Béla Kocsis

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.

The Antibacterial Effect of a Proline-Rich Antibacterial Peptide A3-APO

Antimicrobial resistance is an emerging worldwide concern in light of the widespread antimicrobial drug use in humans, livestock and companion animals. The treatment of life-threatening infections is especially problematic because clinical strains rapidly acquire multiple-drug resistance. Antimicrobial peptides have long been considered to be viable alternatives to small molecule antibiotics. However, the peptides parenteral use is frequently hampered by inadequate safety margins and rapid renal clearance leaving them suitable only for topical applications. The proline-rich peptide A3-APO represents a family of a new class of synthetic dimers that kill bacteria by a dual mode of action and carry domains for interaction with both the bacterial membrane and an intracellular target. From a series of designer antibacterial peptides, A3-APO emerged as a viable preclinical candidate by virtue of its superior ability to disintegrate the bacterial membrane, inhibit the 70-kDa heat shock protein DnaK alone or in synergy with small molecule antibiotics, lack of eukaryotic toxicity and withstand proteolytic degradation in body fluids. As many other proline-rich peptides, A3-APO binds to the C-terminal helical lid of bacterial DnaK and inhibits chaperone-assisted protein folding in bacteria but not in mammalian Hsp70. In this review, the structure, pharmacokinetic properties, antimicrobial spectrum of peptide A3-APO and its in vivo metabolite are summarized and the in vitro and in vivo antimicrobial effects (antimicrobial susceptibilities, postantibiotic effects, resistance induction) are discussed in detail.

Identification of a blaVIM-4 gene in the internationally successful Klebsiella pneumoniae ST11 clone and in a Klebsiella oxytoca strain in Hungary

1 Clermont O, Lavollay M, Vimont S et al. The CTX-M-15-producingEscherichia coli diffusing clone belongs to a highly virulent B2phylogenetic subgroup. J Antimicrob Chemother 2008; 61: 1024–8.2 Nicolas-Chanoine MH, Blanco J, Leflon-Guibout V et al. Intercontinentalemergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15.J Antimicrob Chemother 2008; 61: 273–81.3 Johnson JR, Menard M, Johnston B et al. Epidemic clonal groups ofEscherichia coli as a cause of antimicrobial-resistant urinary tractinfections in Canada, 2002 to 2004. Antimicrob Agents Chemother2009; 53: 2733–9.4 Clinical and Laboratory Standards Institute. Performance Standardsfor Antimicrobial Susceptibility Testing: Seventeenth InformationalSupplement M100-17. CLSI, Wayne, PA, USA, 2007.5 Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination ofthe Escherichia coli phylogenetic group. Appl Environ Microbiol 2000; 66:4555–8.6 Clermont O, Dhanji H, Upton M et al. Rapid detection of theO25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J Antimicrob Chemother 2009; 64: 274–7.7 Sidjabat HE, Paterson DL, Adams-Haduch JM et al. Molecularepidemiology of CTX-M-producing Escherichia coli isolates at a tertiarymedical center in western Pennsylvania. Antimicrob Agents Chemother2009; 53: 4733–9.8 Lau SH, Cheesborough J, Kaufmann ME et al. Rapid identification ofuropathogenic Escherichia coli of the O25:H4-ST131 clonal lineageusing the DiversiLab repetitive sequence-based PCR system. ClinMicrobiol Infect 2009; 16: 232–7.

Polymer structure and antimicrobial activity of polyvinylpyrrolidone-based iodine nanofibers prepared with high-speed rotary spinning technique.

Poly(vinylpyrrolidone)/poly(vinylpyrrolidone-vinylacetate)/iodine nanofibers of different polymer ratios were successfully prepared by a high-speed rotary spinning technique. The obtained fiber mats were subjected to detailed morphological analysis using an optical and scanning electron microscope (SEM), while the supramolecular structure of the samples was analyzed by positron annihilation lifetime spectroscopy (PALS). The maximum dissolved iodine of the fiber samples was determined, and microbiological assay was carried out to test their effect on the bacterial growth. SEM images showed that the polymer fibers were linear, homogenous, and contained no beads. The PALS results, both the o-positronium (o-Ps) lifetime values and distributions, revealed the changes of the free volume holes of fibers as a function of their composition and the presence of iodine. The micro- and macrostructural characterisation of polymer fiber mats enabled the selection of the required composition from the point of their applicability as a wound dressing.

Fitness cost associated with resistance to fluoroquinolones is diverse across clones of Klebsiella pneumoniae and may select for CTX-M-15 type extended-spectrum β-lactamase

Lowered fitness cost associated with resistance to fluoroquinolones was recently demonstrated to influence the clonal dynamics of methicillin-resistant Staphylococcus aureus (MRSA) in the health care setting. We investigated whether or not a similar mechanism impacts Klebsiella pneumoniae. The fitness of K. pneumoniae isolates from major international hospital clones (ST11, ST15, ST147) already showing high-level resistance to fluoroquinolones and of strains from three minor clones (ST25, ST274, ST1028) in which fluoroquinolone resistance was induced in vitro was tested in a propagation assay. Strains from major clones showed significantly less fitness cost than three of four fluoroquinolone-resistant derivatives of minor clone isolates. In addition, plasmids with CTX-M-15 type extended-spectrum β-lactamase (ESBL) genes were all retained in both major and minor clone isolates, irrespective of the strains’ level of fluoroquinolone resistance, while each plasmid harboring SHV-type ESBLs had been lost during the induction of resistance. Major clone K. pneumoniae strains harbored more amino acid substitutions in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes than minor clone isolates. The presence of an active efflux system could be demonstrated in all fluoroquinolone-resistant derivatives of originally SHV-producing minor clone isolates but not in any CTX-M-15-producing strain. Further investigations are needed to expand and confirm our findings on a larger sample. In addition, a long-term observation of our ciprofloxacin-resistant minor clone isolates is required in order to elucidate whether or not they are capable of restoring their fitness while concomitantly retaining high minimum inhibitory concentration (MIC) values.

Description and plasmid characterization of qnrD determinants in Proteus mirabilis and Morganella morganii

We investigated the presence of qnrC and qnrD among 756 non-replicate Enterobacteriaceae isolated in Italy, selected for being non-susceptible to fluoroquinolones and/or resistant to third-generation cephalosporins. Four Proteus mirabilis and one Morganella morganii (0.66% of the total) presented a qnrD gene, located in a 2687-base-pair plasmid that was entirely sequenced. The plasmid is un-typable, and contains no known coding region other than qnrD. That the qnrD gene was found in four unrelated P. mirabilis and in one M. morganii isolate might suggest a frequent association of this gene with the tribe Proteeae.

First detection of plasmid-mediated, quinolone resistance determinants qnrA, qnrB, qnrS and aac(6′)-Ib-cr in extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in Budapest, Hungary

Sir, Quinolone resistance in Enterobacteriaceae usually results from mutations in genes coding for chromosomally encoded type II topoisomerases, efflux pumps or porin-related proteins. Recently, transferable, plasmid-borne, quinolone resistance genes—qnrA, qnrB, qnrS, cr variant of aac(60)-Ib, qepA and oxqB—have been observed in clinical isolates, more frequently among strains producing plasmid-mediated, extended-spectrum b-lactamases (ESBLs). The objective of this study was to determine the prevalence of qnrA, qnrB, qnrS and aac(60)-Ib-cr genes in ESBL-producing clinical isolates of 70 Escherichia coli, 101 Klebsiella pneumoniae, 5 Citrobacter freundii and 61 Enterobacter spp., isolated in two microbiological laboratories in Budapest by collecting samples from seven different hospitals and clinics from 2002 until 2006. The prevalence of ESBL production among Enterobacteriaceae was between 1% and 28% during this period. All of the ESBL-producing organisms were tested for the presence of qnrA, qnrB, qnrS and aac(60)-Ib genes by PCR. To distinguish the qnr gene alleles, restriction enzymes were used and the result was further confirmed by sequencing. The aac(60)-Ib-cr was identified by digestion with BstF5I. The prevalence of qnr determinants was relatively high among K. pneumoniae (8%) isolates and low in the case of E. coli (1.4%) and in Enterobacter spp. (0%). One ESBL-producing C. freundii isolate (out of five) was qnr-positive. qnrA1, qnrB2 and qnrS1 were detected and qnrA1 was the most prevalent (3%) (Table 1). Sixty-three (26.6%) of all ESBL-producing isolates were positive for aac(60)-Ib, of which 19 (8% of all)—16 K. pneumoniae and 3 E. coli—had the aac(60)-Ib-cr variant. None of the qnr-positive strains harboured the aac(60)-Ib-cr variant. The MICs of quinolones were determined by a broth microdilution method according to the CLSI. Conjugation assays were performed for all of the qnr-positive isolates. The qnrA1-positive strains—six K. pneumoniae and one E. coli— were resistant to nalidixic acid, norfloxacin, ciprofloxacin and levofloxacin with very high MIC values (Table 1). The conjugation assay, which was successful in three qnrA1-positive strains, confirmed that the presence of qnrA1 gene alone causes only low-level quinolone resistance. The MIC values for the transconjugants were more than 8-fold higher for nalidixic acid, 132-fold higher for norfloxacin and 66to 132-fold higher for ciprofloxacin, compared with E. coli J53 Rif (nalidixic acid, 4 mg/L; norfloxacin, 0.06 mg/L; and ciprofloxacin, 0.12 mg/L). However, the transconjugants still showed only low-level quinolone resistance, suggesting that the high-level quinolone resistance in the original isolates may be related to the coordinating action of several resistance mechanisms. The qnrB gene was detected in two isolates: one K. pneumoniae and one C. freundii. K. pneumoniae M95 harboured both qnrA1 and qnrB2 genes, but only qnrA1 was transferable by conjugation, suggesting that the two genes have different genetic backgrounds. The ciprofloxacin and levofloxacin MIC values were the highest in K. pneumoniae M95, compared with the other qnrA1-positive strains, which might be explained by the parallel qnrA1 and qnrB2 production of the original isolate. The transfer of qnrA1 alone did not result in transconjugants with greatly altered quinolone susceptibility. The C. freundii M141 harbouring only the qnrB2 gene was susceptible to the fluoroquinolones tested with MICs at least 8-fold lower than the susceptibility breakpoints recommended by the CLSI. Other authors have also observed that QnrB is present in isolates with a wide range of quinolone MIC values, including full susceptibility. The wide range of MIC values for the qnr-positive strains highlights that the detection of the qnr genes in clinical strains might be very difficult phenotypically. The qnrS1 gene was detected only in a K. pneumoniae strain, which was resistant to most of the tested fluoroquinolones. However, the norfloxacin (16 mg/L) and ciprofloxacin (4 mg/L) MIC values were the breakpoints recommended by the CLSI. The levofloxacin MIC value (4 mg/L) was between the two CLSI breakpoints (8 and 2 mg/L). Of the 19 aac(60)-Ib-cr-positive isolates, 6 (32%) were susceptible to both ciprofloxacin and levofloxacin. The presence of aac(60)-Ib-cr does not result in large increases in MIC values of quinolones; however, it substantially increases the frequency of selection of chromosomal mutants. The characteristics of the b-lactamase enzymes—isoelectric points, PCR and sequencing data—produced by the qnr-positive strains were determined. In our study, the presence of qnrA1 gene was connected to SHV-5, SHV-12 and CTX-M-15 enzymes (Table 1). The presence of qnrB2 was connected with SHV-12. We could not determine the genotype of the ESBL enzyme in the qnrB2-positive C. freundii strain. The qnrS1 gene was found together with SHV-2 ESBL. Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkn206

The Renaissance of Polymyxins

Polymyxins are polypeptide antibiotics, with a primary effect of membrane damaging due to their selective binding to the lipopolysaccharide of Gram-negative bacteria. Their nephro- and neurotoxic side effects limited their use, however, in the last decade the emergence of multidrug-resistant Gram-negative bacteria led to the reintroduction of polymyxins into clinical practice. This review provides an overview about the history and the latest developments of polymyxins. We describe the antimicrobial effects, pharmacodynamics, pharmacokinetics and different routes of administration. We highlight natural classic polymyxins, namely polymyxin B and E, the non-classic agents polymyxin M, S and T. Novel polymyxin chemical structure derivatives will be listed including NAB739, NAB740, NAB741 and NAB7061, that can have important therapeutical role in the future.

Dibromodulcitol-based combined postoperative chemotherapy of malignant astrocytomas and glioblastomas

SummaryContinuing our earlier studies with dibromodulcitol (DBD), in a series of 38 evaluable consecutive patients who were operated on for malignant supratentorial gliomas, radiotherapy with smaller daily but higher total doses of DBD has been started 3–5 weeks after surgery. This was followed alternately by a combination chemotherapy of CCNU and DBD or CCNU and Procarbazine. No severe myelotoxicity occurred. Survivals were compared with a group of patients who got irradiation alone. Statistical analysis showed a significantly better survival in the presently treated group: median survival was 55 weeks, p=0.02. These values were very similar to those groups which were treated by intermittent DBD schedule during irradiation. This study seems to confirm our previous suggestion that the concurrent use of DBD during irradiation might be an important factor in improving survival times.