A new rapid and cost-effective method for detection of phages, ICEs and virulence factors encoded by Streptococcus pyogenes

Streptococcus pyogenes (group A Streptococcus, GAS) is a human pathogen that causes diseases of various intensity, from mild strep throat to life threatening invasive infections and postinfectional sequelae. S. pyogenes encodes multiple, often phage encoded, virulence factors and their presence is related to severity of the disease. Acquisition of mobile genetic elements, carrying virulence factors, as phages or ICEs (integrative and cojugative elements) has been shown previously to promote selection of virulent clones. We designed the system of eight low volume multi- and one singleplex PCR reactions to detect genes encoding twenty virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) and twenty one phage and ICE integration sites described so far for S. pyogenes. Classification of strains based on the phage and virulence factors absence or presence, correlates with PFGE MLST and emm typing results. We developed a novel, fast and cost effective system that can be used to detect GAS virulence factors. Moreover, this system may become an alternative and effective system to differentiate between GAS strains.     

Cyclic enkephalin-deltorphin hybrids containing a carbonyl bridge: structure and opioid activity

Six hybrid N-ureidoethylamides of octapeptides in which an N-terminal cyclic structure related to enkephalin was elongated by a C-terminal fragment of deltorphin were synthesized on MBHA resin. The synthetic procedure involved deprotection of Boc groups with HCl/dioxane and cleavage of the peptide resin with 45 % TFA in DCM. d-Lys and d-Orn were incorporated in position 2, and Lys, Orn, Dab, or Dap in position 5. The side chains of the dibasic amino function were protected with the Fmoc group. This protection was removed by treatment with 55 % piperidine in DMF, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate. Using various combinations of dibasic amino acids, peptides containing a 17-, 18-, 19- or 20-membered ring structure were obtained. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid activities were observed, depending on the size of the ring. Extension of the enkephalin sequence at the C-terminus by a deltorphin fragment resulted in a change of receptor selectivity in favor of the δ receptor. The conformational propensities of selected peptides were determined using the EDMC method in conjunction with data derived from NMR experiments carried out in water. This approach allowed proper examination of the dynamical behavior of these small peptides. The results were compared with those obtained earlier with corresponding N-(ureidoethyl)pentapeptide amides.     

Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives

We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.

Methods

ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla _CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla _CTX-M-94 and bla _CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla _CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.

Results

The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla _CTX-M-94 and bla _CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.

Conclusions

This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.     

Tryptic hydrolysis of hGH-RH(1-29)-NH2 analogues containing Lys or Orn in positions 12 and 21.

Two analogues of the 29 amino acid sequence of human growth hormone-releasing hormone, namely [Nle27]hGH-RH(1-29)-NH2 and [Orn(12,21),Nle27]hGH-RH(1-29)-NH2, have been synthesized and subjected to digestion by trypsin. The course of degradation was followed using RP-HPLC and ESI-MS. Several intermediates and final products of degradation were identified and conclusions regarding the rate of cleavages at different positions occupied by Lys and Arg residues were drawn. The analogue containing ornithine was found to be less susceptible to hydrolysis by trypsin: the 12-13 and 21-22 peptide bonds were completely resistant to the cleavage. The results show that by replacing Lys with Orn, a possibility exists to design new peptides, which could be more stable in biological fluids.     

The Social Burden of Resilience: A Historical Perspective

We examine the social burden associated with resilience to environmental shocks in pre-modern societies. We argue that analyses of state-level interventions to mitigate the consequences of catastrophic events tend to isolate these measures from their larger social contexts and thereby overlook the uneven distribution of their burden across different groups. We use three cases of pre-modern societies in the northeastern Mediterranean - the sixth century Roman Empire, the tenth century Byzantine Empire, and the sixteenth century Ottoman Empire. We demonstrate how the adaptive processes that reinforced resilience at the state level incurred different burdens for those at lower levels of the social hierarchy. We found that some groups sustained losses while others gained unexpected benefits in the context of temporary systemic instability. We also found that although elites enjoyed enhanced buffers against the adverse effects in comparison with non-elites, this did not consistently guarantee them a better outcome. We conclude that the differentiated burden of resilience could in some cases entrench existing political or economic configurations, and in other cases, overturn them. Our case studies indirectly address the pressing issue of environmental justice.     

Multilocus variable number tandem repeat analysis (MLVA) of Streptococcus pyogenes

We tested 21 polymorphic loci encoded by the genome of Streptococcus pyogenes (group A Streptococcus, GAS). Seven of them were chosen for the MLVA scheme. The primer pairs, designed for selected loci, detect from few to several alleles, and the method has a Simpson's Index of diversity of 0.957. To test the overall performance of the method, multiplex PCR reactions were carried out for over 700 GAS strains. Using the method we were able to detect differences between highly clonal strains that share the same emm, MLST and PFGE types. The most diverse strains were M4, M2, M3 and M28.We developed a typing method that can be employed to differentiate between GAS strains. The method has high resolution and measures diversity of the GAS core chromosome, on the contrary to methods such as PFGE.     

Synthesis of peptidomimetics: An evaluation of the p-nitrophenyl carbamate of ethylenediamine

Summary The application of p-nitrophenyl carbamate of Boc-ethylenediamine for the solid-phase synthesis of peptidomimetrics was examined. The per step yield of coupling was estimated using mass spectrometry, based on the repeated coupling of the same monomer in the synthesis of alkylurea oligomers. Introduction of the urea moiety at the terminus of the chain adjacent to the resin was accomplished by the use of the BHA resin in the assembly of the chain and liquid HF in the cleavage step. In addition, an alkylurea oligomer was treated with bis(p-nitrophenyl) carbonate followed by ammonolysis in order to obtain a urea moiety at the terminus distant from the resin. Aside from the expected oligomer, a product was obtained in which the terminal alkylurea had undergone cyclization. Finally, four peptidomimetics, analogues of 1–4 enkephalin fragment, containing up to four alkylurea units instead of glycine residues, were synthesized. Two of these peptidomimetics were examined for opioid activity and turned out to be active in the guinea pig ileum assay.     

Synthesis and biological activity of homoarginine-containing opioid peptides.

Two tris-alkoxycarbonyl homoarginine derivatives, Boc-Har{omega,omega'-[Z(2Br)]2}-OH and Boc-Har{omega,omega'-[Z(2Cl)]2}-OH, were prepared by guanidinylation of Boc-Lys-OH, and used for the synthesis of neo-endorphins and dynorphins. The results were compared with that obtained in the synthesis in which Boc-Lys(Fmoc)-OH was incorporated into the peptide chain, and after removing Fmoc protection, the resulting peptide-resin was guanidinylated with N,N'-[Z(2Br)]2- or N,N'-[Z(2Cl)]2-S-methylisourea. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. The results indicated that replacement of Arg by Har may be a good avenue for the design of biologically active peptides with increased resistance to degradation by trypsin-like enzymes.     

Synthesis of a novel side-chain to side-chain cyclized enkephalin analogue containing a carbonyl bridge

A novel type of cyclic opiod peptide analogue, cyclo(N^ϵ,N^ϵ′-carbonyl- D -Lys²,Lys⁵)enkephalinamide, was prepared from a linear precursor peptide. The peptide was synthesized on the Merrifield resin and also by a combination of the solid-phase technique and the classical method in solution. In both cases the cyclization was performed by reaction of bis(4-nitrophenyl)carbonate with the free side-chain amino groups of the two lysine residues. The described method permits the convenient preparation of novel peptide analogues cyclized via a ureido group incorporating the side-chain amino groups of two α,ω-diamino acid residues. The cyclic enkephalin analogue containing a 21-membered ring structure showed preference for μ over δ opioid receptors in opioid bioassays in vitro. © 1997 European Peptide Society and John Wiley & Sons, Ltd.