Immunocompetent rabbits infected with Cryptosporidium cuniculus as an animal model for anti-cryptosporidial drug testing

Cryptosporidium is one of the leading causes of diarrheal disease in humans and animals, which can be severe and deadly in neonates and immunocompromised hosts. Studies on the biology of Cryptosporidium and drug discovery efforts have been hindered by a number of factors including the limited availability of animal models. Here, we report the establishment and characterization of an immunocompetent rabbit model for infection with Cryptosporidium cuniculus. By testing four known anti-cryptosporidial compounds (nitazoxanide, baicalein, curcumin and matrine), we showed that the rabbit could be used as an alternative animal model for evaluating anti-cryptosporidial drug efficacy in vivo.     

A chemiluminescence immunoassay for evaluation of Cryptosporidium parvum growth in vitro

Abstract A chemiluminescence immunoassay (CLIA) was developed to detect Cryptosporidium parvum growth in Madin-Darby canine kidney (MDCK) cell cultures. Optimal results were obtained when MDCK cells were plated at a density of 1 × 10⁴ cells/well (96-well plate) and maintained as a monolayer for 4 days prior to infection with 2 × 10⁴ parasites/well. Two compounds (paromomycin and maduramicin) were evaluated and shown to have selective activity against C. parvum in a dose-dependent manner. There was excellent correlation between CLIA and immunofluorescence assay when assessing anti- C. parvum agents in MDCK cells. CLIA offers advantages over conventional enzyme-linked immunosorbent assay and immunofluorescence assay methods in that it is more sensitive and efficient. The combination of CLIA and MDCK culture provides an efficient tool for evaluating potential anti-cryptosporidial compounds prior to testing in animal models.     

Synthesis and antibacterial activity of novel 4-aryl-[1,2,3]-triazole containing macrolides

Two series of novel triazole containing 14-member macrolides having either a cladinose or a 3-pyridyl acetate group at the 3-position of the macrolide ring were synthesized. The in vitro antibacterial activities against S. aureus, S. pneumoniae, S. pyogenes and E. faecalis were determined. Macrolide 7a and the fluoroketolide 1 (CEM-101) were evaluated in vivo in murine systemic infection models. All of the macrolide analogs were less active in vitro and in vivo than the fluoroketolide 1 (CEM-101).     

Phospholipases and cationic peptides inhibit Cryptosporidium parvum sporozoite infectivity by parasiticidal and non-parasiticidal mechanisms

The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, β-defensin 1, β-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, β-defensin 1, and β-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.     

Evaluation of modern antibiotics efficacy at experimental Northern Asia rickettsiosis]

Comparative investigation of antibiotics therapy efficacy at experimental murine Northern Asia rickettsiosis was performed. The efficacy was evaluated by the determination of protective activity in per cent and by the pathogen erradication. The investigated antibiotics may be ranged in the following order (by the diminishing efficacy): ansamycins (azorif, rifapentine, rifampicin), tetracyclines (doxycycline), macrolides (azitromycin, sumamed), carbapenems (imipenem/cilastatin), fluoroquinolones (lomefloxacin, pefloxacin). Rifampicin and its derivatives--azorif, rifapentine, along with doxycycline and azitromycm can be recommended for clinical trials at experimental rickettsiosis profilaxy and treatment in natural infection focus.     

Macrolide use in the previous years is associated with failure to eradicate Helicobacter pylori with clarithromycin‐containing regimens

Background

There is some evidence that prior use of macrolide antibiotics is a useful predictor of the likelihood of standard triple therapy failure in Helicobacter pylori eradication. In this study, we have evaluated whether previous intake of macrolides correlates with failure to eradicate H. pylori using two different first‐line clarithromycin‐containing regimens.

Materials and Methods

Retrospective study of 212 patients with H. pylori infection treated with one of two first‐line clarithromycin‐containing regimens: 108 patients treated with triple therapy for 10 days and 104 patients treated with concomitant therapy for 10 days. The intake of macrolides (clarithromycin, azithromycin, and other macrolides) prior to the eradication therapy was obtained from the electronic medical record, which contains information regarding all the medication prescribed to the patients since the year 2004.

Results

One hundred of 212 patients (47.2%) had received at least one treatment with macrolides during the years prior to the eradication therapy. H. pylori eradication rates were significantly lower in patients with previous use compared to patients without previous use of macrolides, both with triple therapy (60.8% vs 92.9%; P < .0001) and with concomitant therapy (85.7% vs 98.2%; P = .024).

Conclusions

Previous use of macrolides correlates with a low H. pylori eradication rate with triple and concomitant clarithromycin‐containing regimens. In addition, our study shows that in patients without previous use of macrolides, triple therapy achieves per‐protocol eradication rates over 90%.     

A Screening Pipeline for Antiparasitic Agents Targeting Cryptosporidium Inosine Monophosphate Dehydrogenase

Background

The protozoan parasite Cryptosporidium parvum is responsible for significant disease burden among children in developing countries. In addition Cryptosporidiosis can result in chronic and life-threatening enteritis in AIDS patients, and the currently available drugs lack efficacy in treating these severe conditions. The discovery and development of novel anti-cryptosporidial therapeutics has been hampered by the poor experimental tractability of this pathogen. While the genome sequencing effort has identified several intriguing new targets including a unique inosine monophosphate dehydrogenase (IMPDH), pursuing these targets and testing inhibitors has been frustratingly difficult.

Methodology and Principal Findings

Here we have developed a pipeline of tools to accelerate the in vivo screening of inhibitors of C. parvum IMPDH. We have genetically engineered the related parasite Toxoplasma gondii to serve as a model of C. parvum infection as the first screen. This assay provides crucial target validation and a large signal window that is currently not possible in assays involving C. parvum. To further develop compounds that pass this first filter, we established a fluorescence-based assay of host cell proliferation, and a C. parvum growth assay that utilizes automated high-content imaging analysis for enhanced throughput.

Conclusions and Significance

We have used these assays to evaluate C. parvum IMPDH inhibitors emerging from our ongoing medicinal chemistry effort and have identified a subset of 1,2,3-triazole ethers that exhibit excellent in vivo selectivity in the T. gondii model and improved anti-cryptosporidial activity.     

Macrolides decrease the minimal inhibitory concentrations of anti-pseudomonal agents against Pseudomonas aeruginosa from cystic fibrosis patients in biofilm

ABSTRACT: BACKGROUND: Biofilm production is an important mechanism for bacterial survival and its association with antimicrobial resistance represents a challenge for the patient treatment. In this study we evaluated the in vitro action of macrolides in combination with anti-pseudomonal agents on biofilm-grown Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patient. RESULTS: A total of 64 isolates were analysed. The (BIC) results were consistently higher than those minimal inhibitory concentration (MIC) for most anti-pseudomonal agents tested (ceftazidime: P = 0.001, ciprofloxacin: P = 0.234, tobramycin: P = 0.001, imipenem: P < 0.001, meropenem: P = 0.005). When macrolides were associated with the anti-pseudomonal agents, the BIC values were reduced significantly for ceftazidime (P < 0.001) and tobramycin (P < 0.001), regardless the concentration of macrolides. Strong (IQ) was observed when azithromycin at 8 mg/L was associated with all anti-pseudomonal agents tested in biofilm conditions. CONCLUSIONS: P. aeruginosa from CF patients within biofilms are highly resistant to antibiotics but macrolides proved to augment the in vitro activity of anti-pseudomonal agents.     

Use of macrolides in pregnancy and lactation according to evidence-based medicine: pro et contra

The use of macrolides in the treatment of chlamydial infection during pregnancy and breast feeding is reviewed from the viewpoint of evidence-based medicine. The clinical experience and research data suggest azithromycin to be safe and effective in the obstetric practice.     

Lipase-catalyzed ring-opening polymerization of lactones to polyesters and its mechanistic aspects.

Lipase catalysis induced a ring-opening polymerization of lactones with different ring-sizes. Small-size (four-membered) and medium-size lactones (six- and seven-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were subjected to lipase-catalyzed polymerization. The polymerization behaviors depended primarily on the lipase origin and the monomer structure. The macrolides showing much lower anionic polymerizability were enzymatically polymerized faster than epsilon-caprolactone. The granular immobilized lipase derived from Candida antartica showed extremely efficient catalysis in the polymerization of epsilon-caprolactone. Single-step terminal functionalization of the polyester was achieved by initiator and terminator methods. The enzymatic polymerizability of lactones was quantitatively evaluated by Michaelis-Menten kinetics.     

Macrolides: new therapeutic perspectives in lung diseases

There is strong clinical evidence for the effectiveness of macrolides in the treatment of a number of chronic airway diseases through their immunomodulatory effects. Recently, new information has been released supporting the view that macrolides may also be beneficial in pathologic situations associated with altered repair of the alveolar structure, such as those observed in interstitial lung diseases and fibrosis. It is proposed that macrolides may contribute to lung regeneration through their actions on several components of the remodeling process. The present review provides new insights on the effects of macrolides on the regenerative response of alveolar epithelium to injury. It also discusses novel findings which suggest that macrolides may contribute to alveolar surfactant homeostasis.     

Drug treatment and novel drug target against Cryptosporidium

Cryptosporidiosis emergence triggered the screening of many compounds for potential anti-cryptosporidial activity in which the majority were ineffective. The outbreak of cryptosporidiosis which occurred in Milwaukee in 1993 was not only the first significant emergence of Cryptosporidium spp. as a major human pathogen but also a huge waterborne outbreak thickening thousands of people from a major city in North America. Since then, outbreaks of cryptosporidiosis are regularly occurring throughout the world. New drugs against this parasite became consequently urgently needed. Among the most commonly used treatments against cryptosporidiosis are paromomycin, and azithromycin, which are partially effective. Nitazoxanide (NTZ)'s effectiveness was demonstrated in vitro, and in vivo using several animal models and finally in clinical trials. It significantly shortened the duration of diarrhea and decreased mortality in adults and in malnourished children. NTZ is not effective without an appropriate immune response. In AIDS patients, combination therapy restoring immunity along with antimicrobial treatment of Cryptosporidium infection is necessary. Recent investigations focused on the potential of molecular-based immunotherapy against this parasite. Others tested the effects of probiotic bacteria, but were unable to demonstrate eradication of C. parvum. New synthetic isoflavone derivatives demonstrated excellent activity against C. parvum in vitro and in a gerbil model of infection. Newly synthesized nitro- or non nitro- thiazolide compounds, derived from NTZ, have been recently shown to be at least as effective as NTZ against C. parvum in vitro development and are promising new therapeutic agents.     

Genetic architecture of the polyketide synthases for methymycin and pikromycin series macrolides

The methymycin and pikromycin series of antibiotics are structurally related macrolides produced by several Streptomyces species, including Streptomyces venezuelae ATCC 15439, which produces both 12-membered ring macrolides methymycin, neomethymycin, and 14-membered ring macrolides pikromycin and narbomycin. Cloning and sequencing of the biosynthetic gene clusters for these macrolides from three selected Streptomyces strains revealed a common genetic architecture of their polyketide synthases (PKSs). Unlike PKS clusters of other 14-membered ring macrolides such as erythromycin and oleandomycin, each of the pikromycin series producers harbors a six module PKS cluster, in which modules 5 and 6 are encoded on two separate proteins instead of one bimodular protein, as well as a thioesterase II gene immediately downstream of the main PKS gene. The results shed new light on the evolution of modular PKSs and provide further evidence on the regulation of methymycin and pikromycin production in S. venezuelae ATCC 15439.     

Macrolide resistance and <Emphasis Type="Italic">In Vitro</Emphasis> selection of resistance to antibiotics in <Emphasis Type="Italic">Lactobacillus</Emphasis> isolates

Spreading of resistance to antibiotics is of great concern due to the increasing rate of isolation of multiresistant pathogens. Since commensal bacteria may transfer determinants of resistance to pathogens, studies on development of resistance should include also lactobacilli. Resistance to macrolides, penicillins and tetracycline was determined in 40 isolates of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus crispatus, and Lactobacillus casei isolated from faeces of apparently healthy volunteers. Frequency of mutation and changes in susceptibility after serial exposure to these antibiotics at concentrations of 4× and 8× MIC were evaluated in susceptible isolates. Acquired resistance was defined as an increment in MIC values of at least four times in respect to the pre-selection values. Resistance to macrolides and/or tetracycline was identified in 14 and 4 isolates, respectively. ermB gene and A2058G mutation in 23S rRNA were detected in macrolide resistant isolates. Frequencies of mutation of susceptible isolates (n=26) were lower for ampicillin and erythromycin than for tetracycline. Serial exposure to antibiotics led to selection of resistant mutants. However, acquired resistance was rather unstable and was lost after subcultures in antibiotic-free medium in most mutants. Resistance to erythromycin was associated to a A2058G mutation in 23S rRNA. In conclusion, results indicate that resistance to macrolides and tetracycline is present among intestinal lactobacilli. Decrease in susceptibility following serial exposure to antibiotics might occur in lactobacilli, in a strain- and antibiotic-dependent way. Since lactobacilli are often used as probiotics, their ability to acquire resistance should be evaluated for isolates candidate to be included in probiotics based products.     

Characterization of anti-Cryptosporidium IgA antibodies in sera from immunocompetent individuals and HIV-infected patients.

Human antibody response to Cryptosporidium parvum has been previously shown as involving immunoglobulin (Ig)M and IgG isotypes. The interest in anti-cryptosporidial IgA antibody response has been recently stimulated by studies on the therapeutic effects of secretory IgA antibodies to Cryptosporidium in animal models and in patients. In the present study, isotypes of serum anti-Cryptosporidium antibodies have been characterized in donors of the following categories: (a) healthy adults, (b) healthy children, (c) immunocompetent children with transient cryptosporidial diarrhea, (d) HIV-infected patients without clinical and parasitological evidence of Cryptosporidium infection and (e) AIDS patients with cryptosporidial diarrhea. Antibodies were detected using C. parvum oocysts purified by density gradient centrifugation from bovine faeces. The IgA antibodies were revealed using alpha-chain specific antibodies. Indirect immunofluorescence analysis with oocysts was used as control. Although high levels of serum antibodies of the IgA class were detected in some donors in the group of healthy adults, elevated values were consistently found in HIV-infected patients. Higher values were found in HIV patients with clinical cryptosporidiosis. The presence of a secretory component in serum IgA antibodies in these patients has been documented. Data indicate that IgA serum antibodies are produced as well as IgM and IgG antibodies upon contact with the parasite, and suggest that elevated IgA serum antibodies to Cryptosporidium are not associated with protection in HIV patients.     

Anti-cryptosporidial drug activity screened with an immunosuppressed rat model

Cryptosporidium parvum, a coccidian parasite can cause mild or severe self-limiting diarrhea in immunocompetent humans, but chronic and life-threatening in immunocompromised individuals. An immunosuppressed rat model with persistent cryptosporidiosis was used to investigate the anti-cryptosporidial activity of drugs. Using curative procedures, no activity was found with 6 antibiotics assayed, including spiramycin (31-100%). Mepacrine at a dose of 100 mg/kg had mild activity (19%), while lasalocid (10 mg/kg) and sulfadimethoxine (60 mg/kg) exhibited a 0.1% and 13%, activity respectively. This experimental immuno-suppressed rat model appears suitable for screening candidate drugs either for preventive or curative treatment of cryptosporidiosis.     

Macrolide antibiotics for oral intake in the treatment and prevention of suppurative surgical infection]

The authors studied the clinical efficacy of antibiotics of the macrolides group--erythromycin, oleandomycin, triacetylalleandomycin and leukomycin--in complex treatment of 280 patients with suppurative surgical infection caused by a gram-positive, mainly staphylococcal, microbial flora. Various medicinal forms were used: tablets, capsules, granules, syrup. Efficacy of treatment comprised 70-78 per cent. Tolerance of antibiotics was good in the majority of patients.     

Inhibition of the ribosomal peptidyl transferase reaction by the mycarose moiety of the antibiotics carbomycin, spiramycin and tylosin

Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the peptidyl transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit peptidyl transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the peptidyl transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of peptidyl transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the peptidyl transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA.     

Hemolytic properties of lytic peptides active against the sporozoites of Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that causes mildto-severe diarrheal disease in animals and humans. There are currently no effective chemotherapeutic agents available for the treatment of cryptosporidiosis. Recently, small, naturally occurring antimicrobial lytic peptides with anti-protozoal activities have been described. In the present study, we compare the in vitro anti-cryptosporidial activities of synthetic lytic peptides and their corresponding hemolytic activities after a 30 min incubation at 37 degrees C. Sporozoite viability was assessed microscopically by the uptake of the vital dyes fluorescein diacetate (FDA) and propidium iodide (PI). Hemolysis was assessed spectrophotometrically by the release of soluble hemoglobulin. The most active peptide, Hecate-1, reduced sporozoite viability by 85.5% with a corresponding hemolytic activity of 21.5% at a concentration of 10 microM.     

Antiviral action of carbonyl-conjugated pentaene macrolides

Clear antiviral activity of carbonyl-conjugated pentaene macrolides, such as flavofungin, mycothicin, brunefungin and flavopentin was shown on models with infectious and oncogenic viruses. The antibiotics were active against influenza A and B virus. The effect was most pronounced in the in vitro and in ovo systems. On a model of experimental influenza infection of mice with the lethal outcome, antiinfluenzal activity of flavofungin was comparable to that of remantadin. However, unlike the latter one flavofungin and brunefungin inhibited the growth of influenza B virus. The drugs had a pronounced inhibitory effect on variolavaccine virus and prevented formation of foci of cell neoplastic transformation infected with various strains of Rous sarcoma virus.