell signaling. Full article

Globally, there has been an increase in squamates (particularly lizards and snakes) being kept as pets. Additionally, urban spread has resulted in greater human encroachment and interaction with the natural habitat of wild squamates. A potential consequence of increasing human interaction with squamates is the increased potential for disease transfer. This review collates the literature describing clinical salmonellosis cases that were definitively linked to a squamate through testing of the animal and population-based studies which investigate the risk of salmonellosis linked to pet squamates. It was demonstrated that although squamate-acquired salmonellosis accounted for a small percentage of total cases, children under five were at greatest risk, with the clinical manifestations tending to be more severe. In many cases, it was noted that the patient was unaware of the risks associated with keeping squamates and did not practice proper hand hygiene after handling the animals or cleaning cages. This highlights the need for more education focused on informing the general public of ways to reduce the risk of salmonellosis from pet squamates. There is also the need for future research into the role of wild squamates in the spread of human salmonellosis, both directly and indirectly through cross contamination. Full article

Abstract: Group C rotavirus (RVC) has been described to be a causative agent of gastroenteritis in humans and animals including pigs, cows, and dogs. Fecal samples collected from asymptomatic pigs in smallholder swine farms in Kenya and Uganda were screened for the presence of group C rotaviruses (RVC) using a reverse transcription-polymerase chain reaction assay. A total of 446 samples were tested and 37 were positive (8.3%). A significantly larger (p < 0.05) number of RVC-positive samples was detected in groups of older pigs (5–6 months) than in younger piglets (1–2 months). There were no significant differences in the RVC detection rate between the pigs that were full time housed/tethered and those that were free range combined with housing/tethering. After compiling these data with diagnostic results for group A rotaviruses (RVA), 13 RVC-positive samples were also positive for RVA. This study provides the first evidence that porcine group C rotavirus may be detected frequently in asymptomatic piglets (aged < 1–6 months) in East Africa. The occurrence of RVC in mixed infections with RVA and other enteric pathogens requires further research to investigate the pathogenic potential of RVC in pigs. Full article

Identifying environmental sources of Pseudomonas aeruginosa (Pa) related to hospital-acquired infections represents a key challenge for public health. Biofilms in water systems offer protection and favorable growth conditions, and are prime reservoirs of microorganisms. A comparative genotyping survey assessing the relationship between Pa strains recovered in hospital sink biofilm and isolated in clinical specimens was conducted. Environmental strains from drain, faucet and sink-surface biofilm were recovered by a culture method after an incubation time ranging from 48 to 240 h. The genotyping of 38 environmental and 32 clinical isolates was performed using a multiple-locus variable-number of tandem repeats analysis (MLVA). More than one-third of Pa isolates were only cultivable following ≥48 h of incubation, and were predominantly from faucet and sink-surface biofilms. In total, 41/70 strains were grouped within eight genotypes (A to H). Genotype B grouped a clinical and an environmental strain isolated in the same ward, 5 months apart, suggesting this genotype could thrive in both contexts. Genotype E grouped environmental isolates that were highly prevalent throughout the hospital and that required a longer incubation time. The results from the multi-hospital follow-up study support the drain as an important reservoir of Pa dissemination to faucets, sink surfaces and patients. Optimizing the recovery of environmental strains will strengthen epidemiological investigations, facilitate pathway identification, and assist in identifying and controlling the reservoirs potentially associated to hospital-acquired infections. Full article

Since chronic wasting disease (CWD) was first identified nearly 50 years ago in a captive mule deer herd in the Rocky Mountains of the United States, it has slowly spread across North America through the natural and anthropogenic movement of cervids and their carcasses. As the endemic areas have expanded, so has the need for rapid, sensitive, and cost effective diagnostic tests—especially those which take advantage of samples collected antemortem. Over the past two decades, strategies have evolved from the recognition of microscopic spongiform pathology and associated immunohistochemical staining of the misfolded prion protein to enzyme-linked immunoassays capable of detecting the abnormal prion conformer in postmortem samples. In a history that parallels the diagnosis of more conventional infectious agents, both qualitative and real-time amplification assays have recently been developed to detect minute quantities of misfolded prions in a range of biological and environmental samples. With these more sensitive and semi-quantitative approaches has come a greater understanding of the pathogenesis and epidemiology of this disease in the native host. Because the molecular pathogenesis of prion protein misfolding is broadly analogous to the misfolding of other pathogenic proteins, including Aβ and α-synuclein, efforts are currently underway to apply these in vitro amplification techniques towards the diagnosis of Alzheimer’s disease, Parkinson’s disease, and other proteinopathies. Chronic wasting disease—once a rare disease of Colorado mule deer—now represents one of the most prevalent prion diseases, and should serve as a model for the continued development and implementation of novel diagnostic strategies for protein misfolding disorders in the natural host. Full article

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of two human cancers, Kaposi’s Sarcoma (KS) and primary effusion lymphoma (PEL), and a lymphoproliferation, Multicentric Castleman’s Disease (MCD). Progression to tumor development in KS is dependent upon the reactivation of the virus from its latent state. We, and others, have shown that the Replication and transcriptional activator (Rta) protein is the only viral gene product that is necessary and sufficient for viral reactivation. To induce the reactivation and transcription of viral genes, Rta forms a complex with the cellular DNA binding component of the canonical Notch signaling pathway, recombination signal binding protein for Jk (RBP-Jk). Formation of this Rta:RBP-Jk complex is necessary for viral reactivation to occur. Expression of activated Notch has been shown to be dysregulated in KSHV infected cells and to be necessary for cell growth and disease progression. Studies into the involvement of activated Notch in viral reactivation have yielded varied results. In this paper, we review the current literature regarding Notch dysregulation by KSHV and its role in viral infection and cellular pathogenesis. Full article

Egypt has a unique geographical location connecting the three old-world continents Africa, Asia and Europe. It is the country with the highest population density in the Middle East, Northern Africa and the Mediterranean basin. This review summarizes the prevalence, reservoirs, sources of human infection and control regimes of common bacterial, parasitic and viral zoonoses in animals and humans in Egypt. There is a gap of knowledge conerning the epidemiology of zoonotic diseases at the human-animal interface in different localities in Egypt. Some zoonotic agents are “exotic” for Egypt (e.g., MERS-CoV and Crimean-Congo hemorrhagic fever virus), others are endemic (e.g., Brucellosis, Schistosomiasis and Avian influenza). Transboundary transmission of emerging pathogens from and to Egypt occurred via different routes, mainly importation/exportation of apparently healthy animals or migratory birds. Control of the infectious agents and multidrug resistant bacteria in the veterinary sector is on the frontline for infection control in humans. The implementation of control programs significantly decreased the prevalence of some zoonoses, such as schistosomiasis and fascioliasis, in some localities within the country. Sustainable awareness, education and training targeting groups at high risk (veterinarians, farmers, abattoir workers, nurses, etc.) are important to lessen the burden of zoonotic diseases among Egyptians. There is an urgent need for collaborative surveillance and intervention plans for the control of these diseases in Egypt. Full article

Over the last decade new species of Protozoa have been sequenced and deposited in GenBank. Analyzing large amounts of genomic data, especially using Next Generation Sequencing (NGS), is not a trivial task, considering that researchers used to deal or focus their studies on few genes or gene families or even small genomes. To facilitate the information extraction process from genomic data, we developed a database system called ProtozoaDB that included five genomes of Protozoa in its first version. In the present study, we present a new version of ProtozoaDB called ProtozoaDB 2.0, now with the genomes of 22 pathogenic Protozoa. The system has been fully remodeled to allow for new tools and a more expanded view of data, and now includes a number of analyses such as: (i) similarities with other databases (model organisms, the Conserved Domains Database, and the Protein Data Bank); (ii) visualization of KEGG metabolic pathways; (iii) the protein structure from PDB; (iv) homology inferences; (v) the search for related publications in PubMed; (vi) superfamily classification; and (vii) phenotype inferences based on comparisons with model organisms. ProtozoaDB 2.0 supports RESTful Web Services to make data access easier. Those services were written in Ruby language using Ruby on Rails (RoR). This new version also allows a more detailed analysis of the object of study, as well as expanding the number of genomes and proteomes available to the scientific community. In our case study, a group of prenyltransferase proteinsalready described in the literature was found to be a good drug target for Trypanosomatids. Full article

Staphylococcus aureus is a major pathogen causing bone infections that can become chronic and difficult to treat. Recently, we described the mechanism employed by S. aureus to switch to small colony variants (SCVs) and trigger intracellular bacterial persistence through the global stress regulator SigB. Here, we studied the role of SigB in the formation of chronic osteomyelitis. We used a murine hematogenous osteomyelitis model, where the mice were infected via the tail vein and subsequently developed chronic osteomyelitis. Mice were infected with S. aureus LS1, LS1ΔsigB and LS1ΔsigB complemented and kidney and bone tissues were analyzed six weeks after infection. S. aureus LS1ΔsigB formed a high rate of abscesses in kidneys, but the bacterial loads and the weight loss of the animals were lower in comparison with animals infected with the wild type and the complemented strain, indicating a more rapid and efficient bacterial clearing by the host immune system. Moreover, the sigB-mutant was not able to form SCV phenotypes either in kidney or in bone tissue. Our results demonstrate that staphylococcal SigB is important to avoid bacterial elimination by the host immune response, establish a bone infection and mediate bacterial adaptation (SCV-formation) for persistent infections Full article

Periodontitis originates from a microbial synergy causing the development of a mouth microbial imbalance (dysbiosis), consisting of a microbial community composed of anaerobic bacteria. Most studies concerning the treatment of periodontitis have primarily take into account the Gram-negative bacterium Porphyromonas gingivalis, because it is a prominent component of the oral microbiome and a successful colonizer of the oral epithelium. Here, we focus our attention on the study of the carbonic anhydrases (CAs, EC 4.2.1.1) encoded in the genome of this pathogen as a possible drug target. Carbonic anhydrases are a superfamily of metalloenzymes, which catalyze the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Bacterial CAs have attracted significant attention for affecting the survival, invasion, and pathogenicity of many microorganisms. The P. gingivalis genome encodes for two CAs belonging to β-CA (PgiCAβ) and γ-CA (PgiCAγ) families. These two enzymes were cloned, heterologously expressed in Escherichia coli, and purified to homogeneity. Moreover, they were subject to extensive inhibition studies using the classical CA inhibitors (sulfonamides and anions) with the aim of identifying selective inhibitors of PgiCAβ and PgiCAγ to be used as pharmacological tools for P. gingivalis eradication. Full article

Neuroinvasive microbes are capable of applying their influences on the autonomic nervous system (ANS) of the host followed by the involvement of central nervous system (CNS) by releasing extracellular metabolites that may cause alterations in the biochemical and neurophysiological environment. Consequently synaptic, neuroendocrine, peripheral immune, neuro-immune, and behavioural responses of the host facilitate the progression of infection. The present study was designed to extrapolate the effects of crude and purified extracellular peptides of neuropathogenic bacteria on behavioural responses and neurotransmission of Sprague Dawley (SD) models. Listeria monocytogenes (Lm) and Neisseria meningitides (Nm) were isolated from the 92 cerebrospinal fluid (CSF) samples collected from mentally compromised patients. Bacillus cereus (Bc) and Clostridium tetani (Ct) were also included in the study. All bacterial strains were identified by the standard biochemical procedures. Filter sterilized cell free cultural broths (SCFBs) were prepared of different culture media. Behavioural study and neurotransmitter analysis were performed by giving an intraperitoneal (i.p.) injection of each bacterial SCFB to four groups (Test; n = 7) of SD rats, whereas two groups each (Control; n = 7) received a nutrient broth (NB) control and sterile physiological saline control, respectively. Extracellular bioactive peptides of these bacteria were screened and purified. All experiments were repeated using purified bacterial peptides on SD rat cohorts. Our study indicated promising behavioural changes, including fever, swelling, and hind paw paralysis, in SD rat cohorts. Purified bacterial peptides of all bacteria used in the present study elicited marked changes in behaviour through the involvement of the autonomic nervous system. Furthermore, these peptides of meningitis bacteria were found to potently affect the dopaminergic neurotransmission in CNS. Full article

African trypanosomes cause devastating disease in sub-Saharan Africa in humans and livestock. The parasite lives extracellularly within the bloodstream of mammalian hosts and is transmitted by blood-feeding tsetse flies. In the blood, trypanosomes exhibit two developmental forms: the slender form and the stumpy form. The slender form proliferates in the bloodstream, establishes the parasite numbers and avoids host immunity through antigenic variation. The stumpy form, in contrast, is non-proliferative and is adapted for transmission. Here, we overview the features of slender and stumpy form parasites in terms of their cytological and molecular characteristics and discuss how these contribute to their distinct biological functions. Thereafter, we describe the technical developments that have enabled recent discoveries that uncover how the slender to stumpy transition is enacted in molecular terms. Finally, we highlight new understanding of how control of the balance between slender and stumpy form parasites interfaces with other components of the infection dynamic of trypanosomes in their mammalian hosts. This interplay between the host environment and the parasite’s developmental biology may expose new vulnerabilities to therapeutic attack or reveal where drug control may be thwarted by the biological complexity of the parasite’s lifestyle. Full article

All herpes viruses establish lifelong infections (latency) in their host, and herpes simplex viruses (HSVs) are highly prevalent worldwide. Recurrence of HSV infections contributes to significant disease burden in people and on rare occasion can be fatal. Cell culture models that recapitulate latent infection provide valuable insight on the host processes regulating viral establishment and maintenance of latency. More robust and rapid than infections in live animal studies, advancements in neuronal culture techniques have made the systematic analysis of viral reactivation mechanisms feasible. Only recently have human neuronal cell lines been available, but models in the natural host cell are a critical addition to the currently available models. Full article