Presence Of Subinhibitory Concentrations Research Articles

Effect of sparfloxacin on Staphylococcus aureus adhesiveness and phagocytosis.

We investigated the effect of sparfloxacin, a new broad-spectrum fluoroquinolone, on the morphology, adhesiveness and phagocytosis of a clinical isolate of Staphylococcus aureus sensitive to this compound (MIC = 0.06 mg/L). The strain was tested for its adherence to human buccal epithelial cells, measured by interference contrast microscopy, and for phagocytosis by guinea-pig peritoneal macrophages, measured by fluorescence microscopy. Accumulation of sparfloxacin by macrophages was studied by means of a velocity-gradient centrifugation technique. The S. aureus strain, grown in the presence of sub-inhibitory concentrations of sparfloxacin, exhibited an increased cell diameter and a markedly reduced capacity to adhere to buccal epithelial cells. The phagocytic capacity and activity of macrophages were greater with the treated strain than with an untreated control. A reduction in numbers of intracellular cocci was also observed 2 h after postphagocytic treatment of macrophages with sparfloxacin at 10 x MIC. This intracellular bactericidal activity may result from accumulation of sparfloxacin in macrophages, evidenced by a high ratio of cellular to extracellular concentration. It was concluded that sparfloxacin reduces adherence to epithelial cells, increases phagocytosis and facilitates the intracellular killing of S. aureus.

Morphological response of Bilophila wadsworthia to imipenem: correlation with properties of penicillin-binding proteins

The penicillin-binding protein (PBP) patterns of six strains of Bilophila wadsworthia were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and subsequent fluorography of membrane preparations labelled with [3H]benzylpenicillin. The PBP profiles among the strains were similar; generally, seven to nine PBP-reactive bands could be visualized; their molecular weights ranged from 31 to 137 kDa. The relative affinities of the PBPs of four strains of B. wadsworthia for imipenem were examined and correlated with the morphological responses of the cells to imipenem. Morphological changes were examined by light and electron microscopies. Light microscopy revealed that at low concentrations (less than the MIC), imipenem induced the formation of rounded and bulging cells; rarely, elongation without filamentation was observed. In the presence of imipenem at the MIC, spheroplast formation was observed. Scanning and transmission electron microscopies revealed round forms together with larger, multilobate cells in the presence of subinhibitory concentrations of imipenem, suggesting that new growth sites were initiated while cell division was inhibited. Peeling of the outer membrane was also seen. Spheroplasts were very large (up to 30 microns in diameter) and stable in aqueous solution. Inhibition of the PBPs could be seen in the presence of low imipenem concentrations.

The Use of Salicylic Acid to Prevent the Adherence of Escherichia Coli to Silastic Catheters

We studied the effect of salicylic acid on the attachment of Escherichia coli to silastic catheters. Silastic catheters were exposed to organisms grown in the presence of subinhibitory concentrations (1 and 5mM.) of salicylic acid. An agar rolling technique demonstrated 59% and 79% inhibition of adherence with the 2 concentrations, respectively. Silastic catheters were also pretreated by heating and then incubating in 200mM. and 600mM. salicylic acid in 95% ethanol at —20C. After incubation in 104CFU/ml. E. coli for 5 hours at 37C, 62% and 93% inhibition of adherence was observed. Acetylsalicylic acid and ibuprofen did not demonstrate similar results. Similar inhibition (82% and 95%) was observed despite preincubation of the treated catheters in sterile urine for 4 days. A bioluminescent assay of bacterial adherence also revealed inhibition only with salicylic acid. Studies using 3H-leucine demonstrated a decrease in adherence with higher concentrations of salicylic acid. Finally, tridodecylmethylammonium was used to bind salicylic acid to silastic catheters. After a 5-hour incubation in 104CFU/ml. at 37C, 94% and 99% inhibition were observed with 200 and 600mM. salicylic acid. Salicylic acid decreases adherence of E. coli to silastic catheters. This observation may be of value in designing catheters less likely to cause urinary tract infection.

Selection of variants of the TEM-1 beta-lactamase, encoded by a plasmid of clinical origin, with increased resistance to beta-lactamase inhibitors.

A TEM-1 derived variant beta-lactamase with increased resistance to beta-lactamase inhibitors was selected in vitro. The variant beta-lactamase was obtained from the parent strain Escherichia coli J62-2 containing resistance plasmid R1 which encodes the TEM-1 beta-lactamase. The variants were obtained by repeated subculture of E. coli J62-2 (R1) for five days in the presence of sub-inhibitory concentrations of amoxycillin plus clavulanic acid. Comparison of this variant beta-lactamase with a clinically isolated TEM derived beta-lactamase, with increased resistance to beta-lactamase inhibitors, suggests that they are the same enzyme. Both beta-lactamases have the same pI at 5.25 and have similar ID50 values for the beta-lactamase inhibitors clavulanic acid, sulbactam and tazobactam.

Investigation of potential genotoxic effects of low frequency electromagnetic fields on Escherichia coli.

Exposure of growing cells of Escherichia coli strain AB1157 to a frequency of 1 Hz with field strengths of 1 or 3 kV m-1 did not affect spontaneous or ultraviolet light (UV)-induced mutation frequencies to rifampicin resistance. Neither did growth in the presence of charge alter the sensitivities of strains AB1157, TK702 umuC or TK501 umuC uvrB to UV. Similarly, although the resistance of strains TK702 umuC and TK501 umuC uvrB to UV was increased by the presence of plasmid pKM101, which carries DNA repair genes, pregrowth of plasmid-containing strains in electric fields did not increase UV resistance. Finally, growth in a low frequency field in the presence of sub-inhibitory concentrations of mitomycin C did not affect mitomycin C-induced mutation frequencies. It is concluded that low frequency electromagnetic fields do not increase spontaneous mutation, induce DNA repair or increase the mutagenic effects of UV or mitomycin C.

Trimethoprim, sulphamethoxazole, bacterial adhesion and polymorphonuclear leucocyte function

Culturing Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae in the presence of subinhibitory concentrations (1/4 MIC) of trimethoprim, sulphamethoxazole or their combination, resulted in reduced adherence of all the above strains. The number of phagocytosed bacteria pre-exposed to subinhibitory concentrations of the above antibiotics was not significantly changed, but a significant increase of bactericidal activity of the polymorphonuclear leucocytes was observed. Furthermore, filtrates of K. pneumoniae and P. mirabilis grown in the presence of trimethoprim alone or in combination with sulphamethoxazole induced an increased chemotactic response of human polymorphonuclear leucocytes.

Effect of antifungal agents on the adherence of Candida albicans to murine gastrointestinal mucosal surfaces

The in-vitro adherence of Candida albicans to murine gastrointestinal mucosal surfaces was investigated in the presence of sub-inhibitory concentrations of amphotericin B, ketoconazole and itraconazole. Each antifungal drug showed a significant ability to reduce the adherence of C. albicans to gastric and jejunal mucosa. However, the outcome of an adherence assay was dependent on pH, drug concentration and C. albicans strain. The type of mucosal surface and its cellular arrangement also had an important role to play. Sub-inhibitory concentrations of amphotericin B, ketoconazole and itraconazole significantly reduced the adherence of an azole sensitive strain C. albicans 3436. These effects were influenced by the type of mucosal surface and the concentration of the antifungal drug. Sub-inhibitory concentrations of amphotericin B inhibited the adherence of an azole resistant strain. C. albicans 3310, to stomach and jejunal mucosal surfaces while sub-inhibitory concentrations of ketoconazole only significantly reduced this strain's adherence to gastric mucosal explants. Sub-inhibitory concentrations of itraconazole had little effect. Amphotericin B reduced the adherence of C. albicans 3436 to gastric mucosal explants when assays were performed at physiological pH levels, an effect dependent on concentration. This drug also reduced the adherence of strain 3436 to jejunal mucosal cells and explants at physiological pH levels, though results obtained from assays involving jejunal mucosal explants, incubated at pH 8.0, were concentration dependent.

Comparison of cefodizime with various cephalosporins for their indirect effect on the human neutrophil oxidative burst in vitro

Cefodizime, a 2-amino-thiazolyl cephalosporin, is reported to display in-vitro, ex-vivo and in-vivo immunomodulatory properties; in particular, it enhances the survival of mice infected with cefodizime-resistant pathogens. We have used an in-vitro model to assess the indirect effect of this drug (compared with other cephalosporins) on the neutrophil (PMN) oxidative response. Pseudomonas aeruginosa was employed as the bacterial target for cefodizime and cefotaxime (MICs greater than 128 mg/l), cefsulodin (MIC 16 mg/l) and ceftazidime (MIC 32 mg/l). After overnight growth in the presence of subinhibitory concentrations of each drug (10 mg/l), the altered filamentous P. aeruginosa induced a stronger oxidative response of PMN than untreated control bacteria. For all cephalosporins this was related to alterations of bacterial structure leading to increased deposits of antibodies and/or complement. Furthermore, increased non-opsonin dependent stimulation of the PMN oxidative burst was obtained; the strongest response was observed with cefodizime-treated P. aeruginosa in the case of low responder PMN, which displayed a deficient response after stimulation by control bacteria. The possibility that cefodizime could enhance this PMN function in opsonin-deficient patients requires further investigation.

Inducible and constitutive expression of pMOL28-encoded nickel resistance in Alcaligenes eutrophus N9A.

The nickel and cobalt resistance plasmid pMOL28 was transferred by conjugation from its natural host Alcaligenes eutrophus CH34 to the susceptible A. eutrophus N9A. Strain N9A and its pMOL28-containing transconjugant M220 were studied in detail. At a concentration of 3.0 mM NiCl2, the wild-type N9A did not grow, while M220 started to grow at its maximum exponential growth rate after a lag of 12 to 24 h. When grown in the presence of subinhibitory concentrations (0.5 mM) of nickel salt, M220 grew actively at 3 mM NiCl2 without a lag, indicating that nickel resistance is an inducible property. Expression of nickel resistance required active growth in the presence of nickel salts at a concentration higher than 0.05 mM. Two mutants of M220 were isolated which expressed nickel resistance constitutively. When the plasmids, pMOL28.1 and pMOL28.2, carried by the mutants were transferred to strains H16 and CH34, the transconjugants expressed constitutive nickel resistance. This indicates that the mutation is plasmid located. Both mutants expressed constitutive resistance to nickel and cobalt. Physiological studies revealed the following differences between strain N9A and its pMOL28.1-harboring mutant derivatives. (i) The uptake of 63NiCl2 occurred more rapidly in the susceptible strain and reached a 30- to 60-fold-higher amount that in the pMOL28.1-harboring mutant; (ii) in intact cells of the susceptible strain N9A, the cytoplasmic hydrogenase was inhibited by 1 to 5 nM NiCl2, whereas 10 mM Ni2+ was needed to inhibit the hydrogenase of mutant cells; (iii) the minimal concentration of nickel chloride for the derepressed synthesis of cytoplasmic hydrogenase was lower in strain N9A (1 to 3 microM) than in the constitutive mutant (8 to 10 microM).

Repeated exposition to subinhibitory concentrations of antibiotic in vitro readily decreases susceptibility of Neisseria gonorrhoeae to rifampicin, but not to new cephalosporins and penicillin G.

Repeated subcultivation of Neisseria gonorrhoeae in the presence of subinhibitory concentrations of antibiotic has turned out as a reliable model to predict the low potential for development of resistance with respect to the beta-lactam antibiotic penicillin. Before large-scale introduction of the new cephalosporins we exposed 5 N. gonorrhoeae strains of different susceptibility to penicillin repeatedly to subinhibitory concentrations of cefotiam, ceftizoxime, rifampicin and penicillin G incorporated into chocolate agar. each time the most resistant representatives of a strain were propagated, on the whole 25 times. While resistance to rifampicin increased readily (all strains became relatively resistant, MIC = 4 micrograms/ml), the same was not true of the cephalosporins. Although their susceptibility decreased, too, no strain acquired partial or even total resistance (final MIC less than or equal to 0.128 with cefotiam and ceftizoxime). The cephalosporins thus rather parallelled penicillin G which hardly induced any increase of resistance. Thus, a quick loss of clinical efficacy need not be feared after large-scale introduction of the new cephalosporins into the therapy of gonorrhea.

Effects of subinhibitory concentrations of ketoconazole on in vitro adherence of Candida albicans to vaginal epithelial cells.

The in vitro adherence of multiple 14C-glucose labeled isolates of Candida albicans to exfoliated vaginal epithelial cells in the presence of subinhibitory concentrations of ketoconazole was studied with a new method utilizing differential centrifugation in a gelatin-PBS solution as well as by the standard method of direct microscopy measurement. Pre-incubation of stationary-phase candida in ketoconazole at concentrations of 0.002 to 0.1 microgram/ml for 4 h at 37 degrees C had no effect on adherence. The addition of ketoconazole to logarithmic phase Candida albicans failed to reduce the total number of cell-associated adherent yeast but exposure to subinhibitory concentrations of ketoconazole was associated with a dystrophic morphology of the blastospores, extensive clumping and reduced germination resulting in fewer individual candida blastospores directly attached to the cell membranes. Germination inhibition and a marked reduction in adherent candida was observed when 0.01 microgram/ml ketoconazole was added to Candida albicans incubated in germination promoting medium. The diminished adherence of Candida albicans to vaginal epithelial cells after exposure to subinhibitory concentrations of ketoconazole may have clinical relevance in preventing recurrent candida vaginitis.

A novel synergistic effect of alanosine and guanine on adenine nucleotide synthesis in mammalian cells. Alanosine as a useful probe for investigating purine nucleotide metabolism.

A novel synergistic effect of the antitumor agent alanosine (2-amino-3-(hydroxynitrosoamino) propionic acid), which specifically inhibits the enzyme adenylosuccinate synthetase (ASS) and guanine on the growth of Chinese hamster ovary (CHO) cells and human diploid fibroblasts (HDF) has been observed. In the presence of subinhibitory concentrations of alanosine, both CHO cells and the HDF show excessive sensitivity to exogenous guanine--a phenotype which closely resembles that seen with some of the mutants containing reduced enzymatic activity of ASS. The growth inhibitory effects of alanosine, or alanosine and guanine, on CHO cells are completely reverted by the addition of adenine to the culture medium, and the synergistic effect of guanine is not observed in mutants which lack the enzyme hypoxanthine-guanine phosphoribosyl transferase. These resuls suggest that guanine nucleotides exert a regulatory effect on the activity of the enzyme adenylosuccinate synthetase. The ability to confer the guanine-sensitive phenotype and its modulation by subinhibitory concentrations of alanosine in different cell types indicates that alanosine provides a useful probe for investigating the regulation of purine nucleotide metabolism in mammalian cells.

Effect of leukocyte hydrolases on bacteria

The bactericidal and bacteriolytic effects of lysolecithin (LL) and egg-white lysozyme (LYZ) on Staph. aureus and group A streptococci and the solubilization of phospholipids from the bacterial membranes by these agents was studied. Low concentrations of lysolecithin (1--10 microgrames/ml) are highly bactericidal for Steph. aureus and group A streptococci, but induce neither bacteriolysis nor solubilization of a substantial amount of membrane phospholipids. On the other hand, while LL at greater than 50 micrograms/ml causes substantial lipid release, a combination of LL and LYZ is absolutely needed to solubilize lipids from streptococci. This combination is, however, not bacteriolytic for this microrganism. The solubilization of lipids from staphylococci by LL is much faster than that induced in streptococci by LL + LYZ. The solubilization of the bulk of membrane lipids from staphylococci can also be achieved by Triton X-100 and by sodium lauryl sulfate and from group A streptococci by Triton X-100 plus LYZ. A variety of other detergents (e.g., Cetavlon, sodium taurocholate, cetyl pyrdinium chloride) have no lipid-releasing properties even in the presence of LYZ. The release of lipids by LYZ (in the presence of LL) from group A streptococci is related to its enzymatic activity, on a still unknown substrate, but not to its cationic nature as this muramidase cannot be replaced by a variety of cation substances (histone, polylysin, leukocyte cationic proteins, polymyxin B, and spermidine). The release of lipids from staphylococci by LL is not inhibited by a variety of anionic and cationic polyelectrocytes (heparin, liquoid, chondroitin sulfate, DNA histone, and polylysine) which markedly inhibit the release of lipids from group A streptococci by LL and LYZ. Streptococci that had been cultivated in the presence of subinhibitory concentrations of penicillin G lose their membrane phospholipids to a larger extent and by much smaller concentrations of LL and LYZ, as compared to controls, suggesting that the interference with the synthesis of the peptidoglycan increases the accessibility of the cell membrane to the lipid-releasing agents. The mechanism by which LL collaborates with LYZ in lipid release is still not known. The possible role of bacterial lipids and lyso compounds in the control of bacterial survival in inflammatory sites is briefly discussed.

PRODUCTION OF A POLYSACCHARIDE BY STAPHYLOCOCCUS AUREUS. II. EFFECT OF TEMPERATURE AND ANTIBIOTICS.

SummaryIt has been shown that S. aureus grown in the presence of subinhibitory concentrations of nafcillin produced a disturbance or disorganization of the cell wall fabric resulting in a marked accumulation of a polysaccharide within the cell. An appreciable polysaccharide response was also found for oxacillin, methicillin and erythromycin. This conclusion is based on data which demonstrate a clear differentiation between such an inhibitor of bacterial wall synthesis as penicillin which exerted a pronounced effect on polysaccharide synthesis and other antibacterial antibiotics which failed to increase the quantity of polysaccharide formed. In addition, treatment with nafcillin rendered the normally lysozyme resistant cells of S. aureus susceptible to partial lysis by lysozyme and the concomitant addition of trypsin resulted in further dissolution of the cell bodies. Electron micrographs of such treated cells subjected to thermal stress provide further evidence of the relationship between accumulation of ...