Effects of Microplate Type and Broth Additives on Microdilution MIC Susceptibility Assays

ABSTRACTThe determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50 years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity. For example, daptomycin requires medium supplemented with Ca2+, and the lipoglycopeptides dalbavancin and oritavancin require Tween 80 to be added to the growth medium to prevent the depletion of free drug via adsorption to the plastic microplate. In this report, we examine systematically the effects of several different plate types on microdilution broth MIC values for a set of antibiotics against Gram-positive and Gram-negative bacteria, both in medium alone and in medium supplemented with the commonly used additives Tween 80, lysed horse blood, and 50% human serum. We observed very significant differences in measured MICs (up to 100-fold) for some lipophilic antibiotics, such as the Gram-positive lipoglycopeptide dalbavancin and the Gram-negative lipopeptide polymyxins, and found that nonspecific binding plates can replace the need for surfactant additives. Microtiter plate types and any additives should be specified when reporting broth dilution MIC values, as results can vary dramatically for some classes of antibiotics.

Myeloperoxidase Selectively Binds and Selectively Kills Microbes

ABSTRACTMyeloperoxidase (MPO) is reported to selectively bind to bacteria. The present study provides direct evidence of MPO binding selectivity and tests the relationship of selective binding to selective killing. The microbicidal effectiveness of H2O2and of OCl−was compared to that of MPO plus H2O2. Synergistic microbicidal action was investigated by combiningStreptococcus sanguinis, a H2O2-producing microbe showing low MPO binding, with high-MPO-bindingEscherichia coli,Staphylococcus aureus, orPseudomonas aeruginosawithout exogenous H2O2, with and without MPO, and with and without erythrocytes (red blood cells [RBCs]). Selectivity of MPO microbicidal action was conventionally measured as the MPO MIC and minimal bactericidal concentration (MBC) for 82 bacteria includingE. coli,P. aeruginosa,S. aureus,Enterococcus faecalis,Streptococcus pyogenes,Streptococcus agalactiae, and viridans streptococci. Both H2O2and OCl−destroyed RBCs at submicrobicidal concentrations. Nanomolar concentrations of MPO increased H2O2microbicidal action 1,000-fold. Streptococci plus MPO produced potent synergistic microbicidal action against all microbes tested, and RBCs caused only a small decrease in potency without erythrocyte damage. MPO directly killed H2O2-producingS. pyogenesbut was ineffective against non-H2O2-producingE. faecalis. The MPO MICs and MBCs forE. coli,P. aeruginosa, andS. aureuswere significantly lower than those forE. faecalis.The streptococcal studies showed much higher MIC/MBC results, but such testing required lysed horse blood-supplemented medium, thus preventing valid comparison of these results to those for the other microbes.E. faecalisMPO binding is reportedly weak compared to binding ofE. coli,P. aeruginosa, andS. aureusbut strong compared to binding of streptococci. Selective MPO binding results in selective killing.

Effect of Polysorbate 80 on Oritavancin Binding to Plastic Surfaces: Implications for Susceptibility Testing

ABSTRACT Oritavancin, a semisynthetic lipoglycopeptide with activity against gram-positive bacteria, has multiple mechanisms of action, including the inhibition of cell wall synthesis and the perturbation of the membrane potential. Approved guidelines for broth microdilution MIC assays with dalbavancin, another lipoglycopeptide, require inclusion of 0.002% polysorbate 80. To investigate the potential impact of polysorbate 80 on oritavancin susceptibility assays, we quantified the recovery of [14C]oritavancin from susceptibility assay plates with and without polysorbate 80 and examined the effect of the presence of polysorbate 80 on the oritavancin MICs for 301 clinical isolates from the genera Staphylococcus, Enterococcus, and Streptococcus. In the absence of polysorbate 80, [14C]oritavancin was rapidly lost from solution in susceptibility assay test plates: 9% of the input drug was recovered in broth at 1 h when [14C]oritavancin was tested at 1 μg/ml. Furthermore, proportionately greater losses were observed at lower oritavancin concentrations, suggesting saturable binding of oritavancin to surfaces. The inclusion of 0.002% polysorbate 80 or 2% lysed horse blood permitted the recovery of 80 to 100% [14C]oritavancin at 24 h for all drug concentrations tested. Concordantly, oritavancin MIC90s for streptococcal isolates, as determined in medium containing 2% lysed horse blood, were identical with and without polysorbate 80. In stark contrast, polysorbate 80 reduced the oritavancin MIC90s by 16- to 32-fold for clinical isolates of enterococci and staphylococci, which are typically cultured without blood. The results presented here provide evidence that the MIC data for oritavancin in the current literature significantly underestimate the potency of oritavancin in vitro. Moreover, the combination of data from MIC and [14C]oritavancin recovery studies supports the revision of the oritavancin broth microdilution method to include polysorbate 80 throughout the assay.

Activities of Trovafloxacin, Gatifloxacin, Clinafloxacin, Sparfloxacin, Levofloxacin, and Ciprofloxacin against Penicillin-Resistant Streptococcus pneumoniae in an In Vitro Infection Model

ABSTRACT We adapted an in vitro pharmacodynamic model of infection to incorporate infected fibrin clots. The bactericidal activities of various fluoroquinolones against two strains of penicillin-resistantStreptococcus pneumoniae were studied over a 48-h period. Bacteria were prepared in Muller-Hinton broth by using colonies from a 24-h tryptic soy agar plus 5% sheep blood plate and were added to a mixture of cryoprecipitate (80%) and thrombin (10%) to achieve approximately 106 CFU of organism per fibrin clot. The fibrin clots were suspended into the models and removed, in triplicate, at various time points over 48 h. Control models were also conducted to characterize the growth of S. pneumoniae in the growth medium without antibiotic. Trovafloxacin, gatifloxacin, clinafloxacin, sparfloxacin, levofloxacin, and ciprofloxacin were administered to simulate their pharmacokinetic profiles in humans. Fibrin clot samples were also plated onto antibiotic-containing tryptic soy agar plus 5% lysed horse blood to detect resistance. The newer fluoroquinolones demonstrated better activity than ciprofloxacin against both isolates. In conclusion, the newer quinolones demonstrated significant activity against penicillin-resistant S. pneumoniae, with standard dosing resulting in area under the concentration-time curve/MIC ratios and peak concentration/MIC ratios that resulted in 99.9% killing against these isolates.

Susceptibility of Group A Beta-Hemolytic Streptococci in the Lower St Lawrence Region, Quebec

OBJECTIVE: To determine the susceptibility of group A beta-hemolytic streptococci (GABHS) in the lower St Lawrence region, Quebec to different antibiotics, particularly macrolides, and to compare different antibiogram methods (disk diffusion, E-test and microdilution) and incubation atmospheres (ambient air and 5% carbon dioxide).METHODS: A total of 384 strains of GABHS isolated from 377 patients (throat 335; other sites 49) from three hospitals in the lower St Lawrence region were analyzed for their susceptibility to erythromycin, clarithromycin, azithromycin, penicillin, clindamycin, cephalothin, rifampin and vancomycin by disk diffusion on Mueller-Hinton (MH) agar supplemented with 5% defibrinated sheep blood (MHB) at 35ºC in 5% carbon dioxide. Strains that were found to be intermediately resistant or resistant to one of the antibiotics by disc diffusion, strains from sites other than throat, and a sample of 97 pharyngeal strains were evaluated by E-test on MHB (35ºC, 5% carbon dioxide) for their susceptibility to the antibiotics erythromycin, clarithromycin, azithromycin, penicillin, clindamycin and ceftriaxone. In addition, minimum inhibitory concentrations (MICs) were determined for erythromycin and azithromycin by broth microdilution using MH broth supplemented with 2.5 % of lysed horse blood (35ºC, ambient air) on strains that were resistant or intermediately resistant to the macrolides (erythromycin, clarithromycin, azithromycin). An evaluation was also carried out on these strains to determine the influence of the incubating atmosphere (ambient air versus 5% carbon dioxide) on susceptibility results obtained by disk diffusion (erythromycin, clarithromycin and azithromycin) and E-test (erythromycin and azithromycin) methods.RESULTS: Nine strains (2%) from nine patients (throat eight, pus one) were resistant to all macrolides as tested by three different techniques (disk diffusion, E-test and microdilution). All strains were susceptible to all the other antibiotics tested. For the strains intermediately resistant or resistant to macrolides, incubation in a 5% carbon dioxide atmosphere was associated with a reduction in the diameter of inhibition determined by disk diffusion (P<0.001) with frequent minor variations in interpretation, and with an increase in the MIC by E-test (P<0.001), which had no impact on interpretation.CONCLUSIONS: Resistance of GABHS to macrolides was not common (2%) in the lower St Lawrence Region. GABHS susceptibility to erythromycin seemed to predict the susceptibility to the other macrolides. Significant variation in antibiogram results (disk diffusion and E-test) of GABHS susceptibility to macrolides was related to the incubation atmosphere and may have an impact on the interpretation of disk diffusion results.

The Antimicrobial Implications of the Pharmacokinetics of Cotrimoxazole in CAPO Patients

Cotrimoxazole (TMP/SMX) has been used to treat continuous ambulatory peritoneal dialysis (CAPD) associated peritonitis. It is considered bactericidal for some species. The most common single organism responsible for this type of peritonitis is Staph. epidermidis (SE). When the drug is given orally, the typical ratio of TMP to SMX achieved in the peritoneal fluid is 1:5, which is different from the optimal combination for antimicrobial synergy of 1:19. This study investigated the antimicrobial activity of TMP alone and TMP/SMX by agar dilution at ratios of 1:19, 1:10 and 1:5 against 99 strains of Staphylococcus epidermis (SE). The majority of strains were susceptible to TMP and to all ratios of TMP/SMX by the routine agar dilution methods. We studied the bactericidal activity of TMP/SMX against one strain each of SE and of Staph. aureus (SA) in pooled uninfected spent dialysate by killing curve experiments. TMP at concentrations of 0.5 to 2.0 μg/ml was bacteriostatic. TMP/SMX became slowly bactericidal when 5 % lysed horse blood, a source of thymidine phosphorylase, was added to the dialysate. It is concluded that susceptibility tests of inhibition do not predict bactericidal activity of TMP/SMX in recovered dialysate. Inhibition of TMP/ SMX is most likely due to thymidine present in dialysate. TMP alone was as effective against SE as TMP/ SMX. Until it is shown that bactericidal activity is not required for cure of CAPD-related peritonitis, cotrimoxazole should not be used routinely for its treatment.

Counts on viableBacteroides fragilis:a modification of the microdroplet technique

SummaryA modification of Sharpe's microdroplet counting technique is presented which permits viable counting ofBacteroides fragilis. Lysed horse blood was added to 0·1 ml soy digest agar droplets and 2% was found to be the optimal concentration which combined adequate intradroplet colony size with acceptable visibility on the counting screen of the Colworth Droplette machine.