Fritz D. Schoenknecht

Anaerobic Bacteria in Nonspecific Vaginitis

To study the cause of nonspecific vaginitis, we analyzed vaginal fluid from normal women and from 53 women with nonspecific vaginitis, using quantitative anaerobic cultures and gas-liquid chromatography for short-chained organic-acid metabolites of the microbial flora. In normal vaginal fluid, lactate was the predominant acid, and the predominant organisms were lactobacillus and streptococcus species (lactate producers). In nonspecific vaginitis, lactate was decreased, whereas succinate, acetate, butyrate, and propionate were increased, the predominant flora included Gardnerella (Haemophilus) vaginalis (acetate producer), and anaerobes, which included bacteroides species (succinate producers) and peptococcus species (butyrate and acetate producers). After metronidazole therapy, symptoms and signs of nonspecific vaginitis cleared, butyrate and propionate disappeared, and lactate and lactate-producing organisms became predominant. We conclude that certain anaerobes act with G. vaginalis as causes of nonspecific vaginitis, and that a high ratio of succinate to lactate in vaginal fluid is a useful indicator in the diagnosis of this condition.

Neonatal Escherichia coli septicemia--bacterial counts in blood.

Bacterial counts in blood of E. coli (number of viable organisms per milliliter) are reported for 30 neonates whowere cultured because of suspected septicemia. Fifteen of the infants died; 13 of the deaths occurred within 48 hours after cultures were obtained and appropriate antibiotic therapy had been initiated. Thirty-five positive cultures were obtained from the 30 neonates; in 11 (31%) of the cultures the colony counts were in excess of 1,000/ml; in 5 (14%), the counts ranged from 50 to 1,000/ml; in 11 (31%), from 5 to 49/ml; in 8 (23%), from 0 to 4/ml. Seventy-three per cent (8/11) of the neonates with colony counts greater than 1,000/ml died.

Laboratory evaluation of a rapid, automatic susceptibility testing system: report of a collaborative study.

Seven laboratories participated in a collaborative study to evaluate the Autobac 1 system. Results obtained with this assay system were compared to those obtained by the standardized Bauer-Kirby disk diffusion test, and each of these two methods was compared to the agar dilution technique. Comparison of the Autobac 1 and the disk diffusion results from the seven laboratories showed an overall average of 91.5% interpretive agreement with the 17 antimicrobial agents tested. The distribution in the levels of Autobac 1/disk diffusion agreement was such that with 13 antimicrobial drugs agreement was 90% or higher; with three, between 85 and 90%; and with one, 77% (nitrofurantoin). Comparison of the Autobac 1 and disk diffusion tests with the International Collaborative Study agar dilution test showed that both methods gave levels of agreement with the International Collaborative Study agar dilution technique that were generally high and equivalent. The average overall agreement between the agar dilution test and each of the other two methods was approximately 90%. Disagreements that did occur tended to involve organisms that were drug susceptible by the Autobac 1 system but intermediate or resistant by the other two methods. This was in part due to the narrow intermediate interpretive zone of the Autobac 1 test. In reproducibility studies with the Autobac 1 and disk diffusion methods, no significant differences were observed between the interpretive reproducibility of the two methods. Images

Effect of Several Components of Anaerobic Incubation on Antibiotic Susceptibility Test Results

The factors influencing the in vitro activity of antibiotics during anaerobic incubation were studied by the disc method with a facultative organism, Escherichia coli. We observed the effects of incubation aerobically, anaerobically (Torbal jars), in a CO2 incubator, and aerobically and anaerobically with all CO2 removed. We also monitored pH changes during incubation and observed the effect of two different initial agar pH values (7.4 and 8.3). With aminoglycosides, zones were larger at pH 8.3 and, in each agar pH group, zones were decreased by incubation with increased CO2 (anaerobically and CO2 incubator). A fall in agar pH took place during the first 5 to 7 hr of incubation when increased CO2 was present. Decreased aminoglycoside zones in the presence of increased CO2 were due to fall in agar pH. Erythromycin showed the same zone size changes as the aminoglycosides. Chloramphenicol zones were somewhat smaller at the lower medium pH. Zones around tetracycline discs were largest after incubation anaerobically. Further aerobic (or CO2) incubation of plates after anaerobic incubation resulted in large “zones of relative inhibition” around the aminoglycoside discs. This suggests that these antibiotics had become more active after exposure to aerobic conditions. Our studies indicate that antibiotic susceptibility test results can be significantly altered by several components of anaerobic incubation including changes in agar pH and CO2 concentration as well as anaerobiosis per se. Images

Cation components of Mueller-Hinton agar affecting testing of Pseudomonas aeruginosa susceptibility to gentamicin.

Seven lots of Mueller-Hinton agar were examined for calcium and magnesium contents and their distribution in pools or compartments. Gel disruption and centrifugation yielded the soluble cations, which varied from 9 to 113% of the total calcium and from 76 to 102% of the total magnesium. Throughout the experiments, a standardized disk diffusion test, using Pseudomonas aeruginosa (ATCC 27852) and a 10-μg gentamicin disk, served as an indicator for medium performance. Zone diameters correlated well with the sums of the soluble calcium and magnesium values in the different lots (r = −0.85). Ionized calcium, presumably the biologically active ion, was measured with a calcium-specific electrode. It represented only a fraction of the soluble calcium pool in three lots. Autoclaving resulted in shifts of the cations between the different pools. Addition of magnesium to one medium lot resulted in shifts of soluble and ionized calcium, indicating an interdependence of calcium and magnesium, and zone diameters correlated with soluble magnesium (r = −0.98), soluble calcium (r = −0.96), and ionized calcium (r = −0.96) in this experiment. Manipulation of one medium to match the performance of another showed that excess amounts of both ions were required to obtain similar performance. Satisfactory performance of an individual medium can be obtained by cation supplementation, but simple adjustment will not suffice for all media. The interaction of the other cation pool components must also be evaluated.

Relationship of Early Readings of Minimal Inhibitory Concentrations to the Results of Overnight Tests

Broth dilution minimal inhibitory concentration (MIC) readings were compared after different incubation periods and with different inoculum concentrations. The purpose was to determine the best conditions for obtaining early results as close as possible to overnight readings. Initially, 76 antibiotic-organism combinations were tested using the International Collaborative Study technique and inoculum and were read after 3, 8, and 18 h of incubation. Approximately 28% of tests showed fourfold or greater increases in MICs after 18 h of incubation compared with the 3-h readings. No overnight MICs were lower than early readings. MICs of single antibiotics against seven organisms were also read with an automatic particle counter to confirm the validity of the visual readings. Experiments were made to determine whether inoculum manipulation could reconcile the differences between 3- and 18-h MIC results. One hundred and eight organism-antibiotic combinations were tested comparing 3-h MIC readings using an inoculum of 107 organisms per ml with overnight readings using 105 per ml. In 71 cases, readings with both inocula were within the range tested and 57 (86%) were within ±1 log2 of each other and followed an approximately normal distribution. Improved comparability between early read and overnight MICs thus may be achieved by inoculum manipulation, and this may be a suitable approach in the future development of automated procedures.

Effect of Different Lots of Mueller-Hinton Agar on the Interpretation of the Gentamicin Susceptibility of Pseudomonas aeruginosa

Population distributions and quality control data for strains of Pseudomonas aeruginosa tested for gentamicin susceptibility on six lots of Mueller-Hinton agar were analyzed. The lots of agar were used in three University of Washington hospitals from April 1975 through October 1977. The analyses indicated that the performance of members of the P. aeruginosa populations in each hospital closely followed the performance of the quality control strain, P. aeruginosa ATCC 27853, when tested on each lot of Mueller-Hinton medium. The variability of zone diameters with the P. aeruginosa populations and the quality control strain indicated that a fixed indeterminate range (13 to 16 mm) of gentamicin susceptibility was not applicable to these organisms as it was with the Enterobacteriaceae. Variability in gentamicin susceptibility results was demonstrated in both minimal inhibitory concentration and disk diffusion tests when eight selected P. aeruginosa strains and the quality control strain were tested on each lot of medium. This variation in susceptibility to gentamicin was not related to the total Ca2+, Mg2+, or Zn2+ content of each lot of medium. The data demonstrated that a moving indeterminate range of gentamicin susceptibility, 3 to 6 mm below the mean zone diameter of the quality control strain, was a suitable criterion for strains tested on a single medium lot. These results illustrate the importance of defining stringent performance standards for media used in the susceptibility testing of P. aeruginosa with gentamicin and other aminoglycoside antibiotics.

Interpretation of the Disk Diffusion Susceptibility Test for Amikacin: Report of a Collaborative Study

Because excessively high rates of false resistance have been encountered with the 10-μg amikacin disk in diffusion susceptibility tests, a study was performed to examine existing zone diameter interpretative criteria and to compare the accuracy of 10- and 30-μg amikacin disks by the error rate-bounded classification scheme. Although current zone diameter interpretative criteria eliminate false susceptibles, there is an unacceptably high rate of false resistants. This problem can be resolved in most instances by revising the zone diameter interpretative criteria for the 10-μg disk (resistant, ≤9 mm; indeterminate, 10 to 11 mm; susceptible, ≥12 mm) or, preferably, by replacing the 10-μg disk with a 30-μg disk and adopting new interpretative criteria (resistant, ≤14 mm; indeterminate, 15 to 16 mm; susceptible, ≥17 mm). Because of significant differences in performance among media, it is necessary to include Pseudomonas aeruginosa ATCC 27853 among controls routinely tested and to exclude from use lots of Mueller-Hinton agar yielding results outside the 75% tolerance (90% confidence) limits for amikacin.

Emergence in a Burn Center of Populations of Bacteria Resistant to Gentamicin, Tobramycin, and Amikacin: Evidence for the Need for Changes in Zone Diameter Interpretative Standards

From July 1974 through June 1976, a number of isolates of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa from the Burn Center exhibited a shift to smaller zone diameters with gentamicin than did isolates from the general hospital population. Although many had zone diameters ≥13 mm and would have been considered susceptible by this breakpoint, they were found to have minimal inhibitory concentrations (MICs) of ≥8 μg of gentamicin per ml by agar dilution testing. Zone diameters and MICs of gentamicin, tobramycin, and amikacin were subsequently compared for 168 isolates from both the Burn Center and general hospital. The results revealed many isolates that fell into presently used gentamicin- and tobramycin-“susceptible” categories by disk diffusion tests but were resistant by MIC. The data indicated that criteria for gentamicin disk diffusion testing should include an intermediate or indeterminate category, and that the limits of the intermediate category for tobramycin and amikacin should be expanded.

Stopcock Contamination in an ICU

Because Propionibacterium acnes and Staphylococcus epidermidis are the predominant organisms found on human skin, it seemed probable that st phylococci, at least, would be found in the open stopcocks (1). Other organisms normally present on the skin are Staphylococcus aureus, nonhemolytic streptococci, enterococci (Streptococcus faecalis), and Candida. Gram-negative coliforms and mimeae may be found. Some of the normal flora of the