In vitro activity of RU 64004, a new ketolide antibiotic, against gram-positive bacteria

The comparative in vitro activity of RU 64004 (also known as HMR 3004), a new ketolide antibiotic, was tested by agar dilution against approximately 500 gram-positive organisms, including multiply resistant enterococci, streptococci, and staphylococci. All streptococci were inhibited by < or = 1 microg of RU 64004 per ml. The ketolide was more potent than other macrolides against erythromycin A-susceptible staphylococci and was generally more potent than clindamycin against erythromycin A-resistant strains susceptible to this agent. Clindamycin-resistant staphylococci (MIC, > 128 microg/ml) proved resistant to the ketolide, but some erythromycin A- and clindamycin-resistant enterococci remained susceptible to RU 64004.     

Antimicrobial activity of RU-66647, a new ketolide

A new macrolide subclass called ketolides, possess a mode of action similar to the macrolide-lincosamide-streptogramin (MLS) compounds. Utilizing reference in vitro tests, the in vitro activity of RU-66647 (a ketolide) was compared to other MLS compounds against 376 Gram-positive organisms and over 400 representative strains of Gram-negative bacilli. The ketolide's spectrum was most similar to clindamycin and an earlier drug in the series (RU-64004 or RU-004) against staphylococci and streptococci. However, RU-66647 was more active than erythromycin and azithromycin against oxacillin-resistant Staphylococcus spp. and vancomycin-resistant enterococci. Ketolide activity was more potent than other MLS drugs against vancomycin-susceptible enterococci (MIC90, 0.25-4 micrograms/ml) and all streptococci (MICs, < or = 0.25 microgram/ml). Erythromycin-resistant (constitutive) strains were generally inhibited by < or = 2 micrograms RU-66647/ml (staphylococci, 31 to 36%; streptococci, 100%; enterococci, 72%). RU-66647 was active against Haemophilus influenzae (MIC90, 2 micrograms/ml), Moraxella catarrhalis (MIC90, 0.12 microgram/ml), and pathogenic Neisseria spp. (MIC90 0.5 microgram/ml). The ketolide failed to inhibit Enterobacteriaceae, nonfermentative Gram-negative bacilli, and Bacteriodes fragilis group strains. RU-66647 was observed to be a promising new compound directed toward some organisms resistant to other MLS-class drugs.     

Immunochemical analysis of an unusual cell wall polysaccharide from animal coagulase-positive staphylococci. 1. Fragments obtained after hydrolysis in hydrofluoric acid and alkali

Polysaccharide P (poly P) of canine coagulase-positive staphylococci contains glycerol, glucose, glucosamine, muramic acid, phosphate, and the usual peptidoglycan amino acids, but does not cross-react serologically with standard teichoic acids. Products from hydrolyses in hydrofluoric acid and alkali contained phosphates of glycerol and glucose as well as combinations of these, but neither glucosyl-glycerol units nor glucosamine-phosphates were observed. The teichoic acid of poly P is probably a polymer of a repeating unit consisting of alternating glycerol, phosphate and glucose.     

Regulation of D-alanine carboxypeptidase and peptidoglycan cross-linkage in amino acid-deprived Escherichia coli

The effect of amino acid deprivation on the activities of D-alanine carboxypeptidase (CPase) and peptidoglycan transpeptidase in Escherichia coli was determined. Enzymes were assayed in ether-treated bacteria (ETB) which were permeable to peptidoglycan nucleotide precursors. ETB were prepared at intervals from cultures grown in the presence and absence of a required amino acid. The specific activity of CPase in ETB decreased 50 to 85% during amino acid deprivation. This was paralleled by a 60 to 70% decrease in the specific activity of peptidoglycan transpeptidase. Both enzymes reached their lowest level of activity about 40 min after the onset of amino acid deprivation. The decrease in CPase activity apparently was not due to degradation of the enzyme, since full activity was restored after disruption of ETB by sonication. A decrease in CPase activity was associated with an enhancement of transpeptidation. The peptidoglycan synthesized in vitro by amino acid-deprived ETB was 1.7 times more cross-linked than the peptidoglycan synthesized by control ETB These results support the proposal that CPase may be involved in regulating transpeptidation in E. coli.     

Identification of two binding domains, one for peptidoglycan and another for a secondary cell wall polymer, on the N-terminal part of the S-layer protein SbsB from Bacillus stearothermophilus PV72/p2

First studies on the structure-function relationship of the S-layer protein from B. stearothermophilus PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP). The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain. The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content.     

Deletion analysis of MotA and MotB, components of the force-generating unit in the flagellar motor of Salmonella

MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.     

Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs

The monofunctional penicillin-binding DD-peptidases and penicillin-hydrolyzing serine beta-lactamases diverged from a common ancestor by the acquisition of structural changes in the polypeptide chain while retaining the same folding, three-motif amino acid sequence signature, serine-assisted catalytic mechanism, and active-site topology. Fusion events gave rise to multimodular penicillin-binding proteins (PBPs). The acyl serine transferase penicillin-binding (PB) module possesses the three active-site defining motifs of the superfamily; it is linked to the carboxy end of a non-penicillin-binding (n-PB) module through a conserved fusion site; the two modules form a single polypeptide chain which folds on the exterior of the plasma membrane and is anchored by a transmembrane spanner; and the full-size PBPs cluster into two classes, A and B. In the class A PBPs, the n-PB modules are a continuum of diverging sequences; they possess a five-motif amino acid sequence signature, and conserved dicarboxylic amino acid residues are probably elements of the glycosyl transferase catalytic center. The PB modules fall into five subclasses: A1 and A2 in gram-negative bacteria and A3, A4, and A5 in gram-positive bacteria. The full-size class A PBPs combine the required enzymatic activities for peptidoglycan assembly from lipid-transported disaccharide-peptide units and almost certainly prescribe different, PB-module specific traits in peptidoglycan cross-linking. In the class B PBPs, the PB and n-PB modules cluster in a concerted manner. A PB module of subclass B2 or B3 is linked to an n-PB module of subclass B2 or B3 in gram-negative bacteria, and a PB module of subclass B1, B4, or B5 is linked to an n-PB module of subclass B1, B4, or B5 in gram-positive bacteria. Class B PBPs are involved in cell morphogenesis. The three motifs borne by the n-PB modules are probably sites for module-module interaction and the polypeptide stretches which extend between motifs 1 and 2 are sites for protein-protein interaction. The full-size class B PBPs are an assortment of orthologs and paralogs, which prescribe traits as complex as wall expansion and septum formation. PBPs of subclass B1 are unique to gram-positive bacteria. They are not essential, but they represent an important mechanism of resistance to penicillin among the enterococci and staphylococci. Natural evolution and PBP- and beta-lactamase-mediated resistance show that the ability of the catalytic centers to adapt their properties to new situations is limitless. Studies of the reaction pathways by using the methods of quantum chemistry suggest that resistance to penicillin is a road of no return.     

Quinupristin/dalfopristin: spectrum of activity, pharmacokinetics, and initial clinical experience

In recent years, the prevalence of multiple drug-resistant strains of common Gram-positive pathogens has grown in many regions of the world. Increasingly, methicillin-resistant Staphylococcus aureus, coagulase-negative staphylococci, vancomycin-resistant enterococci, and penicillin-resistant pneumococci have been identified as causative organisms in serious and life-threatening infections. This increase in resistance highlights the need for new antimicrobial agents to expand the therapeutic armamentarium. Quinupristin/dalfopristin is the first of a unique class of antibiotics called streptogramins. It is characterized by a unique mechanism of action, intracellular activity, synergistic activity of its components, broad spectrum of activity against most Gram-positive cocci, common respiratory pathogens, and anaerobes, and demonstrated postantibiotic effect. Clinical evidence to date indicates that quinupristin/dalfopristin may be effective for the treatment of multidrug-resistant infections, especially those due to vancomycin-resistant enterococci and methicillin-resistant staphylococci. This article reviews the pharmacology, microbiology, and clinical experience with quinupristin/dalfopristin to date.     

A new method of inference of ancestral nucleotide and amino acid sequences

A statistical method was developed for reconstructing the nucleotide or amino acid sequences of extinct ancestors, given the phylogeny and sequences of the extant species. A model of nucleotide or amino acid substitution was employed to analyze data of the present-day sequences, and maximum likelihood estimates of parameters such as branch lengths were used to compare the posterior probabilities of assignments of character states (nucleotides or amino acids) to interior nodes of the tree; the assignment having the highest probability was the best reconstruction at the site. The lysozyme c sequences of six mammals were analyzed by using the likelihood and parsimony methods. The new likelihood-based method was found to be superior to the parsimony method. The probability that the amino acids for all interior nodes at a site reconstructed by the new method are correct was calculated to be 0.91, 0.86, and 0.73 for all, variable, and parsimony-informative sites, respectively, whereas the corresponding probabilities for the parsimony method were 0.84, 0.76, and 0.51, respectively. The probability that an amino acid in an ancestral sequence is correctly reconstructed by the likelihood analysis ranged from 91.3 to 98.7% for the four ancestral sequences.     

Sequence and cognitive analyses of two virulence-associated markers of bluetongue virus serotype 17

Genome segments 2 and 3 were completely sequenced for one virulent and one avirulent bluetongue serotype 17 (BLU-17). These two segments were previously shown to exhibit virulence-associated markers. The marker on segment 2 was characterized as a change in the neutralization domain on its protein product, VP2. The nucleotide sequences for segments 2 were 94.5% identical, and their predicted proteins differed by 34 amino acids or 3.7%. Three clusters of variability were identified which may be involved with viral neutralization. These variable regions were compared to mutations for published monoclonal antibody-resistant variants of BLU. The marker on segment 3 was characterized as a mobility shift in polyacrylamide gel electrophoresis (PAGE). The nucleotide sequences were 95.0% identical, and their predicted proteins differed by four amino acids or 0.4%. These amino acid changes were relatively conserved; therefore, they are not likely responsible for virulence. The segment 3 sequences were compared to published sequences, and evidence was found to suggest that the virulent isolate had naturally reassorted between a BLU-17 and BLU-10 isolate.     

Isolated systolic hypertension: an important cardiovascular risk factor

BACKGROUND: The rising number of vancomycin-resistant enterococci (VREs) is a major concern to modern medicine because vancomycin is currently the 'last resort' drug for life-threatening infections. The D-alanyl-D-X ligases (where X is an hydroxy or amino acid) of bacteria catalyze a critical step in bacterial cell-wall peptidoglycan assembly. In bacteria that produce glycopeptide antibiotics and in opportunistic pathogens, including VREs, D-, D-ligases serve as switches that confer antibiotic resistance on the bacteria themselves. Peptidoglycans in vancomycin-sensitive bacteria end in D-alanyl-D-alanine, whereas in vancomycin-resistant bacteria they end in D-alanyl-D-lactate or D-alanyl-D-serine. RESULTS: We demonstrate that the selective utilization of D-serine by the Enterococcus casseliflavus VanC2 ligase can be altered by mutagenesis of one of two residues identified by homology to the X-ray structure of the Escherichia coli D-alanyl-Dalanine ligase (DdlB). The Arg322-->Met (R322M) and Phe250-->Tyr (F250Y) ligase mutants show a 36-44-fold decrease in the use of D-serine, as well as broadened specificity for utilization of other D-amino acids in place of D-serine. The F250Y R322M double mutant is effectively disabled as a D-alanyl-D-serine ligase and retains 10% of the catalytic activity of wild-type D-alanyl-D-alanine ligases, reflecting a 6,000-fold switch to the D-alanyl-D-alanine peptide. Correspondingly, the Leu282-->Arg mutant of the wild-type E. coli DdlB produced a 560-fold switch towards D-alanyl-D-serine formation. CONCLUSIONS: Single-residue changes in the active-site regions of D-, D-ligases can cause substantial changes in recognition and activation of hydroxy or amino acids that have consequences for glycopeptide antibiotic efficacy. The observations reported here should provide an approach for combatting antibiotic-resistant bacteria.     

Desiderata for product labeling of medical expert systems

The ability of seventy clinical laboratories in nine European countries to detect glycopeptide resistance in Gram-positive bacteria was investigated. Results of routine tests were compared with those on the same strains by a reference method in national co-ordinating laboratories. In addition, control strains were tested by some of the participants. Errors in reporting susceptibility of Staphylococcus aureus to teicoplanin and vancomycin and coagulase-negative staphylococci to vancomycin were < 1%. With coagulase-negative staphylococci however, 44 (3.4%) teicoplanin susceptible isolates were reported intermediate and six (0.4%) resistant; 18 (58.1%) of 31 teicoplanin intermediate isolates were reported susceptible and five (16.1%) resistant; and six of nine teicoplanin resistant isolates were reported susceptible and two intermediate. All seven isolates of enterococci intermediate to vancomycin were reported susceptible. Distribution of a known vancomycin intermediate strain of E. gallinarum indicated problems with vancomycin susceptibility testing (44.4% reported susceptible, 32.7% intermediate, 32.1% resistant) and identification (only 34.1% correct) of this organism. Two of 28 teicoplanin resistant enterococci and three of 30 vancomycin resistant isolates were reported susceptible. Among other organisms, one resistant Lactobacillus sp. was reported susceptible to teicoplanin and vancomycin. In reporting teicoplanin susceptible organisms, there were fewer errors with comparative/Stokes methods than with most other methods and more errors with the ATB and Sceptor methods than most other methods. None of the methods used were reliable for testing teicoplanin intermediate and resistant coagulase-negative staphylococci or low-level vancomycin resistant enterococci. Alternative methods, such as breakpoint screening, should be considered for detecting glycopeptide resistance.     

The femR315 gene from Staphylococcus aureus, the interruption of which results in reduced methicillin resistance, encodes a phosphoglucosamine mutase

The femR315 gene was recently identified by Tn551 insertional mutagenesis as one of the new auxiliary genes, the alteration of which resulted in a drastically reduced methicillin resistance of the Staphylococcus aureus strain COL. femR315 (also known as femD) theoretically encoded a protein of 451 amino acids showing significant amino acid sequence homology with phosphoglucomutases and similar enzymes catalyzing the isomerization of hexoses and hexosamine phosphates (S. Wu, H. de Lencastre, A. Sali, and A. Tomasz, Microb. Drug Resist. 2:277-286, 1996). We describe here the overproduction and purification of the FemR315 protein as well as its identification as the phosphoglucosamine mutase which catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, the first step in the reaction sequence leading to the essential peptidoglycan precursor UDP-N-acetylglucosamine. On the basis of these findings, we propose to change the names femR315 and femD to the functionally more appropriate name glmM.     

Water-borne hepatitis E virus epidemic in Islamabad, Pakistan: a common source outbreak traced to the malfunction of a modern water treatment plant

Following the digestion of chromosomal DNA of Deinococcus radiodurans with a restriction enzyme a partial genomic library was constructed using lambda phage as a vector. A phage clone whose DNA can complement the deficiency in a radiation-sensitive mutant of D. radiodurans was isolated. Following the subcloning using phasmid vector, a hybrid plasmid containing 1.2 kb inserted DNA was obtained. After the determination of nucleotide sequence, the deduced amino acid sequence showed close homology to RuvB protein of Escherichia coli; approximately 81% of the amino acids (310 residues in total) was homologous (152 were identical and 100 amino acids were similar). The putative protein has a conserved ATP binding domain characteristic of DNA helicases. However, we could not find an SOS promoter and ORF for RuvA protein in the sequence upstream of ruvB in contrast to the E. coli homologue. The mutant was transformed with exogenous DNA at the same rate as the wild-type cells, but it was moderately sensitive to UV, gamma-rays and to interstrand cross-linking reagents.     

Antimicrobial effect of enterocin CCM 4231 in the cattle slurry environment

The antagonistic effect of enterocin CCM 4231 towards enterococci, staphylococci, Escherichia coli, listeriae and pseudomonads in the cattle slurry environment was assessed during periods of 1 and 2 weeks. The maximum decrease in the viable cells of enterococci and staphylococci (5.39 to 1.1 log CFU ml-1, and 4.3 to 2.3 log CFU ml-1, respectively) was detected on the second day after enterocin CCM 4231 addition to cattle slurry. E. coli cells, listeriae and pseudomonads decreased insignificantly. After 1 week, enterococci were completely inhibited. Staphylococci were suppressed by reaching a 1.8 log CFU ml-1 difference between the experimental and the control samples. A stable suppressive effect of enterocin CCM 4231 on the growth of listerial cells became significant with 2.59 log CFU ml-1 between the experimental and the control samples in the second week of bacteriocin addition. This was demonstrated in an experiment with enterocin addition to slurry which was sterilized and then inoculated with Listeria monocytogenes Ohio culture. Further possibilities of using bacteriocins for the treatment of animal waste are discussed.     

Activities of the semisynthetic glycopeptide LY191145 against vancomycin-resistant enterococci and other gram-positive bacteria

LY191145 is the prototype of a series of compounds with activities against vancomycin-resistant enterococci derived by modification of the glycopeptide antibiotic LY264826. LY191145 had MICs for vancomycin- and teicoplanin-resistant enterococci of < or = 4 micrograms/ml for 50% of isolates and < or = 16 micrograms/ml for 90% of isolates. Its MICs for vancomycin-resistant, teicoplanin-susceptible enterococci were 1 to 8 micrograms/ml. LY191145 retains the potent activities of its parent compound against staphylococci and streptococci. In vivo studies in a mouse infection model confirmed these activities. This compound indicates the potential of semisynthetic glycopeptides as agents against antibiotic-resistant gram-positive bacteria.     

Isolation of endothelial cells from human umbilical cords and development of low-cost culture medium

A one-day point prevalence of infection analysis was undertaken in 1417 intensive care units (ICUs) (10,038 patients) in 17 western European countries. The prevalence of ICU-acquired infection was 20.6% (2064 patients), representing almost half the cases of infection. Pneumonia was the most commonly reported infection (46.9%), followed by infection of the lower respiratory tract (17.8%), urinary tract (17.6%), and blood (13.0%). Staphylococcus aureus was the most frequently isolated organism (30.1%), followed by Pseudomonas aeruginosa (28.7%), coagulase-negative staphylococci (19.1%), yeasts (17.1%), and enterococci (11.7%). As a group, the Enterobacteriaceae were the most commonly isolated organisms (34.4%). The study also revealed that resistance to antimicrobial agents is common among Staphylococcus aureus, Pseudomonas aeruginosa, and coagulase-negative staphylococci.     

Recovery of Streptococcus mutans after amino acid deprivation

The recovery of Streptococcus mutans FA-1 in a complete, chemically defined medium was examined after 1, 3, and 6 h of essential amino acid deprivation. Amino acids could be divided into two groups based on their effect on the relative rates of recovery: those amino acids (leucine and cystine) that are precursors of protein only, and amino acids (glutamate/glutamine or lysine) that are incorporated into both protein and cell wall peptidoglycan. Culture turbidity, deoxyribonucleic acid, ribonucleic acid, protein and cell wall peptidoglycan measurements indicated rapid recovery after leucine/cystine starvation periods. However, a 6-h leucine/cystine deprivation resulted in a slower exponential rate of growth (180-min doubling time compared to the normal doubling time of 85 to 90 min) after recovery. Glutamate/glutamine starvation, on the contrary, resulted in greatly extended recovery periods, especially after 3- and 6-h amino acid deprivations. Macromolecular synthesis was most severely affected by 6-h glutamate/glutamine starvation and required 6 to 10 h for recovery of an exponential rate. A delay in the recovery of deoxyribonucleic acid and cell wall peptidoglycan synthesis beyond that of the other macromolecules was observed after 1 and 3 h of deprivation with either leucine/cystine or glutamate/glutamine. However, after a 6-h amino acid deprivation, deoxyribonucleic acid synthesis recovered more rapidly than that of the other macromolecules studied. The results are discussed in terms of the nutritional environment of the oral cavity and its effect on the growth and survival of S. mutans.