Post-operative infection by pathogenic micro-organisms in the oral cavity of patients with prostatic carcinoma.

The aim of this study was to analyse the change in the oral cavity microflora of 14 patients who had undergone a radical prostatectomy for prostatic carcinoma. The detection of micro-organisms in the oral cavity was compared before and after the surgical procedure. Post-operative infection, defined as those patients who had increased Candida species counts and/or pathogenic bacteria only at the post-operative examination, was observed in 10 patients. Six patients showed increased Candida species counts at the post-operative examination compared with the pre-operative examination. In five patients, pathogenic bacterial species were detected at the post-operative examination but not at the pre-operative examination. One patient had detectable pathogenic bacterial species only at the post-operative examination along with increased Candida species counts. Our findings suggest that pre-operative oral hygiene to remove bacterial and Candida species from patients who are scheduled for surgical procedures is important for satisfactory clinical outcomes.

[Combination therapy against vancomycin-resistant enterococci].

The therapeutic options for patients infected with vancomycin-resistant enterococci are limited. Optimal therapy for serious enterococcal infections requires the use of synergistic combinations of a cell wall-active agent plus an aminoglycoside. Enterococci have acquired aminoglycoside resistance genes that mediate production of aminoglycoside-modifying enzymes, which eliminate this synergistic bactericidal effect. Traditional therapy has been compromised due to the increasing prevalence of enterococci with beta-lactam, glycopeptide, and high-level gentamicin resistance. Arbekacin, a semi-synthetic aminoglycoside, shows excellent activity against a wide variety of bacteria that produce aminoglycoside modifying enzymes, including AAC(6')-APH(2"). In recent studies, the combination of ampicillin and arbekacin demonstrated inhibitory activity against vancomycin-resistant enterococci, and may prove useful in the treatment of enterococcal infections where treatment options are limited.

In-vitro synergistic activity of the combination of ampicillin and arbekacin against vancomycin-and high-level gentamicin-resistant Enterococcus faecium with the aph(2")-Id gene.

The combination of ampicillin plus arbekacin produced synergistic killing against 8 of 13 vancomycin-, ampicillin-, gentamicin-, and streptomycin-resistant Enterococcus faecium isolates that possess the high-level gentamicin resistance gene, aph(2")-Id. This combination may prove useful in treating infections caused by multiresistant enterococci.

Efficacy of ampicillin plus arbekacin in experimental rabbit endocarditis caused by an Enterococcus faecalis strain with high-level gentamicin resistance.

Enterococcus faecalis LC40 is an ampicillin-susceptible clinical isolate with high-level gentamicin resistance due to the aac(6')-Ie-aph(2")-Ia aminoglycoside resistance gene. The combination of ampicillin plus arbekacin reduced mean bacterial vegetation counts significantly more than ampicillin alone or ampicillin plus gentamicin in a rabbit model of aortic-valve endocarditis caused by E. faecalis LC40.

Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci.

Conditions have been optimized for the use of a multiplex PCR for the detection of vancomycin-resistant enterococci in nosocomial surveillance specimens. Seven primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 Enterococcus faecalis-specific, Enterococcus faecium-specific, and rrs (16S rRNA) were used in one reaction tube. The PCR method developed in the present study is simple and reliable for the rapid characterization of vancomycin-resistant enterococci.

In-vitro activity of arbekacin alone and in combination with vancomycin against gentamicin- and methicillin-resistant Staphylococcus aureus.

In-vitro susceptibility studies were performed on 99 clinical Staphylococcus aureus isolates. A total of 68 of 73 methicillin-resistant S. aureus and 2 of 26 methicillin-susceptible S. aureus were gentamicin-resistant (gentamicin MIC range 16 to 1,024 microg/mL). All 70 gentamicin-resistant isolates contained the aac(6')-Ie-aph(2'')-Ia aminoglycoside resistance gene, and none possessed the aph(2'')-Ic or aph(2'')-Id aminoglycoside resistance genes. The arbekacin MIC for the 70 gentamicin-resistant isolates ranged from 0.25 to 4 microg/mL. The combination of arbekacin plus vancomycin produced synergistic killing against 12 of 13 gentamicin-resistant MRSA isolates. The combination of gentamicin plus vancomycin produced synergistic killing against 7 of the same 13 isolates. Arbekacin may prove useful when used in combination with vancomycin in treating infections caused by gentamicin-resistant MRSA.

Evidence that the PBP 5 synthesis repressor (psr) of Enterococcus hirae is also involved in the regulation of cell wall composition and other cell wall-related properties.

psr has been reported by M. Ligozzi, F. Pittaluga, and R. Fontana, (J. Bacteriol. 175:2046-2051, 1993) to be a genetic element located just upstream of the structural gene for the low-affinity penicillin-binding protein 5 (PBP 5) in the chromosome of Enterococcus hirae ATCC 9790 and to be involved in the repression of PBP 5 synthesis. By comparing properties of strains of E. hirae that contain a full-length, functional psr with those of strains that possess a truncated form of the gene, we have obtained data that indicate that psr is involved in the regulation of several additional surface-related properties. We observed that cells of strains that possessed a truncated psr were more sensitive to lysozyme-catalyzed protoplast formation, autolyzed more rapidly in 10 mM sodium phosphate (pH 6.8), and, in contrast to strains that possess a functional psr, retained these characteristics after the cultures entered the stationary growth phase. Cellular lytic properties did not correlate with differences in the cellular contents of muramidase-1 or muramidase-2, with the levels of PBP 5 produced, or with the penicillin susceptibilities of the strains. However, a strong correlation was observed with the amounts of rhamnose present in the cell walls of the various strains. All of the strains examined that possessed a truncated form of psr also possessed approximately one-half of the rhamnose content present in the walls of strains that possessed a functional psr. These data suggest that psr is also involved in the regulation of the synthesis of, or covalent linkage to the cell wall peptidoglycan of, a rhamnose-rich polysaccharide. These differences in cell wall composition could be responsible for the observed phenotypic differences. However, the multiple effects of psr suggest that it is part of a global regulatory system that, perhaps independently, affects several cell surface-related properties.

Bacterial walls, peptidoglycan hydrolases, autolysins, and autolysis.

Knowledge of the chemistry, ultrastructure, biosynthesis, assembly, and function of bacterial cell walls has expanded enormously since the opening of this field of research approximately 40 years ago, primarily by the early work of Milton Salton. It has become abundantly clear that, in most environments, walls are essential to the survival and growth of bacteria and in many ways are structurally and functionally unique. A common but not universal feature of bacterial walls is the presence of peptidoglycan (PG; murein, or in the case of certain Archae the analogous structure-pseudomurein). PGs are considered to be primarily responsible for the protective and shape-maintaining properties of walls. They are a biologically unique class of macro-molecules in that they are not linear or even branched macromolecules. Instead they are two- or three-dimensional net like polymers that are linked together by three different chemical bonds (glycosidic, amide, and peptide). In addition, they contain the D-isomers of some amino acids and therefore may possess DL, LD, and DD linkages. Furthermore, the exact chemical structure of a PG may vary depending on environmental factors, however, retaining the essential protective and shape maintaining properties of the wall. Thus, the overall architectural plan of the wall may be more important than the exact shape of the bricks used for the construct. Another somewhat unique feature of PGs (and walls) is their final assembly in situ on the outside of the cellular permeability barrier. A broad variety of bacteria have been shown to possess enzymes that can hydrolyze bonds in the wall PG. Hydrolysis of a sufficient number of bonds can result in the weakening of, or serious damage to, the protective properties of the PG. Frequently, a bacterial strain may possess more than one PG hydrolase activity. A commonly believed, but as yet unproven, hypothesis is that PG hydrolases play one or more roles in PG assembly and/or surface growth and cell division. At a minimum, such potentially suicidal activities must be exquisitely well regulated. Currently we know little concerning the regulation of these activities, or how they communicate with, and integrate with, chromosome replication, synthesis of cytoplasmic macromolecules, cell growth, and division, although such, probably two-way, communications must occur in growing and dividing cells. Recent data indicate that the psr element in Enterococcus hirae described by Fontana and collaborators as a genetic element that is involved in the regulation of the synthesis of PBP 5, also is involved in the regulation of several other surface properties. These properties include (1) autolysis rates of exponential phase. cells, (2) the retention of this property after cells enter the stationary phase, (3) lysozyme sensitivity, and (4) the ratio of rhamnose-containing wall polysaccharide to PG in the walls. Thus the psr element may be a part of a "global" regulation and communication system in E. hirae.

Penicillin resistance and autolysis in enterococci.

Comparison of several cell wall-related properties of the ATCC 9790 strain and the R40 strain, a penicillin-resistant, PBP5 overproducing strain, and Rev14, a penicillin-hypersensitive, PBP5-deficient strain, is consistent with a role of the genetic element, psr, in the global regulation of lysozyme sensitivity, autolytic capacity, and wall-rhamnose-containing polysaccharide content. These parameters appear to be independently regulated by a system that involves psr in a currently unknown manner.

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790.

A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.

Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin.

The mature forms of the extracellular muramidase-2 of Enterococcus hirae and Streptococcus faecalis autolysin have very similar primary structures. Each consists of an active-site-containing N-terminal domain fused to a multiple-repeat C-terminal domain. Polypeptide segments occurring at equivalent places in these two bacterial wall lytic enzymes have homologues in two phage lysozymes and in three functionally unrelated proteins, illustrating the principle that protein molecules frequently are constructed from modules that are linked in a single polypeptide chain.

Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.

Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCl extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage lambda gt11. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The nucleotide sequence of each subclone was determined. The sequence data indicated an open reading frame encoding a polypeptide of 666 amino acid residues, with a calculated molecular mass of 70,678 Da. The first 24 N-terminal amino acids of purified extracellular muramidase-2 were in very good agreement with the deduced amino acid sequence after a 49-amino-acid putative signal sequence. Analysis of the deduced amino acid sequence showed the presence at the C-terminal region of the protein of six highly homologous repeat units separated by nonhomologous intervening sequences that are highly enriched in serine and threonine. The overall sequence showed a high degree of homology with a recently cloned Streptococcus faecalis autolysin.

Properties of cell wall-associated DD-carboxypeptidase of Enterococcus hirae (Streptococcus faecium) ATCC 9790 extracted with alkali.

DD-Carboxypeptidase (DD-CPase) activity of Enterococcus hirae (Streptococcus faecium) ATCC 9790 was extracted from intact bacteria and from the insoluble residue (crude cell wall fraction) of mechanically disrupted bacteria by a brief treatment at pH 10.0 (10 mM glycine-NaOH) at 0 degrees C or by extraction with any of several detergents. Extractions with high salt concentrations failed to remove DD-CPase activity from the crude wall fraction. In contrast to N-acetylmuramoylhydrolase (both muramidase 2 and muramidase 1) activities, DD-CPase activity failed to bind to insoluble cell walls or peptidoglycan matrices. Thus, whereas muramidase 1 and muramidase 2 activities can be considered to be cell wall proteins, the bulk of the data are consistent with the interpretation that the DD-CPase of this species is a membrane protein that is sometimes found in the cell wall fraction, presumably because of hydrophobic interactions with other proteins and cell wall polymers. The binding of [14C]penicillin to penicillin-binding protein 6 (43 kilodaltons) was proportional to DD-CPase activity. Kinetic parameters were also consistent with the presence of only one DD-CPase (penicillin-binding protein 6) in E. hirae.

The effect of head group structure on phase transition of phospholipid membranes as determined by differential scanning calorimetry.

The phase transition characteristics of cardiolipin and phosphatidylglycerol suspensions were investigated by differential scanning calorimetry. The phase transition temperatures for dimyristoylphosphatidylglycerol, tetramyristoylcardiolipin, dipalmitoylphosphatidylglycerol and tetrapalmitoylcardiolipin were 25.0, 47.0, 40.5 and 62.2 degrees C, respectively. The phase transition temperature for a mixture of two analogous phospholipids was higher than that for phosphatidylglycerol alone, but lower than that for cardiolipin alone. It increased along with cardiolipin content. The phase transition temperature for cardiolipin was increased in the presence of divalent cations, particularly Ca2+. The results indicate that the head group of cardiolipin by itself can increase the phase transition temperature.

Characterization of a glycophospholipid of Streptococcus pyogenes-derived L-form.

A glycophospholipid was detected as a major lipid in the L-form derived from Streptococcus pyogenes. The glycophospholipid was purified and chemically analyzed. From its phosphorus:glucose:glycerol molar ratio of 1:2:2 and mass spectrum, the deacylated glycophospholipid was identified as glycerophosphoryldiglucosylglycerol. This structural assignment was confirmed by thin layer chromatography of the lipid itself, which comigrated with known phosphatidyldiglucosyldiglyceride from S. faecalis. A possible mechanism for biosynthesis of the glycophospholipid in the L-form that invokes known biosynthetic processes involved in glycophospholipid metabolism in related bacteria is proposed.

Increase of cardiolipin content in Staphylococcus aureus by the use of antibiotics affecting the cell wall.

Effect of antibiotics affecting cell wall synthesis on phospholipid composition in Staphylococcus aureus 209P was examined. Each antibiotic was added in the middle exponential growth phase and the growth was followed turbidimetrically. Penicillin, fosfomycin, cycloserine, moenomycin and cefazolin caused a leveling off of turbidity and growth to cease without lysis. Enramycin and bacitracin were bacteriolytic. Bacteriolytic antibiotics caused a greater increase of cardiolipin content than those that were non-bacteriolytic. The amount of phosphatidylglycerol decreased in proportion to the increment of cardiolipin content. Since bacteriolytic antibiotics bind to undecaprenol, the role of cardiolipin was discussed in relation to the mechanism of synthesis of cell surface materials.

Effect of glucose and oxygen on the structure of the plasma membrane of Staphylococcus aureus.

The effects of glucose and oxygen on the formation of the plasma membrane of Staphylococcus aureus were studied. Phospholipids were consistent components of the membrane and were not affected by glucose or oxygen. Phospholipid fatty acids in cells grown in glucose containing media were rich in Ceven (C18, C20) fatty acid chains, whereas cells grown in glucose deficient media (normal broth) had anteiso Codd (C15,C17) fatty acid chains in place of Ceven chains. This may indicate increased membrane rigidity of the cells grown in glucose containing media. Cytochromes and ATPase were present in the membrane from cells grown in normal broth, but were deficient in the cells grown in glucose containing media. Polypeptide analysis of the membrane proteins showed a deficiency of the bands corresponding to these enzymes. They were not induced by the additionof oxygen to cells grown in glucose containing media. It was concluded that glucose was the dominant factor inhibiting the formation of these membrane enzymes.

Lipid composition of Staphylococcus aureus and its derived L-forms.

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L-forms were cultured in serum-containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L-forms, but for less than 25% in parent bacteria. The cardiolipin content of L-forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L-forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L-forms but not in the parent strains when they were cultured in serum-free broth. To examine the ability of L-forms to synthesize cholesterol, the cholesterol content of L-forms cultured in serum-free broth was compared with that of the medium. The results indicated that staphylococcal L-forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptational change in response to the disappearance of the cell wall.