Ferenc Rozgonyi

Alternative stabilities of a proline-rich antibacterial peptide in vitro and in vivo

The proline‐rich designer antibacterial peptide dimer A3‐APO is currently under preclinical development for the treatment of systemic infections caused by antibiotic‐resistant Gram‐negative bacteria. The peptide showed remarkable stability in 25% mouse serum in vitro, exhibiting a half‐life of ∼100 min as documented by reversed‐phase chromatography. Indeed, after a 30‐min incubation period in undiluted mouse serum ex vivo, mass spectrometry failed to identify any degradation product. The peptide was still a major peak in full blood ex vivo, however, with degradation products present corresponding to amino‐terminal cleavage. When injected into mice intravenously, very little, if any unmodified peptide could be detected after 30 min. Nevertheless, the major early metabolite, a full single‐chain fragment, was detectable until 90 min, and this fragment exhibited equal or slightly better activity in the broth microdilution antimicrobial assay against a panel of resistant Enterobactericeae strains. The Chex1‐Arg20 metabolite, when administered three times at 20 mg/kg to mice infected with a sublethal dose (over LD50) of an extended spectrum β‐lactamase‐producing Escherichia coli strain, completely sterilized the mouse blood, similar to imipenem added at a higher dose. The longer and presumably more immunogenic prodrug A3‐APO, injected subcutaneously twice over a 3‐wk period, did not induce any antibody production, indicating the suitability of this peptide or its active metabolite for clinical development.

The designer proline-rich antibacterial peptide A3-APO is effective against systemic Escherichia coli infections in different mouse models.

Antimicrobial peptides are considered to be viable alternatives to conventional antibiotics. However, they rarely show systemic efficacy in animal models when added at non-toxic doses. The dimer A3-APO was designed to attack both the bacterial membrane and the Enterobacteriaceae-specific domain of the heat shock protein DnaK in order to reduce toxicity whilst maintaining activity. The peptide exhibited a minimal inhibitory concentration (MIC) range of 2-128 mg/L against 28 clinical Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium strains, with a median MIC of 30 mg/L. At this concentration, A3-APO was bactericidal to E. coli 5770, a fluoroquinolone-resistant extended-spectrum beta-lactamase-producing strain. The No Observed Adverse Effect Limit (NOAEL) at repeated intraperitoneal peptide administration was 20mg/kg. When administered at this dose three times starting immediately after E. coli Neumann infection, A3-APO cured 100% of mice in a standard bacteraemia model used by the pharmaceutical industry. In a more stringent assay, when treatment started after E. coli 5770 bacteraemia had already been established, three doses of 10mg/kg A3-APO prolonged early survival at a rate similar to that of imipenem and reduced the bacterial counts to base level. When the second assay was repeated in kidney clearance conditions resembling those in humans, 10mg/kg A3-APO was as efficacious as imipenem in the long-term. The increased in vivo efficacy compared with the in vitro bactericidal figures can potentially be explained by the generally observable immunostimulatory properties of antimicrobial peptides. Peptide A3-APO shows promising features as a member in our antibiotic arsenal against multidrug-resistant bacterial pathogens.

Extended-spectrum beta-lactamase-producing Klebsiella spp. in a neonatal intensive care unit: risk factors for the infection and the dynamics of the molecular epidemiology

The extended-spectrum beta-lactamase (ESBL)-producing Klebsiella spp. cause worldwide problems in intensive care units. The aim of this study was to investigate the molecular epidemiology of ESBL-producing Klebsiella pneumoniae and K. oxytoca strains in a neonatal intensive care unit (NICU) in Budapest, Hungary and to determine the risk factors of the infections and the epidemiological features. Infections with Klebsiella spp. were analyzed retrospectively by reviewing the medical records between January 2001 and December 2005. Antibiotic susceptibility tests, isoelectric focusing, pulsed field gel electrophoresis, plasmid analysis, PCR for blaTEM and blaSHV and DNA sequencing analysis were performed on ESBL-producing Klebsiella isolates. A total of 45 babies were found to be infected with non-ESBL-producing Klebsiella spp. and 39 with ESBL-producing Klebsiella spp. Of the parameters analyzed, including sex, gestational age, twin pregnancy, birth weight, presence of central vascular catheter, mechanical ventilator use, parenteral nutrition, polymicrobial infection, caesarean section, transfusion and mortality, we found no statistically significant difference between the ESBL and the non-ESBL groups, or between the K. pneumoniae and K. oxytoca species. Further characterization of the ESBL-producing K. pneumoniae and K. oxytoca strains isolated between February 2001 and January 2003 revealed three distinct PFGE patterns of SHV-5-producing K. pneumoniae (A, B, E) and two distinct patterns of SHV-12-producing K. oxytoca (C,D) isolates; these had different plasmid profiles. From July to November 2005, a new SHV-5 producing K. oxytoca (F) was isolated. The molecular epidemiology of ESBL-producing organisms in a NICU over time shows substantial shifts in predominant strains. The ESBL production of the infected organisms has an impact on the survival of newborn babies with infections caused by Klebsiella spp.

Broad-spectrum antimicrobial efficacy of peptide A3-APO in mouse models of multidrug-resistant wound and lung infections cannot be explained by in vitro activity against the pathogens involved.

Although the designer proline-rich antimicrobial peptide A3-APO has only modest activity against Escherichia coli and Acinetobacter baumannii in vitro, in mouse models of systemic and wound infections it shows superior efficacy compared with conventional antibiotics. In this study, the efficacy of A3-APO in several additional mouse models was investigated, including Staphylococcus aureus wound infection, mixed Klebsiella pneumoniae-A. baumannii-Proteus mirabilis wound infection and K. pneumoniae lung infection, mimicking blast wound infections, foot ulcers and ventilator-induced nosocomial infections, respectively. Whilst the peptide practically did not kill the strains in vitro, when administered intramuscularly or as an aerosol it significantly improved mouse survival and reduced bacterial counts at the infection site and in blood. In the lung infection study, the blood bacterial counts following A3-APO treatment were as low as after treatment with colistin and were lower than after treatment with imipenem or amikacin. The wounds of treated animals, unlike their untreated counterparts, lacked pus and signs of inflammation. In human peripheral blood mononuclear cells, A3-APO upregulated the expression of the anti-inflammatory cytokines interleukin-4 and interleukin-10 by four- to six-fold. One of the mechanisms mediating the in vivo protective effects might be the prevention of inflammation around bacterial infiltration.

Synthesis of osteotropic hydroxybisphosphonate derivatives of fluoroquinolone antibacterials

1-Hydroxybisphosphonate derivatives of ciprofloxacin, gatifloxacin and moxifloxacin have been synthesized using Cu(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction. The 1,2,3-triazol linked hydroxybisphosphonate derivative of ciprofloxacin exhibited antibacterial activity comparable to the parent antibiotic and all fluoroquinolone-bisphosphonates displayed osteotropic properties in a bone model.

Preclinical advantages of intramuscularly administered peptide A3-APO over existing therapies in Acinetobacter baumannii wound infections

OBJECTIVES The designer antibacterial peptide A3-APO is efficacious in mouse models of Escherichia coli and Acinetobacter baumannii systemic infections. Here we compare the efficacy of the peptide with that of imipenem and colistin in A. baumannii wound infections after burn injury. METHODS CD-1 mice were inflicted with burn wounds and different inocula of A. baumannii, isolated from an injured soldier, were placed into the wound sites. The antibiotics were given intramuscularly (im) one to five times. Available free peptide in the blood and the systemic toxicity of colistin and A3-APO were studied in healthy mice. RESULTS While toxicity of colistin was observed at 25 mg/kg bolus drug administration, the lowest toxic dose of A3-APO was 75 mg/kg. In the A. baumannii blast injury models, 5 mg/kg A3-APO improved survival and reduced bacterial counts in the blood as well as in the wounds and improved wound appearance significantly better than any other antibiotic treatment. The free peptide concentration in the blood did not reach 1 µg/mL. CONCLUSIONS Peptide A3-APO, with an intramuscular therapeutic index of 15, is more efficacious and less toxic than any existing burn injury infection therapy modality against multidrug-resistant Gram-negative pathogens. A3-APO administered by the im route probably binds to a biopolymer that promotes the peptides biodistribution.

In Vitro and In Vivo Activities of Amikacin, Cefepime, Amikacin plus Cefepime, and Imipenem against an SHV-5 Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Strain

ABSTRACT The in vitro and in vivo effectiveness of amikacin, cefepime, and imipenem was studied using a high inoculum of an extended-spectrum β-lactamase-producing Klebsiella pneumoniae strain. An in vitro susceptibility test at the standard inoculum predicted the in vivo outcome of amikacin or imipenem while it did not do so for cefepime due to the inoculum effect.

Diazo Transfer−Click Reaction Route to New, Lipophilic Teicoplanin and Ristocetin Aglycon Derivatives with High Antibacterial and Anti-influenza Virus Activity: An Aggregation and Receptor Binding Study

Semisynthetic, lipophilic ristocetin and teicoplanin derivatives were prepared starting from ristocetin aglycon and teicoplanin psi-aglycon (N-acetyl-D-glucosaminyl aglycoteicoplanin). The terminal amino functions of the aglycons were converted into azido form by triflic azide. Copper catalyzed 1,3-dipolar cycloaddition reaction with lipophilic alkynes resulted in the title compounds. Two of the teicoplanin derivatives showed very good MIC and MBC values against various Gram-positive bacteria, including vanA enterococci. The aggregation and interaction of a n-decyl derivative with bacterial cell wall components was studied. One of the lipophilic ristocetin derivatives displayed favorable anti-influenza virus activity.

Significance of methicillin–teicoplanin resistant Staphylococcus haemolyticus in bloodstream infections in patients of the Semmelweis University hospitals in Hungary

The purpose of this study was to quantify the impact of Staphylococcus haemolyticus in the epidemiology of the blood stream infection (BSI) and to characterize the rates and quantitative levels of resistance to antistaphylococcal drugs. During an eight-year period, 2967 BSIs of the patients hospitalized in different clinical departments of the Semmelweis University, Budapest, Hungary were analyzed. One hundred eighty-four were caused by S. haemolyticus, amounting to 6% of all infections. The antibacterial resistance of S. haemolyticus isolates was investigated by the broth microdilution method, vancomycin agar screen, population analysis profile and PCR for mecA, vanA and vanB genes detection. Epidemiological investigation was processed by determining phenotypic antibiotic resistance patterns and PFGE profiles. Extremely high MIC levels of resistance were obtained to oxacillin, erythromycin, clindamycin, gentamicin and ciprofloxacin. The incidence of teicoplanin reduced susceptibility revealed 32% without possessing either the vanA or vanB gene by the strains. PFGE revealed 56 well-defined genotypes indicating no clonal relationship of the strains. The propensity of S. haemolyticus to acquire resistance and its pathogenic potential in immunocompromised patients, especially among preterm neonates, emphasise the importance of species level identification of coagulase-negative staphylococci and routinely determine the MIC of proper antibacterial agents for these isolates.

Evaluation of the New Micronaut-Candida System Compared to the API ID32C Method for Yeast Identification

ABSTRACT A new system, Micronaut-Candida, was compared to API ID32C to identify 264 yeast (Candida albicans, C. parapsilosis, C. tropicalis, C. krusei, C. inconspicua, C. norvegensis, C. lusitaniae, C. guilliermondii, C. dubliniensis, C. pulcherrima, C. famata, C. rugosa, C. glabrata, C. kefyr, C. lipolytica, C. catenulata, C. neoformans, Geotrichum and Trichosporon species, Rhodotorula glutinis, and Saccharomyces cerevisiae) clinical isolates. Results were in concordance in 244 cases. Eighteen out of the 20 of discordant results were correctly identified by Micronaut-Candida but not by API ID32C, as confirmed by PCR ribotyping.