Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation, and minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media. MICs are used by diagnostic laboratories mainly to confirm resistance, but most often as a research tool to determine the in vitro activity of new antimicrobials, and data from such studies have been used to determine MIC breakpoints. MBC determinations are undertaken less frequently and their major use has been reserved for isolates from the blood of patients with endocarditis. Standardized methods for determining MICs and MBCs are described in this paper. Like all standardized procedures, the method must be adhered to and may not be adapted by the user. The method gives information on the storage of standard antibiotic powder, preparation of stock antibiotic solutions, media, preparation of inocula, incubation conditions, and reading and interpretation of results. Tables giving expected MIC ranges for control NCTC and ATCC strains are also supplied.

A mathematical model is presented of the kinetics of non-native protein aggregation that combines Lumry-Eyring and nucleated polymerization (LENP) descriptions. The LENP model is solved for cases in which aggregation rates are slow compared to folding-unfolding equilibration and is shown to be a generalization of a number of previously proposed nucleation-and-growth models for non-native and native protein aggregation. The model solutions exhibit a number of qualitative kinetic regimes. Each regime has a characteristic set of experimental signatures that are related to the relative rates of growth and nucleation as well as to the threshold size at which aggregates condense to form higher-order structures or other phases. Approximate model solutions provide practical rate equations that can be regressed against typical experimental kinetic data to obtain mechanistic parameters characterizing the aggregation pathway. In all kinetic regimes, it is found that observed rate coefficients (kobs) or half-lives (t50) obtained from extent-of-reaction measurements are convolutions of more than one stage in the pathway unless purely seeded growth occurs. Despite this convolution, the combination of apparent reaction order (time domain) and the scaling of kobs or t50 with initial protein concentration provides a means to determine a value for the dominant nucleus size in each case. Additional information, such as equilibrium unfolding thermodynamics and the limiting aggregate size distribution, are required to further deconvolute kobs into intrinsic contributions from nucleation, growth, and conformational changes. The model and analysis are expected to be generally applicable to a wide range of proteins and polypeptides that form non-native aggregates.

The in vitro activity of Bay 09867, a new quinoline derivative, was compared with those of norfloxacin, nalidixic acid, ceftazidime, cefaclor, cefuroxime, gentamicin, and other antimicrobial agents, when appropriate, against 410 A new cluinoline derivative, 1-cyclopropyl-6-fluro-1, 4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid hydrochloride (Bay 09867) (Fig. 1 Inocula were prepared in the following manner. For all strains except those of B. fragilis, streptococci (including pneumococci), N. gonorrhoeae, and H. influenzae, the organisms were grown overnight in nutrient broth yielding a viable count of about 109 CFU/ml. H. influenzae, B. fragilis, and streptococcal strains were grown overnight in Levintal broth, thioglycolate broth, and Todd-Hewitt broth, respectively, yielding comparable viable counts. Pneumococci were also grown in Todd-Hewitt broth, and the viable count was 108 CFU/ml. N. gonorrhoeae strains were grown overnight on a chocolate agar plate, and the growth was removed just before use and suspended in peptone water to yield a viable count of 106 and 107 CFU/ml. Two inocula of 104 and 106 CFU, respectively, were used to test all the strains except N. gonorrhoeae and the pneumococci and were obtained by transferring 1 ,ul of an appropriate dilution of the overnight culture to the surface of the antibiotic-containing agar by a multipoint inoculating device (Denley-Tech Ltd., Billingshurst, England

The kinetics and structural transitions of non-native aggregation of alpha-chymotrypsinogen (aCgn) were investigated over a wide range of temperature and initial protein concentration at pH 3.5, where high molecular weight aggregates remained soluble throughout the reaction. A comparison of thermodynamic, kinetic, and spectroscopic data shows that aggregation under non-native-favoring conditions proceeds through a molten globule unfolded monomer state, with a nucleation and growth mechanism. Formation of irreversible aggregates and conversion to beta-sheet secondary structures occur simultaneously without detectable intermediates, suggesting that beta-sheet formation may be a commitment step during the nucleation and growth stages. Analysis of the kinetics using a Lumry-Eyring with nucleated polymerization (LENP) model provides the predominant nucleus size and the product of the intrinsic nucleation and intrinsic growth time scales at each state point. We find that the nucleus size depends on both temperature and protein concentration, and in some cases there is competition between two distinct nucleus sizes. The observed rate coefficient (kobs) for aggregation displays a maximum as a function of temperature because of the competition between folding-unfolding thermodynamics and the intrinsic growth and nucleation rates; the latter contribution has a large, negative activation enthalpy that dominates kobs at elevated temperatures. Temperature-jump experiments reveal that aggregates depolymerize at high temperatures, indicating that they are lower in enthalpy than the free monomer. Overall, the results suggest more generally that non-native aggregation may proceed through more than one nucleus size and that intrinsic kinetics of nucleation and growth may have significant entropic barriers.

The in-vitro activity of colistin sulphomethate sodium was compared with that of other commonly used antimicrobial agents against 377 recent clinical isolates of Gram-negative bacteria (including 94 strains of Pseudomonas aeruginosa from patients with cystic fibrosis) and 16 organisms with defined resistance patterns. Colistin was active against most strains of P. aeruginosa (MIC90 4 mg/L), Shigella spp. (MIC90 0.5 mg/L), Salmonella spp. (MIC90 1 mg/L), Acinetobacter spp. (MIC90 2 mg/L), Citrobacter spp. (MIC90 1 mg/L), Escherichia coli (MIC90 1 mg/L), Klebsiella spp. (MIC90 8 mg/L) and Enterobacter spp. (MIC50 1 mg/L). No useful activity was demonstrated against Providentia spp. or Serratia spp. The results show that colistin remains a useful antimicrobial agent against Gram-negative bacteria, particularly those strains which are resistant to more commonly used antibiotics.

The intrinsic time scales for nonnative aggregate nucleation (tau0(n)) and chain growth (tau0(g)) were determined for alpha-chymotrypsinogen A as a function of temperature under acidic conditions where the resulting aggregates do not appreciably condense. Previous results (Andrews and Roberts (2007) Biochemistry 46, 7558) indicated that the product tau0(n)tau0(g) increases with increasing temperature but could not distinguish tau0(n) and tau0(g). Separate experimental values of tau0(n) and tau0(g) are reported here from two approaches based on either (i) combining unseeded monomer loss kinetics with static light scattering of the resulting aggregates or (ii) seeded monomer loss kinetics as a function of number concentration of seed. Values of tau0(n) and tau0(g) from (i) and (ii) agree quantitatively, and indicate that nucleation has a large, negative effective activation energy (ca. -76 kcal/mol) while growth has at most a weak dependence on temperature. The results are consistent with a model in which nucleation requires significant conformational changes within a nonnative oligomer, beyond those for monomer unfolding. The results more generally illustrate the potential utility of approaches (i) and (ii) for quantitatively determining in vitro tau0(n) and tau0(g) values, as well as how the effects of seeding can be predicted purely from unseeded kinetics and static light scattering measurements prior to significant aggregate condensation.

For nearly a decade microbiologists have used the MIC breakpoints published in the BSAC Guide to Susceptibility Testing to interpret susceptibility. Historically, and unlike the rest of Europe, the UK and Ireland have used a comparative method of disc testing to interpret susceptibility rather than one based on a correlation between MIC and zone of inhibition. Although innovative when introduced in the 1970s, Stokes' comparative method has evolved ad hoc and it has become increasingly apparent that there is a need for a standardized method of disc testing that is correlated with BSAC MIC breakpoints. The method described here, like all other standardized methods of disc testing, cannot be adapted by the user, and interpretative criteria are only applicable if the method is adhered to fully. A major advantage of this approach to susceptibility testing is that data from several sources can be combined for surveillance of resistance, a task that has been made much easier by the introduction of this method and coincides with the availability of automated zone measuring devices. It is hoped that the method described here will provide the core document for standard operating procedures; however, changes will necessarily occur over time as the method is developed and refined.

The concentrations of moxifloxacin achieved after a single 400 mg dose were measured in serum, epithelial lining fluid (ELF), alveolar macrophages (AM) and bronchial mucosa (BM). Concentrations were determined using a microbiological assay. Nineteen patients undergoing fibre-optic bronchoscopy were studied. Mean serum, ELF, AM and BM concentrations at 2.2, 12 and 24 h were as follows: 2.2 h: 3.2 mg/L, 20.7 mg/L, 56.7 mg/L, 5.4 mg/kg; 12 h: 1.1 mg/L, 5.9 mg/L, 54.1 mg/L, 2.0 mg/kg; 24 h: 0.5 mg/L, 3.6 mg/L, 35.9 mg/L, 1.1 mg/kg, respectively. These concentrations exceed the MIC(90)s for common respiratory pathogens such as Streptococcus pneumoniae (0.25 mg/L), Haemophilus influenzae (0.03 mg/L), Moraxella catarrhalis (0.12 mg/L), Chlamydia pneumoniae (0.12 mg/L) and Mycoplasma pneumoniae (0. 12 mg/L) and indicate that moxifloxacin should be effective in the treatment of community-acquired, lower respiratory tract infections.