Pharmacodynamic modelling of ß-lactam/ß-lactamase inhibitor checkerboard data: illustration with aztreonam-avibactam.

OBJECTIVES: Checkerboard experiments followed by fractional inhibitory concentration (FIC) index determinations are commonly used to assess in vitro pharmacodynamic interactions between combined antibiotics, but FIC index cannot be determined in case of antibiotic/non-active compound combinations. The aim of this study was to use a simple modelling approach to quantify the in vitro activity of aztreonam-avibactam, a new ß-lactam-ß-lactamase inhibitor combination. METHODS: MIC checkerboard experiments were performed with 12 Enterobacteriaceae with diverse ß-lactamases profiles. Aztreonam MICs in the absence and presence of avibactam at different concentrations (ranging from 0.0625 to 4 mg/L) were determined. Aztreonam MIC versus avibactam concentrations were fitted by an inhibitory Emax model with a baseline effect parameter. RESULTS: A concentration-dependent relationship was observed with a steep initial reduction of aztreonam MIC at low avibactam concentrations and reaching a maximum at higher avibactam concentrations that was adequately fitted by the model. Maximum avibactam effect was characterized by the ratio of aztreonam MICs in the absence of avibactam (MIC0) and when avibactam concentration tends toward infinity (MIC∞), and this ratio ranged between 90 and 10 068 depending on the strain. Avibactam potency was characterized by avibactam concentrations corresponding to 50% of the maximum effect (IC50 values between 0.00022 and 0.053 mg/L). CONCLUSIONS: An inhibitory Emax model with a baseline effect could quantify maximum avibactam effect and potency among various strains. This simple modelling approach can be used to compare the activity of other combinations of antibiotics with non-antibiotic drugs when FIC index is inappropriate.

Pyrazolopyrimidinediones are selective agents for Helicobacter pylori that suppress growth through inhibition of glutamate racemase (MurI).

Pyrazolopyrimidinediones are a novel series of compounds that inhibit growth of Helicobacter pylori specifically. Using a variety of methods, advanced analogues were shown to suppress the growth of H. pylori through the inhibition of glutamate racemase, an essential enzyme in peptidoglycan biosynthesis. The high degree of selectivity of the series for H. pylori makes these compounds attractive candidates for novel H. pylori-selective therapy.

Helicobacter pylori physiology predicted from genomic comparison of two strains.

Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.

Structural characterization of an abnormally cross-linked muropeptide dimer that is accumulated in the peptidoglycan of methicillin- and cefotaxime-resistant mutants of Staphylococcus aureus.

Laboratory mutants of Staphylococcus aureus strain ATCC 8325 (27S) selected for increased minimal inhibitory concentration (MIC) values to methicillin and cefotaxime showed increased rates of cell wall turnover and detergent-induced autolysis in virtual parallel with the increasing MIC for the antibiotic. Also in parallel with the increasing MICs for the particular antibiotic used in the selection was the gradual accumulation of an unusual muropeptide in the peptidoglycan of the mutants, muropeptide 12, which is a minor component of the cell wall of the parental strain. Analysis of muropeptide 12, its peptide derivative, and its lysostaphin degradation products by high pressure liquid chromatography, Edman degradation, and mass spectrometry suggests that muropeptide 12 is a dimer in which the two monomeric components are interlinked by two pentaglycyl cross-bridges, thus generating a 14-member macrocyclic ring structure. This unusual cross-linked structure may be the product of the abnormal activity of penicillin-binding protein 2 which has grossly reduced antibiotic binding capacity in the mutant staphylococci.

Cloning of the Staphylococcus aureus ddh gene encoding NAD+-dependent D-lactate dehydrogenase and insertional inactivation in a glycopeptide-resistant isolate.

The mechanism of low-level glycopeptide resistance among staphylococci is not known. A cytoplasmic protein, provisionally called Ddh (W. M. Milewski, S. Boyle-Vavra, B. Moreira, C. C. Ebert, and R. S. Daum, Antimicrob. Agents Chemother. 40:166-172, 1996), and the RNA transcript that contains the ddh gene, which encodes Ddh, are present in increased amounts in a vancomycin-resistant isolate, 523k, compared with the susceptible parent isolate, 523. Sequence analysis had previously revealed that Ddh is related to NAD+-dependent D-lactate dehydrogenase (D-nLDH) and VanH. This latter protein is essential for high-level glycopeptide resistance in Enterococcus faecium and Enterococcus faecalis by synthesizing the D-lactate needed for biosynthesis of D-lactate-terminating peptidoglycan precursors with low affinity for vancomycin. We now provide the direct evidence that the ddh gene product is Staphylococcus aureus D-nLDH and hereafter refer to the protein as D-nLDH. However, overproduction of this protein in isolate 523k did not result in production of D-lactate-containing peptidoglycan precursors, and susceptibility testing of ddh mutants of 523k demonstrated that S. aureus D-nLDH is not necessary for glycopeptide resistance in this isolate. We conclude that the mechanism of glycopeptide resistance in this isolate is distinct from that in enterococci.

Structural characterization of peptidoglycan muropeptides by matrix-assisted laser desorption ionization mass spectrometry and postsource decay analysis.

In this study we report the development of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)-based methods for the structural characterization of muropeptides derived from peptidoglycan. Prior to analysis, peptidoglycan samples were subjected to enzymatic digestion with muramidase and the resulting muropeptides were purified by HPLC. A new matrix, 5-chloro-2-mercaptobenzothiazole, was employed for the MALDI-MS analysis. The results have demonstrated that sub-picomole to femtomole detection can be achieved in both positive mode and negative mode, allowing unambiguous determination of the molecular masses of monomeric and oligomeric muropeptides. Structural information from monomeric muropeptides was obtained by further postsource decay (PSD) analysis. Fragmentation patterns in positive mode and negative mode PSD were complementary for the elucidation of the peptide chain sequence. Lysostaphin digestion was also incorporated with MALDI mass mapping analysis for determination of peptide chain cross-linking patterns of muropeptide oligomers from Staphylococcus aureus strains.

Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4.

The high level of cross-linking found in Staphylococcus aureus peptidoglycan is dependent on the low-molecular-weight penicillin-binding protein PBP4. Recently, the PBP4 gene, pbpD, was cloned and shown to be adjacent to and divergently transcribed relative to the putative ABC-type transporter gene, abcA. Disruption of abcA (in strain KB400) was previously shown to result in heightened resistance to several antibiotics known to interact with PBP4, suggesting that the regulation of pbpD is affected by abcA. In this report, this hypothesis was confirmed by use of a Northern (RNA) blot analysis which revealed increased accumulation of pbpD-specific transcripts in KB400 compared to that in the wild-type strain, 8325-4. By using reverse-phase high-performance liquid chromatography to examine the structure of the peptidoglycan, it was demonstrated that the increased expression of pbpD resulted in an increased level of peptidoglycan cross-linking in the staphylococcal cell wall. Promoter fusion studies demonstrated that the abcA mutation caused approximately 7-fold and 100-fold increases in pbpD and abcA promoter activities, respectively. Primer extension experiments revealed that these genes have long, untranslated leader sequences that result in a transcriptional overlap of 80 bp. Interestingly, deletion of a 26-bp region containing an inverted repeat sequence resulted in the loss of expression from both the abcA and the pbpD promoters. These data provide evidence that abcA and pbpD are under the control of a common regulatory mechanism that may involve the transport function of the abcA gene product.

Peptidoglycan composition of vancomycin-resistant Enterococcus faecium.

Muropeptide composition of peptidoglycan isolated from isogenic vancomycin-resistant and sensitive Enterococcus faecium strains was analyzed by reverse-phase high-performance liquid chromatography combined with amino acid and fast atom bombardment mass spectrometric analyses. Peptidoglycan of the sensitive and resistant strains was the same and was composed of tri- and tetrapeptides stem peptide subunits with or without aspartate or asparagine substitutions on the epsilon-amino group of the lysine residue. Thus, the synthesis of lactate-terminating peptidoglycan precursors in vancomycin-resistant E. faecium did not affect the chemical composition of peptidoglycan.

Effect of exogenous glycine on peptidoglycan composition and resistance in a methicillin-resistant Staphylococcus aureus strain.

A highly homogeneously methicillin-resistant Staphylococcus aureus strain was grown in the presence of various concentrations of exogenous glycine. Increasing concentrations of glycine in the medium resulted in a decrease in methicillin resistance and the appearance of a heterogeneous resistance phenotype. Parallel to the gradual changes in resistance was an alteration in the muropeptide composition of peptidoglycan. Increasing concentrations of glycine in the medium resulted in peptidoglycan in which muropeptides with a D-alanyl-D-alanine terminus were replaced with D-alanyl-glycine-terminating muropeptides. The disappearance of D-alanyl-D-alanine-terminating muropeptides in peptidoglycan and the concomitant decrease in resistance indicate a central role for D-alanyl-D-alanine-terminating precursors in methicillin resistance.

Altered peptidoglycan composition in vancomycin-resistant Enterococcus faecalis.

The muropeptide compositions of isogenic vancomycin-resistant and -susceptible Enterococcus faecalis strains were analyzed by reverse-phase high-performance liquid chromatography combined with amino acid analysis and fast atom bombardment mass spectrometry. Peptidoglycan of the susceptible strain contained pentapeptides as stem peptides, whereas peptidoglycan of the isogenic resistant strain was composed of muropeptides with tetrapeptide stem peptides. Despite the synthesis of lactate-terminating peptidoglycan precursors, no lactate-containing muropeptides were detected in peptidoglycan of the resistant strain. These findings indicate that either lactate-terminating precursors are not incorporated into peptidoglycan of the resistant strain or that the lactate residues are removed from peptidoglycan during synthesis.

Increased cell size and shortened peptidoglycan interpeptide bridge of NaCl-stressed Staphylococcus aureus and their reversal by glycine betaine.

Staphylococcus aureus cells grown in a defined medium under conditions of high ionic stress (2.5 M NaCl) were significantly larger than cells grown under unstressed conditions, even though the cells grew much more slowly under stressed conditions. Analysis of the structure of peptidoglycan from stressed cells showed a shorter interpeptide bridge than in peptidoglycan from unstressed cells. Glycine betaine inclusion in the high-NaCl medium resulted in cells with sizes and interpeptide bridges similar to those of cells grown under unstressed conditions.

Reduced methicillin resistance in a new Staphylococcus aureus transposon mutant that incorporates muramyl dipeptides into the cell wall peptidoglycan.

Screening of a new Tn551 library constructed in the background of a highly methicillin-resistant Staphylococcus aureus strain identified a new insertion site located on the SmaI B-fragment of the chromosome that reduced the minimal inhibitory concentration of the parent (1600 micrograms/ml) to 25-50 micrograms/ml in the mutant and caused heterogeneous expression of resistance and abnormality in peptidoglycan composition (absence of the unsubstituted pentapeptide and incorporation of alanylglutamate- and alanylisoglutamate-containing muropeptides). There was an accumulation of large amounts of the UDP-linked muramyl dipeptide in the cytoplasmic wall precursor pool of the mutant. Reduced (heterogeneous) antibiotic resistance and all the biochemical abnormalities were reproduced in genetic backcrosses by transduction with phage 80 alpha. Mutant RUSA235 appears to be impaired in the biosynthesis of the staphylococcal cell wall precursor muropeptide before the lysine addition step. We propose to provisionally call the gene inactivated in this mutant femF.

Molecular aspects of methicillin resistance in Staphylococcus aureus.

All clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates examined so far contain the mecA gene, a 2130bp stretch of DNA of non-staphylococcal origin which, together with a larger block (up to 40-60 Kb) of 'foreign' DNA, is incorporated into the staphylococcal chromosome. mecA encodes for the 78 Kd penicillin-binding protein (PBP) 2A, which has very low affinity for beta-lactam antibiotics. The sequence of the mecA gene contains structural motifs characteristic of cell wall synthetic transpeptidases. It is generally assumed that the mecA gene product (PBP 2A) acts as a surrogate enzyme which takes over the task of cell wall synthesis from the normal complement of staphylococcal PBPs, since the latter are inhibited by relatively low (e.g. methicillin) concentrations of beta-lactam antibiotics. While direct biochemical evidence for a transpeptidase activity in PBP 2A is still missing, the essentiality of an intact mecA gene for the expression of high-level methicillin resistance has been clearly established by transposon inactivation experiments. On the other hand, it was already noted some time ago that an intact mecA and its gene product PBP 2A alone cannot be fully in control of the resistant phenotype, since all MRSA isolates, irrespective of their MIC values (from as low as 3 mg/L or as high as 1600 mg/L), were found to contain comparable amounts of PBP 2A. Such major disparities between cellular amounts of PBP 2A and the antibiotic MIC values suggested that a factor or factors of unknown nature ('factor X') other than the mecA gene product also played an essential role in the phenotypic expression of resistance. The same conclusion was reached in early genetic studies in which methicillin resistance could be reduced by insertional inactivation of a chromosomal site (omega 2003) within the so-called femA gene--(factor essential for the expression of methicillin resistance) outside the mecA determinant. More recently, several additional chromosomal sites were identified outside the mecA gene in which transposon inactivation reduced the level of beta-lactam resistance. The importance of these genes becomes clear if one realizes that it is the appropriate functioning of these determinants (in the genetic background of MRSA) rather than the quantity of PBP 2A in the cells that seems to determine the MIC value of an MRSA isolate. It is not clear at the present time how many such 'auxiliary genes' exist and exactly how these gene co-operate with the mecA gene in bringing about high-level beta-lactam resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered muropeptide composition in Staphylococcus aureus strains with an inactivated femA locus.

Tn551 inactivation of femA, a factor involved in methicillin resistance of Staphylococcus aureus, caused the production of peptidoglycan in which the fraction of monoglycyl- and serine-containing muropeptides was increased at the expense of pentaglycyl muropeptides. femA mutants have a specific block in the biosynthesis of pentaglycine cross bridges after the addition of the first glycine residue.

Abnormal peptidoglycan produced in a methicillin-resistant strain of Staphylococcus aureus grown in the presence of methicillin: functional role for penicillin-binding protein 2A in cell wall synthesis.

Upon the addition of methicillin even at relatively low concentrations (5 mug/ml or 0.3% of the MIC) to the medium, methicillin-resistant staphylococci shift to the production of a new peptidoglycan with an abnormal muropeptide composition which may be the synthetic product of penicillin-binding protein 2A.

Peptidoglycan composition in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain.

It was shown that Tn551 inactivation of two chromosomal (so-called auxiliary) loci other than the mec gene result in a dramatic reduction of methicillin resistance and decreased cell wall turnover and autolytic capacity in a methicillin-resistant Staphylococcus aureus strain (de Jonge, B. L. M., de Lencastre, H., and Tomasz, A. (1990) J. Bacteriol. 173, 1105-1110). To understand the mechanistic basis of these phenomena we have examined the status of the autolytic enzymes and the muropeptide composition of peptidoglycan using reversed-phase high-performance liquid chromatography and mass spectral analyses. While no differences could be detected in the number of autolytic hydrolases, the mutants showed major changes in peptidoglycan composition. Nine prominent muropeptides of the parental strain each carrying a pentaglycyl substituent were missing from the cell wall of one group of mutants. The second mutant lacked four parental muropeptides which were composed of the unsubstituted disaccharide pentapeptide and its alanyl-tetraglycine derivative. The auxiliary genes are genetic determinants involved with the biosynthesis of peptidoglycan precursors, the presence of which in the cell wall may be needed for optimal cell wall turnover.

Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A.

All clinical isolates of methicillin-resistant Staphylococcus aureus contain an extra penicillin binding protein (PBP) 2A in addition to four PBPs present in all staphylococcal strains. This extra PBP is thought to be a transpeptidase essential for the continued cell wall synthesis and growth in the presence of beta-lactam antibiotics. As an approach of testing this hypothesis we compared the muropeptide composition of cell walls of a highly methicillin-resistant S. aureus strain containing PBP2A and its isogenic Tn551 derivative with reduced methicillin resistance, which contained no PBP2A because of the insertional inactivation of the PBP2A gene. Purified cell walls were hydrolyzed into muropeptides which were subsequently resolved by reversed-phase high-performance liquid chromatography and identified by chemical and mass spectrometric analysis. The peptidoglycan composition of the two strains were identical. Both peptidoglycans were highly cross-linked mainly through pentaglycine cross-bridges, although other, chemically distinct peptide cross-bridges were also present including mono-, tri-, and tetraglycine; alanine; and alanyl-tetraglycine. Our experiments provided no experimental data for a unique transpeptidase activity associated with PBP2A.

Suppression of autolysis and cell wall turnover in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain.

Isogenic Tn551 mutants of a highly and uniformly methicillin-resistant strain of Staphylococcus aureus were tested for their rates of autolysis and cell wall degradation in buffer and for cell wall turnover during growth. The normal (relatively fast) autolysis and turnover rates of the parent strain were retained in a Tn551 mutant in which the insert was located within the mec gene and which produced undetectable levels of penicillin-binding protein 2A. On the other hand, autolysis and cell wall turnover rates were greatly reduced in auxiliary mutants, i.e., mutants in which the transposon caused conversion of the high-level and uniform resistance of the parent strain to a variety of distinct heterogeneous expression types and greatly decreased resistance levels. All of these mutants contained an intact mec gene and produced normal amounts of penicillin-binding protein 2A, and one of the mutations was located in the femA region of the staphylococcal chromosome (B. Berger-Bachi, L. Barberis-Maino, A. Strassle, and F. H. Kayser, Mol. Gen. Genet. 219:263-269, 1989). Autolysis rates were related to the degree of residual methicillin resistance and to the sites of Tn551 insertion. Fast cell wall turnover may help expression of high-level methicillin resistance by providing a mechanism for the excision of abnormal (and potentially lethal) structural elements of the cell wall synthesized by the bacteria in the presence of methicillin.

Isogenic variants of Escherichia coli with altered morphology have peptidoglycan with identical muropeptide composition.

The peptidoglycan compositions of three isogenic morphological mutants of Escherichia coli were determined by high-pressure liquid chromatography analysis. The muropeptide compositions of the peptidoglycan of these mutants were the same, indicating that the shape of E. coli is not (solely) determined by the chemical composition of the peptidoglycan. Furthermore, it appeared that the muropeptide composition of the peptidoglycan was not affected by growth temperature.