Evaluation of CHROMagar Orientation for differentiation and presumptive identification of gram-negative bacilli and Enterococcus species.

A new chromogenic plate medium, CHROMagar Orientation, was evaluated for use in the differentiation and presumptive identification of gram-negative bacilli and Enterococcus species by a multipoint inoculation (replicator) technique. In this study, 1,404 gram-negative bacilli and 74 enterococcal isolates were tested on CHROMagar Orientation. Six control American Type Culture Collection strains were also included with the testing to ensure quality control of the media. Of the Escherichia coli isolates (n = 588) tested, 99.3% produced a pink-to-red color. Only in four isolates that were O-nitrophenyl-beta-D-galactopyranoside (ONPG) negative did this result differ. Proteus mirabilis and P. vulgaris were well differentiated on this medium. P. mirabilis (n = 184) produced a clear colony with diffusible brown pigment around the periphery. By contrast, 15 of 16 P. vulgaris isolates produced bluish-green colonies with a slight brown background. All Aeromonas hydrophila isolates (n = 26) tested produced clear to pink colonies at 35 to 37 degrees C. This colony color changed to blue after 2 to 3 h of incubation at room temperature. A. hydrophila exhibited stronger color and better growth at 30 degrees C. Serratia marcescens (n = 29) demonstrated an aqua blue color that deepened to a darker blue when exposed to room temperature. All enterococcal isolates (n = 74) resulted in a blue color and gave pinpoint colonies on purity subcultures at 35 to 37 degrees C after 18 h of incubation. Similarity in color resulted in failure to discriminate accurately between Klebsiella, Enterobacter, and Citrobacter species. However, these species could be readily differentiated from other members of the family Enterobacteriaceae. Pseudomonas aeruginosa (n = 151) was easily differentiated from members of the Enterobacteriaceae but was less easily distinguishable from other gram-negative nonmembers of the Enterobacteriaceae. The medium was found to facilitate easy visual detection of mixed bacterial isolates in culture. When used in a replicator system, it easily detected mixed growths of organisms which may have otherwise led to false antibiotic susceptibility results. These mixed growths were not obvious on the routine susceptibility testing medium (Isosensitest).

Comparison of two commercial enzyme immunoassays with cytotoxicity assay and culture for the diagnosis of Clostridium difficile related diarrhea.

184 stool samples were analysed for the presence of Clostridium difficile and toxins using the Meridian Premier Toxin A and TechLab Tox-A EIA kits, selective culture and cytotoxin assay. Of the 184 samples 36 stools tested positive for cytotoxin. In comparison the sensitivity and specificity of the EIAs and culture were as follows: Meridian, 72 and 87, TechLab, 64 and 95, and selective culture, 83 and 96%, respectively. The positive predictive values and negative predictive values for the various methods were: Meridian, 58 and 93, TechLab, 77 and 92, and selective culture, 83 and 96%, respectively. Discrepant results to those obtained by cytotoxicity assay were encountered with both EIA kits evaluated and less so by culture. In this study direct isolation of Clostridium difficile from stool samples most closely paralleled the findings of the "gold standard" cell line cytotoxicity assay. It appears that a single test for the determination of Clostridium difficile disease is adequate, although a second method improves the predictability of the diagnosis. Direct culture of feces provided a reliable secondary procedure to cytotoxicity assay. The EIAs were simple to use, labour efficient and provided a rapid result. However the lack of sensitivity and relative expense did not justify their routine use in our laboratory.

Typing multidrug-resistant Staphylococcus aureus: conflicting epidemiological data produced by genotypic and phenotypic methods clarified by phylogenetic analysis.

An outbreak of an unusual tetracycline-sensitive, rifampicin- and ciprofloxacin-resistant, methicillin-resistant Staphylococcus aureus (MRSA) strain at a large teaching hospital was investigated. Two typing methods, phage typing and restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (RFLP-PFGE), gave conflicting results which were clarified by phylogenetic analysis. Phage typing identified all the "epidemic-associated" strains as identical, while RFLP-PFGE further divided these strains into four pulsotypes. Phylogenetic analysis showed these four pulsotypes were related genetically and also recognized a second strain of MRSA causing a continuing cross-infection problem. Variation in the RFLP-PFGE pattern was shown to occur following lysogenization of phage-sensitive MRSA. These results indicate that in analyzing outbreaks caused by subgroups of clonal organisms like MRSA, it is necessary to use at least two typing methods and that conflicts between these could be resolved by phylogenetic analysis.

Penetration of cefaclor into bronchial mucosa.

Bronchial mucosal biopsy specimens were obtained during fibreoptic bronchoscopy in 30 patients receiving a new oral cephalosporin antibiotic, cefaclor (10 had 250 mg, 10 had 500 mg, and 10 had 1000 mg every eight hours). In 10 patients (from all dosage groups) cefaclor was undetectable in the bronchial mucosa but in every case the serum concentration was low, suggesting incomplete absorption. The mean (SD) bronchial mucosal concentration after 250 mg was 3.78 (1.77) micrograms/g (range 2.1-5.8 micrograms/g, n = 4), after 500 mg 4.43 (2.04) micrograms/g (range 2.0-7.1 micrograms/g, n = 8), and after 1000 mg 7.73 (2.76) micrograms/g (range 5.0-12.7 micrograms/g, n = 6). A significantly higher concentration in the bronchial mucosa was achieved with 1000 mg than with 250 mg (p less than 0.05) or 500 mg (p less than 0.025). These concentrations should be effective against Streptococcus pneumoniae, most strains being inhibited below 1.0 microgram/ml. The concentrations were within one dilution of the minimal inhibitory concentration for Haemophilus influenzae, most strains being inhibited below 4.0 micrograms/ml. Some strains of H influenzae will not be inhibited by the concentrations of cefaclor found in the bronchial mucosa, particularly those that are ampicillin resistant.

Penetration of piperacillin into bronchial mucosa and sputum.

Bronchial mucosal biopsies were obtained during fibreoptic bronchoscopy in 12 patients receiving a new semisynthetic penicillin, piperacillin. The piperacillin levels estimated in bronchial mucosa exceeded those required to eradicate organisms associated with acute bronchitis, Haemophilus influenzae and Streptococcus pneumoniae, and compared favourably with those required for activity against a wide variety of anaerobic and Gram-negative organisms including Pseudomonas aeruginosa. Sputum and serum piperacillin levels were obtained from eight patients with bronchial disease receiving a five to seven day course (8 to 16 g/day). Sputum/serum level ratios were constant for the two dosages (10.7% for 8 g/day; 14.3% for 16 g/day) suggesting a diffusion transfer process, although the presence of pus in the sputum appeared to facilitate penetration. Seven patients achieved sputum levels exceeding those required for activity against Haemophilus influenzae and Streptococcus influenzae, and four for Pseudomonas aeruginosa. This study provides pharmacolinetic support of the use of piperacillin in bronchopulmonary infection.

Pharmacokinetics of piperacillin in renal failure.

Patients with chronic renal failure who received bolus doses of piperacillin maintained high serum levels for up to six hours. The percentage of the injected dose which was detectable in the urine in 24 hours was reduced proportionately to the severity of the renal failure.

New aid to management of septicaemia: rapid direct antibiotic susceptibility testing.

A rapid and reliable method of performing direct antibiotic susceptibility tests on positive blood cultures has been developed. A result is now available five hours after laboratory confirmation of septicaemia. An evaluation involving 55 blood-culture isolates demonstrated an over-all correlation of 97.8% when results obtained by the rapid direct Autobac l method were compared to those obtained by definitive testing procedures. The method is particularly applicable to isolates of Staphyloccus aureus, Pseudomonas aeruginosa, and the Enterobacteriaceae. It is especially significant because of the recent emergence of multiple antibiotic resistance amongst hospital strains of these bacteria with many isolates showing resistance to antibiotics frequently used in the empirical treatment of septicaemia. The rapid availability of a reliable antibiogram is important in allowing early appropriate chemotherapy and, hence, in the reduction of septicaemia-associated morbidity and mortality, and the length of stay in hospital.

Australian evaluation of Autobac I with suggested interpretive and technical modifications.

Autobac I, a recently introduced semiautomated method for rapid antibiotic susceptibility testing, has been evaluated by comparison with the calibrated dichotomous sensitivity disk diffusion technique, which is routinely used in many Australian hospitals. Only the most common clinical isolates, Staphylococcus aureus, Escherichia coli, Klebsiella sp., and Proteus mirabilis, were included in this evaluation, and an overall interpretive agreement of 93% was obtained. However, an unusually high rate of discrepancy was noted in several organism-antibiotic combinations, in particular E. coli and P. mirabilis with ampicillin, S. aureus with penicillin, and methicillin-resistant S. aureus with methicillin, erythromycin, and clindamycin. The discrepancies associated with ampicillin have been reduced from 29 and 24% for E. coli and P. mirabilis, respectively, to less than 5% after the utilization of commercial 10-micrograms diffusion disks, in preference to the lower antibiotic content disks supplied by the Autobac manufacturer. Furthermore, modifications in the interpretive procedure have eliminated discrepancies associated with S. aureus and penicillin.

The photobioluminometer, an instrument for the study of ecological factors affecting photosynthesis.

An instrument for measurement of bioluminescence and photosynthesis is described. It may be used to detect chemicals toxic to luminous bacteria or to measure photosynthetic oxygen production of algae in mixed culture with bacteria. The latter technique has been used to study the effect on algal photosynthesis of environmental factors such as light quality, temperature, and salinity, and to study the factors affecting chlorophyll synthesis. The technique is ideal for rapid detection of photosynthesis-inhibiting herbicides and other toxic substances in water.

The effect of high pressure on the hemolysis of red blood cells.

Investigations into the effects of pressure on the hypotonic hemolysis of human erythrocytes show that pressures up to 130 atm (1700 psi) do not potentiate the hemolysis as has previously been suggested. Furthermore, such pressures do not remove the protection against hypotonic hemolysis conferred by the presence of general anesthetics to more than a negligible extent.