Osmoprotective activity, urea protection, and accumulation of hydrophilic betaines in Escherichia coli and Staphylococcus aureus.

The hydrophilic betaines, deanol betaine, triethanol betaine, diethanolthetin and methylethanolthetin, and also thioxanium betaine and citrulline betaine, were accumulated by Escherichia coli. All betaines tested had significant osmoprotective activity for E. coli and, with the exception of citrulline betaine and diethanolthetin, also demonstrated urea protection. Staphylococcus aureus accumulated only methylethanolthetin, deanol betaine and thioxanium betaine: the first two had an osmoprotective effect but conferred no urea protection. Diethanolthetin and thioxanium betaine significantly decreased urea tolerance for S. aureus.

Inhibitors of bacterial growth in urine: what is the role of betaines?

It has long been recognised that some individuals produce urine that is inhibitory to uropathogens. This may be partly explained by inhibitors. Several inhibitors have been identified in urine including urea and organic acids. Bacteria adapt to high osmolarity by activating osmoregulated betaine porters and accumulating organic osmolytes intracellularly. The preferred substrate is glycine betaine, which is present in urine, and promotes rapid growth by balancing osmotic forces and stabilising macromolecular structures against the toxicity of urea and low pH. Other dietary betaines such as trigonelline may also be taken but enhance urea toxicity. The importance of such compounds in vivo is unknown.

Osmoprotective properties and accumulation of betaine analogues by Staphylococcus aureus.

Betaines were evaluated as potential antistaphylococcal agents for urinary tract infections. Staphylococcus aureus accumulated all tested betaines except trigonelline. S. aureus transport systems were less sensitive to carbon chain length than those of Escherichia coli. Betaines were accumulated in the absence of osmotic stress, and 10-fold more in hyperosmotic medium. Most betaines increased the osmotolerance of S. aureus in defined minimal medium. Unlike E. coli, S. aureus did not significantly accumulate a second betaine in the presence of glycine betaine. Betaines are less likely to be useful in treating staphylococcal than E. coli urinary infections.

Natural and synthetic betaines counter the effects of high NaCl and urea concentrations.

Escherichia coli was used as a model system to evaluate a range of betaines for their ability to protect against salt and urea stresses. Betaine structure determined the salt and urea protective effects. Dimethylthetin conferred salt protection similar to glycine betaine, whereas dimethylsulfoniopropionate (DMSP) was less effective than either glycine betaine or dimethylthetin, but similar to propionobetaine (its nitrogen analogue). Hydrophobic alpha-substituents altered salt tolerance. Valine betaine with an aliphatic side group conferred salt tolerance similar to glycine betaine. Betaines containing phenyl groups (phenylglycine, phenylalanine and N-phenylglycine betaines) did not confer salt protection, growth being similar to, or less than the control (no betaine). Hydrophobic groups decreased the ability to protect against urea stresses; valine betaine conferred poor urea tolerance. The addition of an hydroxyl group increased the ability of a betaine to protect against urea denaturation. Proline betaine, an effective salt protector, conferred poor urea tolerance. Increasing the charge separation in the betaine molecule decreased the ability to confer urea tolerance. Thiolanium, pyridinium and triethylglycine betaines, with larger cationic functions, conferred no urea tolerance to E. coli.

The effect of aminoglycoside-induced adaptive resistance on the antibacterial activity of other antibiotics against Pseudomonas aeruginosa in vitro.

The effect of gentamicin-induced adaptive resistance on the antibacterial activity of six non-aminoglycoside antibiotics was studied. Adaptive resistance was induced in Pseudomonas aeruginosa in a dynamic in-vitro model of infection. The bactericidal effect of ceftazidime, imipenem, aztreonam, ciprofloxacin, and piperacillin was not altered in the presence of adaptive resistance but the effect of rifampicin was increased.

Is the ability of urinary tract pathogens to accumulate glycine betaine a factor in the virulence of pathogenic strains?

The regulation of intracellular concentrations of organic solutes, including glycine betaine, is an important adaptive response to osmotic stress for Escherichia coli. The clinical significance of glycine betaine to uropathogens is not clear. Clinical isolates of E. coli, Klebsiella pneumoniae, Enterobacter species, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus, S. saprophyticus, and Enterococcus faecalis accumulated glycine betaine from hyperosmotic media. The addition of glycine betaine to hyperosmotic minimal medium accelerated the growth rates of all species tested except P. mirabilis. However, when clinical strains of E. coli were transferred from urine with low osmolality to hyperosmotic urine, there was no slowing of the growth rate. There was no difference in growth rates of E. coli isolates from acute pyelonephritis, cystitis, and asymptomatic bacteriuria nor from fecal isolates. The ability to accumulate osmolytes, although it may be a factor in the adaptation to hypertonic environments, was not related to virulence.

Accumulation of natural and synthetic betaines by a mammalian renal cell line.

Intracellular accumulation of different betaines was compared in osmotically stressed Madin Darby canine kidney (MDCK) cells to model the betaine accumulation specificity of the mammalian inner medulla and to show how this accumulation differed from that of bacteria. All betaines accumulated less than glycine betaine. Arsenobetaine (the arsenic analogue of glycine betaine) accumulated to 12% of the glycine betaine levels and the sulphur analogue dimethylthetin accumulated to >80%. Most substituted glycine betaine analogues accumulated to 2-5% of intracellular glycine betaine concentrations, however, serine betaine accumulated to <0.5% of glycine betaine levels. Inhibition studies to distinguish the betaine ports were performed by the addition of proline. Butyrobetaine and carnitine accumulation was not proline sensitive, whereas that of other betaines was. As with glycine betaine, the accumulation of propionobetaine and dimethylthetin was proline sensitive and osmoregulated. Pyridinium betaine was accumulated by both proline-sensitive and -insensitive systems, with a small increase under osmotic stress. High concentrations (10 times that of glycine betaine) of the dietary betaines proline betaine and trigonelline inhibited total betaine accumulation. Because alpha-substituted betaines are accumulated by bacteria and not by MDCK cells, these betaines may be the basis for design of antimicrobial agents.

Competitive accumulation of betaines by Escherichia coli K-12 and derivative strains lacking betaine porters.

Escherichia coli was grown in hyperosmotic media containing both glycine betaine and one other betaine. E. coli K-12 derivative WG439 (putP- proP- proU-) did not accumulate any of 15 betaines. Strains WG445 (putP- proP- proU+), WG443 (putP- proP+ proU-) and the control strains all accumulated less betaine, (CH3)3N(+)-(CH2)n-COO-, when n was greater than 1. Accumulation was not detectable when n = 5. Both L- and D-isomers of alpha-substituted betaines were accumulated by both strains WG443 and WG445, the D-isomers more slowly. Hydroxylated alpha-substituted betaines were accumulated relatively more through the osmoregulated transport protein ProU than through ProP. In actively growing cultures glycine betaine appeared to be the preferred substrate for accumulation, but the proportion of the second accumulated betaine increased as cultures approached stationary phase.

Relationship between osmoprotection and the structure and intracellular accumulation of betaines by Escherichia coli.

Naturally occurring betaines, especially glycine betaine and proline betaine, were accumulated by Escherichia coli from urine. In synthetic hyperosmotic medium, with an homologous series of added betaines, (CH3)3N(+)-(CH2)n-COO-, osmoprotective activity and intracellular accumulation decreased monotonically as n increased from 1 to 5. In contrast, alpha-substituted glycine betaines were accumulated in a similar manner to glycine betaine, but with different osmoprotective activities. Arsenobetaine, with a quaternary arsonium group, was also accumulated but amino acids which can become negatively charged in a chemically basic environment were not.

Effects of betaines and urine on the antibacterial activity of aminoglycosides.

Urine has long been known to inhibit the activity of aminoglycosides against urinary tract pathogens. Glycine betaine which is present in urine confers resistance against high osmolarity to Gram-negative organisms. We postulated that glycine betaine might contribute to the aminoglycoside resistance found in hypertonic urine. Escherichia coli became extremely resistant to gentamicin (40 x MIC in 0.9 M sodium chloride) when cultured in minimal medium supplemented with 10(-4) M glycine betaine and 0.1-1.0 M sodium chloride. Resistance was increased in the presence of high glucose concentrations but to a lesser extent (3 x MIC in 1.0 M glucose). This effect was not produced by other polyols or urea. These results suggest the observed synergism is mediated by the osmoprotective effects of glycine betaine and the inhibitory effect of sodium chloride or glucose against the aminoglycoside. Other betaines tested had a less marked effect. The betaines in urine permit the expression of increased resistance to aminoglycosides in concentrated urine.

Lomefloxacin compared with trimethoprim for the treatment of women with cystitis.

A prospective, randomised, single-blind study compared the efficacy and safety of the long-acting difluoroquinolone, lomefloxacin, with trimethoprim for the treatment of women with cystitis. Seventy-five patients were treated with either lomefloxacin (400 mg once daily) or trimethoprim (300 mg once daily) for 5 days. Of the 75 patients enrolled, 47 were evaluable for efficacy. All patients treated with lomefloxacin and all but one treated with trimethoprim had a satisfactory clinical response. Twenty-three of 24 patients (96%) treated with lomefloxacin and 22 of 23 (96%) with trimethoprim had a satisfactory bacteriological response. All 75 patients were evaluated for safety. No serious side effects or toxicity were noted and no patients was withdrawn because of an adverse reaction. Both drugs proved highly effective and safe for the treatment of women with cystitis.

Comparison of gentamicin dosing regimens using an in-vitro model.

An in-vitro model which simulates in-vivo pharmacokinetics was used to compare the efficacy against Pseudomonas aeruginosa of dosing regimens of gentamicin which achieve different peak/trough concentrations but use the same total dose over 24 h. First exposure to gentamicin produced a rapid bactericidal effect which was proportional to the initial peak concentration. Subsequent doses of gentamicin produced a smaller bactericidal effect. Regrowth occurred with all dosing regimens, even after very high initial concentrations (26 mg/L). The time to reach bacterial counts above starting values was prolonged in relation to peak concentrations. Regrowth was also demonstrated in continuous infusion experiments which maintained very high concentrations (26 mg/L), although an inhibitory effect was evident compared with single dose experiments and the experiments mimicking in-vitro pharmacokinetics. There was little evidence of a post-antibiotic effect. The data supports the use of larger initial and longer interval bolus dosing compared with current recommendations.

Comparison of ciprofloxacin with netilmicin for the treatment of acute pyelonephritis.

A prospective, randomised trial was undertaken to compare the efficacy of ciprofloxacin and netilmicin for the treatment of acute pyelonephritis. Forty-three patients were enrolled and 34 (29 women) completed the protocol. Fifteen of 17 patients treated with ciprofloxacin and 15 of 17 treated with netilmicin were cured. All patients were treated for five days. One patient relapsed after ciprofloxacin and another had a reinfection, while two relapsed after netilmicin. Five of six patients with a urinary tract abnormality were cured. Side effects were generally mild and rapidly reversible. Patients treated with ciprofloxacin spent a mean of 3.7 days in hospital compared with 5.3 days for those treated with netilmicin. The difference in duration of hospital stay was statistically significant (p less than 0.01). Both drugs proved highly effective and safe for the treatment of severe acute pyelonephritis.

Antimicrobial effects of lomefloxacin in vitro.

The MIC of lomefloxacin was determined for 554 isolates from the urinary tract. Some of the more resistant strains of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa were studied in a dynamic in-vitro model in order to study dosing strategies. The model simulated, in Mueller-Hinton broth, the profile of plasma lomefloxacin concentrations in volunteers. Bacteria were exposed to a range of lomefloxacin concentration profiles achievable by oral dosing. The first dose of lomefloxacin was rapidly bactericidal in a dose-dependent manner for all strains. On re-exposure, a dose-dependent inhibitory effect was observed. Drug-resistant mutants were readily isolated from all bacteria tested on lomefloxacin-containing plates. These occurred at a high frequency in P. aeruginosa (10(-1)), but less frequently in K. pneumoniae (10(-3)). P. mirabilis (10(-5)), and E. coli (10(-6)). The number of resistant mutants isolated tended to be lower with use of higher drug concentrations. The results suggest that lomefloxacin dosing regimens ensuring maintenance of a high serum concentration: MIC ratio will result in maximal antibacterial effects and minimal problems with resistant strains.