Loss of the mitochondrial cox2 intron 1 in a family of monocotyledonous plants and utilization of mitochondrial intron sequences for the construction of a nuclear intron.

The intron content of plant organellar genes is a useful marker in molecular systematics and evolution. We have tested representatives of a wide range of monocotyledonous plant families for the presence of an intron (cox2 intron 1) in one of the most conservative mitochondrial genes, the cox2 locus. Almost all species analyzed were found to harbor a group II intron at a phylogenetically conserved position. The only exceptions were members of a single monocot family, the Ruscaceae: representatives of all genera in this family were found to lack cox2 intron 1, but instead harbor an intron in the 3' portion of the cox2 coding region (cox2 intron 2). The presence of cox2 intron 1 in families of monocotyledonous plants that are closely related to the Ruscaceae suggests that loss of the intron is specific to this family and may have accompanied the evolutionary appearance of the Ruscaceae. Interestingly, sequences that are highly homologous to cox2 intron 2 are found in a nuclear intron in a lineage of monocotyledonous plants, suggesting that the originally mitochondrial group II intron sequence was transferred to the nuclear genome and reused there to build a spliceosomal intron.

Evaluation of serum arabinitol as a diagnostic test for candidiasis.

Gas-liquid chromatography was used to quantitate the arabinitol concentration in the sera of patients with candidiasis and in that of control patients. Serum arabinitol was elevated in 59% (n = 34) of patients with Candida sepsis, in 39% (n = 38) with Candida colonization, in 14% (n = 62) with bacterial sepsis, and in 0% (n = 11) of normal persons. The above patients were subsequently divided on the basis of renal function. Of those with decreased renal function, serum arabinitol was elevated in 89, 92, and 50% of patients with Candida sepsis, Candida colonization, and bacterial sepsis, respectively. Of those with normal renal function, serum arabinitol was elevated in only 23 and 14% of patients with Candida sepsis and Candida colonization, respectively. When serum arabinitol/creatine ratios were calculated for patients with both increased arabinitol and increased creatinine, elevated ratios were obtained in 69, 36, and 0% of patients with Candida sepsis, Candida colonization, and bacterial sepsis, respectively.

Rapid biotyping of Haemophilus influenzae and Haemophilus parainfluenzae with PathoTec strips and spot biochemical tests.

PathoTec strips and spot biochemical tests were evaluated for the ability to biotype Haemophilus influenzae and Haemophilus parainfluenzae. Indole, urease, and ornithine decarboxylase reactions were tested. The results of PathoTec strips compared favorably with those conventional methods; the percent agreements were as follows: indole, 100; urease, 99.5; and ornithine, 95.5. Spot tests were simple and rapid, and the results also compared favorably with those of conventional tests; the percent agreements were as follows: indole, 99; urease, 100; and ornithine, 96.

Medium for selective isolation and presumptive identification of the Bacteroides fragilis group.

We developed and evaluated a new medium (FRAG agar) for the selective isolation and presumptive identification of the Bacteroides fragilis group. This medium contains 1% D-glucuronic acid as a fermentable carbon source, a reduced peptone content, gentamicin, and 20% bile. Presumptive identification of the B. fragilis group was based on growth, fermentation, and typical colony morphology. A total of 75 stock culture isolates of the B. fragilis group grew well on this medium, and 69 showed evidence of fermentation. Of 90 other anaerobes, none grew well or fermented glucuronic acid. In a clinical trial of 100 specimens sent for anaerobic culture, FRAG agar inhibited 71 of 71 anaerobes not belonging to the B. fragilis group, as well as 104 of 110 facultative organisms. A total of 33 isolates of the B. fragilis group were recovered on the selective medium, whereas only 23 were recovered by routine methods. Of 23 cultures positive for the B. fragilis group on routine plates, 22 were positive on FRAG agar.

Rapid identification of enterococci by reduction of litmus milk.

Of 100 strains of enterococci, 83% reduced litmus milk within 4 h. None of the 100 non-enterococcal streptococci tested were positive.

Comparison of the API 20E and Corning N/F systems for identification of nonfermentative gram-negative rods.

A total of 231 strains of nonfermenting gram-negative rods were tested on the API 20E system, the Corning N/F system, and conventional media. When the results of identification to species were compared, the API system agreed with the conventional system on 69% of the isolates, and the Corning system agreed with the conventional system on 79% of the isolates. Both kit systems were deficient in identifying Pseudomonas cepacia and the more unusual isolates.

Comparison of Micro-ID and API 20E systems for identification of Enterobacteriaceae.

The Micro-ID 4-h identification system for Enterobacteriaceae was compared to the API 20E overnight method, using 230 fresh clinical isolates and 74 stock cultures. Agreement was 97.8% for the clinical isolates and 93.2% for the stock cultures. Eighty-seven percent of primary culture plates containing gram-negative rods yielded sufficient growth to perform the 4-h Micro-ID identification on the same day the organisms were isolated.

Comparison of three methods for identifying nonfermenting gram-negative rods.

Seventy-six strains of nonfermenting gram-negative rods were tested on the Analytab Products, Inc. (API) system and on conventional media. In addition, 51 strains were tested on the Oxi-Ferm system. When the identification results were compared, the API agreed with the conventional system on 41% of the isolates and Oxi-Ferm agreed with the conventional system on 72% of the isolates. API had the greatest difficulty in identifying Pseudomonas aeruginosa. API and Oxi-Ferm both had difficulty identifying P. cepacia. Oxi-Ferm had more individual discrepant biochemical reactions than did API when compared to the conventional media, but still maintained a higher percentage agreement with the conventional system.

Use of cerebrospinal fluid lactate levels in the diagnosis of bacterial meningitis.

Ninety-two cerebrospinal fluids were analyzed by a simple gas-liquid chromatography method for lactic acid. The presence of greater than 30 mg/dl of lactic acid correlated with proven bacterial or cryptococcal meningitis. Elevated lactate levels were also found in two patients with suspected but not proven bacterial meningitis. In 78 patients who did not have bacterial meningitis the cerebrospinal fluid contained less than 30 mg/dl of lactic acid.

Rapid deoxyribonuclease test with methyl green.

A 4-h deoxyribonuclease test using methyl green to differentiate Serratia from other Enterobacteriaceae was developed. The tests agreed 100% with an overnight plate test for 100 Serratia, 83 Enterobacter, and 6 Klebsiella species.

Comparison of cream of rice agar and horse serum for differentiating germ tubes of Candida albicans from filaments of Candida tropicalis.

Simple cream of rice agar was superior to horse serum for the demonstration of germ tubes by Candida albicans and in the differentiation of pseudohyphae of Candida tropicalis from germ tubes at 37 degrees C. Mycelium and chlamydospores were also produced on this medium.

Rapid speciation of Haemophilus with the porphyrin production test versus the satellite test for X.

The detection of porphyrins produced from omega-aminolevulinic acid was evaluated for use in speciation of the Haemophilus. Two hundred and forty-eight clinical isolates of nonhemolytic Haemophilus were tested concurrently for porphyrin production and for X and V requirements. The porphyrin production test was more rapid (4 h) and more accurate than the satellite test.

A new type of penicillin resistance of Staphylococcus aureus.

Penicillin--"tolerant" Staphylococcus aureus strains are resistant to the lethal action of penicillins, but are inhibited by normal (low) concentrations. They are deficient in autolytic enzyme activity which appears to be necessary for bacteriolysis and the lethal action of penicillins. This "deficiency" is caused by a large excess of an inhibitor of autolysin. Seven such tolerant strains have been isolated from blood, bone, or sputum of patients who responded poorly to penicillin treatment of endocarditis, osteomyelitis, or staphylococcal pneumonia. These isolates were of different phage-types, and most showed cross-tolerance to the killing action of cephalosporins or vancomycin, antibiotics to which they were sensitive (inhibited). They were killed at normal rates by gentamicin, cycloserine, and rifampicin. Population analysis indicated that the proportion of tolerant organisms within a resistant strain is 7% or less; their ability to inhibit autolytic activity within their own and neighbouring cells appears to account for the net decreased autolytic activity of the entire strain; 44% of the bacteraemic strains studied showed penicillin tolerance. Tolerance is thus a common, clinically important form of penicillin resistance, that differs from previously described forms of pencillin resistance, that due to beta-lactamase, and that due to "intrinsic" (e.g., methicillin resistance) mechanisms.

Inoculation of API-20E from positive blood cultures.

The API-20E system (Analytab Products, Inc., Plainview, N. Y.) was inoculated from 4- to 6-h tryptic soy broth cultures that had been inoculated from positive blood cultures containing gram-negative bacilli. This method gave the same genus and species identification for 139 of 140 organisms (47 patient and 96 simulated positive cultures) when compared to the Analytab Products, Inc., recommended method of inoculation.

Stability of gentamicin in serum.

Patients' sera were divided into three portions when the initial gentamicin level was determined and were stored at -20, 4, and 25 degrees C in plastic or glass tubes. Gentamicin levels were repeated after 1 and 2 days of storage at the respective temperatures. There was no significant difference in gentamicin levels among portions, except those from a patient in renal failure with high serum concentrations of carbenicillin.

Significance of urinary isolates of coagulase-negative Micrococcaceae.

Of 16,347 urine cultures submitted to the hospital laboratory, 68 (0.4%) specimens from 50 patients yielded greater than 10(4) coagulase-negative staphylococci/ml in pure culture. A total of 62 of 63 organisms available for study were staphylococci: 45 Staphylococcus epidermidis (predominantly subgroup 1), 15 Staphylococcus saprophyticus (subgroup 3), and 2 Staphylococcus aureus. Twenty-one patients had "probable" urine infections. Eight patients had two or more positive urine cultures, and all isolates from the same patients were identical (by morphology, antibiotic susceptibility, and hemolytic pattern). Nine (75%) of the 12 isolates of S. saprophyticus, which were novobiocin resistant and nonhemolytic on the synergistic hemolysis test, were from patients with probable urinary infection. Eight were young women with acute symptoms and pyuria. Differences in the glucose and mannitol fermentation tests with different media may lead to difficulties in identification. Novobiocin resistance cannot be relied upon to differentiate isolates of S. saprophyticus from S. epidermidis.

Current taxonomy and identification of nonfermentative gram negative bacilli.

In recent years significant advances have been made in the characterization and taxonomy of nonfermentative bacilli as well as in the recognition of their pathogenic potential for man. In addition, certain other infrequently isolated bacteria that may be fermentative have been recognized, and improved methods for their identification have been developed. This discussion attempts to present a practical approach to the identification of these micro-organisms in the clinical microbiology laboratory.