T-mycoplasmas: a study of the morphology, ultrastructure and mode of division of some human strains.

The morphology of 10 strains of T-mycoplasma was studied in wet preparations of viable cells by darkfield, phase-contrast and interference microscopy, and in fixed preparations by various techniques of electron microscopy. Mycoplasma-like artefacts in the horse-serum component of the medium were eliminated by filtration. All 10 strains were similar. Individual cells were spherical, 0-25-1-0 mum in size, with a bounding trilaminar membrane, 10 nm thick and containing 7-5-12-5-nm particles, and a layer of pilus-like projections, 5-8 nm long, on the outer surface. A possible capsular matrix was observed only by the pseudoreplica technique. The cells contained 12-15-nm ribosomes, nuclear fibroids 7-5-9 nm wide, and vacuoles. During replication, the cell elongated slightly and the ribsomes migrated to the ends of the cell leaving a ribosome-free area into which the bounding membrane invaginated to form a bud. The bud eventually separated by completion of the process of invagination; a cross-septum did not form. Usually only a single bud developed but sometimes two appeared simultaneously.

Mycoplasma hominis, Ureaplasma urealyticum, and Corynebacterium genitalium recovered from the lower genital tracts of adolescent women.

The lower genital tracts of 137 adolescent women were examined for the presence of Mycoplasma hominis, Ureaplasma urealyticum, and Corynebacterium genitalium in relation to sexual activity, previous pregnancy, presence of vaginal discharge and oral contraceptive use. None of the sexually inactive and 10% of the sexually active adolescent females were colonized with U. urealyticum. None of the sexually inactive and 4% of the sexually active adolescent females were colonized with C. genitalium. Nineteen percent of the sexually inactive and 36% of the sexually active adolescent females were colonized with M. hominis. The presence of M. hominis in the lower genital tract was not associated with any clinically identifiable vaginal discharge or inflammatory changes in exfoliated cervical and vaginal epithelial cells. The presence of M. hominis in the lower genital tract did not appear to be related to the use of oral contraceptives or antecedent pregnancy. There was no significant difference in the recovery rates of these microorganisms when we compared women who had non-specific vaginitis with those who did not. There is no evidence from this study that any of these microorganisms is responsible for non-specific vaginitis.

Differential responses of single cells and aggregates of mycoplasms to ultraviolet irradiation.

Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M. pneumoniae, and M. salivarium were inactivated exponentially by ultraviolet (UV) irradiation, in contrast to broth cultures containing clusters of elementary bodies. The susceptibility of the mycoplasmas was unaffected by storage at 2-4 C and at -70 C, by sonication, and by filtration. The rate of inactivation was dependent on the intensity of the radiations but independent of the concentration of the cells. Therefore, single-cell suspensions of these mycoplasmas could be differentiated from aggregates of cells by exponential inactivation of the colony-forming units (CFU). By this criterion, the CFU of M. arthritidis in the exponential phase of growth consisted of single cells, in contrast to the other species in which the CFU contained two or more elementary bodies. Even though the cultures of M. fermentans (PG 18) were grown from single cells, they were not homogeneous in their susceptibility to UV light. Neither were cultures of M. arthritidis and M. orale type 1 grown from single cells which had survived irradiation.

Decontamination of microbiological incubators.

A practical method of employing moist heat for the elimination of bacterial and fungal contaminants in microbiological incubators is described.

T-mycoplasmas: Growth patterns and physical characteristics of some human strains.

Five T-mycoplasmas isolated from patients with nonspecific urethritis and five laboratory strains of T-mycoplasma were examined for differentiating biological properties. The strains differed by the presence or absence of a lag phase and the number of T-mycoplasmas constituting a colony-forming unit (cfu). Most T-mycoplasmas had no lag phase and a cfu consisting of single organisms. All had biphasic ultraviolet inactivation curves typical of suspensions containing both mononucleate and binucleate cells. Binucleate cells probably were in the process of division. They resisted sonication for 3 min. Sonication disrupted multicellular cfu in to single cells within 2 min. Stationary-phase organisms died more rapidly than exponential-phase cells. T-mycoplasmas replicated at 2 C; they grew more slowly in T-broth at 40 C and died in 2.5 min at 56 C. Inactivation curves at 45 C and 50 C differed but insufficiently to permit identification of individual strains. At pressures less than 5 psi, single-cell suspensions passed through filter membranes with 0.65-mum and 0.45-mum pores but were retained by membranes with 0.22-mum pores.

Differences in the growth requirements of some strains of Ureaplasma urealyticum of human origin.

The growth requirements of Ureaplasma urealyticum strains T-McA, T-Pi, T-27, and T-207 were studied. All strains grew in T-broth containing horse serum. Only strain T-McA grew in broth containing serum fraction A, a finding that confirmed that ureaplasmas are not homogeneous and suggested that they can be divided into two species on the basis of their requirement for horse serum. The growth factors required by T-McA are arginine, cystine, methionine, and unidentified metabolites in serum fraction A and trypticase soy broth. The vitamins in Eagle's minimal essential medium are not essential metabolites for T-McA but enhanced its growth. Therefore, we recommend that these vitamins be incorporated in ureaplasma media and that the effect of supplementing media with arginine, cystine, and methionine for the primary isolation of ureaplasmas be ascertained.

Adaptation of Mycoplasma hominis to an obligate parasitic existence in monkey kidney cell culture (BSC-1).

During attempts to eliminate Mycoplasma hominis from a monkey kidney BSC-1 cell line with antibiotics, the mycoplasmas were isolated repeatedly. However, the organisms ultimately failed to grow on medium although electron microscopy confirmed that the cell culture still contained mycoplasmas. Thus, the mycoplasma had adapted to an environment in which viable cells were required for growth. Budding mycoplasmas which are indicative of replication were seen associated with viable cells extracellularly. Moreover, structures resembling cycoplasmas were observed budding from the cells which suggests that the mycoplasmas replicate within the cells and are similar to many viruses in their manner of release from the cells.

Asymptomatic bacteriuria, bacteremia, and other infections due to NSU corynebacteria.

By means of the new medium, nonspecific urethritis (NSU) chocolate agar, NSU corymebacteria were isolated from patients with asymptomatic bacteriuria, bacteremia, cervicitis, conjuctivitis, and pericarditis, and also with bone marrow, wound, and cul-de-sac infections. The NSU corynebacteria were considered the etiologic agents. On the basis of biochemical reactions, antibiotic sensitivity, and complement fixation tests some isolates were the same microorganisms. Both patients with conjunctivitis were infected with the same NSU corynebacteria. A second isolate was cultured from patients with osteomyelitis and cervicitis, while a third was recovered from an infected leg wound and from a patient with pericarditis. Seven of the isolates, when injected into rabbits hypersensitive to four NSU corynebacteria isolated from the inflamed epididymis of patients with epididymitis, elicited delayed hypersensitivity reactions, which indicated that they also were related antigenically. It is suggested that nonspecific urethritis and eididymitis may represent an infection with NSU corynebacteria, or may be an extension of bacteriuria due to these microorganisms, with a delayed hypersensitivity reaction as a possible additional complication. Colony counts on NSU chocolate agar of the bacteria in urines from male and female patients were higher than those obtained on conventional agar media. NSU chocolate agar is superior to other agar media for the isolation of pathogenic and saprophytic bacteria not only from the urogenital tract but also from other foci of infection. It is easily prepared from commercial blood agar plates and its use should be considered when a selective medium is not required.

The preparation of transforming DNA from Mycoplasma hominis strain Sprott tetr and quantitative studies of the factors affecting the genetic transformation of Mycoplasma salivarium strain S9 tets to tetracycline resistance.

DNA extracted by a standard method from Mycoplasma hominis Sprott, resistant to 100 micrograms tetracycline, permitted the quantitative genetic transformation of tetracycline-sensitive Mycoplasma salivarium to resistance. The yield was 1 microgram DNA/10(9) cells. This DNA enabled determination of the optimum conditions for making M. Salivarium competent with CaCl2 and for studying some factors affecting transformation. Mycoplasma salivarium was transformed to resistance to 10, 20, and 30 micrograms tetracycline but not to 40 micrograms. The optimum DNA concentration for transforming resistance to 10, 20, and 30 micrograms tetracycline was the same, i.e., 50 micrograms DNA/10(8) viable cells. Treatment with DNase indicated that DNA uptake took 30 min. Competition between transforming DNA and DNA from calf thymus and M. salivarium tets inhibited transformation.

A diagnostic key employing biological reactions for differentiating pathogenic Corynebacterium genitalium (NSU corynebacteria) from commensals of the urogenital tract.

More than 100 strains of Corynebacterium genitalium, probably responsible for coryneform urethritis and other infections, and 600 commensals of the male and female urogenital tracts have been studied and grouped into five pathogenic types numbered I to V and six saprophytic types designated C-1 to C-6 on the basis of eight biological reactions. This preliminary classification has been based on differences in requirements for oxygen, on the fermentation of fructose, dextrose, sucrose, and starch together with the production of the enzymes gelatinase, lipase, and urease. One criterion differentiated the pathogens from the commensals: All pathogens were nonfructose fermenters whereas every commensal fermented this sugar.

Preparation of competent single-cell suspensions of Mycoplasma hominis tets and Mycoplasma salivarium tets for genetic transformation to tetracycline resistance by DNA extracted from Mycoplamsa hominis tetr.

DNA extracted from Mycomplasma hominis (Sprott strain), resistant to 100 micrograms of tetracycline/ml transformed M. hominis strain H29 and Mycoplasma salivarium strain S9, which are sensitive to 2.5 and 5.0 micrograms of tetracycline/ml, respectively, to resistance. The transformants were selected on agar medium containing 10 micrograms of tetracycline/ml. Some transformants were resistant also to 20 micrograms of tetracycline/ml, a finding confirming that transformation occurred between homologous and heterologous species and that resistance is stepwise and controlled by several genetic loci. Medium containing 10 micrograms of tetracycline/ml was bacteriostatic. Prototype experiments employing mixtures of strains that were tetr and tets (tetracycline-resistant and tetracycline-sensitive, respectively) demonstrated that tetr mutants and transformants formed typical fried-egg colonies when mixtures containing not more than 10(9) mycoplasmas were spread on tetracycline agar plates. No mutants to tetracycline resistance were detected. Both M. hominis and M. salivarium were competent after treatment with MgCl2 and CaCl2, while Mycoplasma orale type 2 was inactivated. During DNA extraction different quantities of DNA formed insoluble precipitates with protein, thus preventing quantitative experiments.

The preparation of membranes of some human T-mycoplasmas and the analysis of their carbohydrate content.

Purified membranes were prepared from seven human T-mycoplasmas of which four are laboratory strains and three isolates from patients with nonspecific urethritis. The T-mycoplasmas were resistant to osmotic shock and sonication. Membranes were obtained only after lysing most of the T-mycoplasmas by four passes through a cell fractionator at 40,000 psi and separating the membranes from the unlysed cells by differential centrifugation. The membranes contained between 1 and 7% carbohydrates by dry weight. Mannose, galactose, and glucose were identified with glucose in the largest concentration.

The urea requirement and urease production of some human species of T-mycoplasmas.

Seven species of human T-mycoplasmas that grow in Fraction A and 20 mug urea/ml died when the urea was omitted. Two species would not grow in Fraction A broth containing 10 mug/urea/ml. The other five strains grew in broth containing 10 mug urea/ml and were adapted by serial passage in broth containing decreasing concentrations of urea to grow in broth containing 2.5 mug/ml urea, but not in broth containing 1.25 mug/ml. Therefore the minimal urea requirement is not the same for the growth of all strains of T-mycoplasmas. In exponential phase broth cultures, urease was detected only intracellularly, none being found in the medium.

Morphological changes induced during fixation of Mycoplasma mycoides for electron microscopy.

Only spherical mycoplasmas 0.6-0.9 mum diam were observed by darkfield microscopy in single cell suspensions prepared from exponential broth cultures of Mycoplasma mycoides var mycoides and Mycoplasma mycoides var capri. Similar cells were seen in pseudoreplicas by electron microscopy and they are considered characteristic of the morphology of M. mycoides. When the mycoplasmas were fixed by the addition of 10% formalin to the suspensions or washed, centrifuged cells were fixed by glutaraldehyde, filamentous and ring forms were observed in electron micrographs. These are not considered typical of the morphology of M. mycoides but are artifacts produced during the preparation of the mycoplasmas for electron microscopy.

Binucleate classical mycoplasmas pathogenic for goats.

The growth of three pathogenic goat mycoplasmas, strains Y, KH1 and Mycoplasma mycoides var. capri (PG3), was studied. They formed classical colonies on agar containing 1/500 thallium acetate. They were inactivated during storage at 2 to 4 C and by freezing and thawing but not by shaking. Only KH1 was killed by sonic treatment. Ultraviolet inactivation curves showed that their colony-forming units were single binucleate cells. Details of their growth curves are given. Filtration through 0.45- or 0.3-mum membrane filters removed up to 97% of the cells. Less than 0.003% passed 0.22-mum membranes. In electron micrographs, the cells were seen replicating by budding and most were 0.6 to 0.9 mum in diameter; but cells between 0.1 and 0.2 mum reproduced. They usually multiplied by producing one bud, a form of binary fission. However, two buds were produced by some synchronized cells, indicating that both nuclei had divided simultaneously to form progeny, an alternate method of multiplication.

Infection of a nonspecific urethritis patient and his consort with a pathogenic species of nonspecific urethritis Corynebacteria, Corynebacterium genitalium, N. SP.

A patient with nonspecific urethritis (NSU) and his consort were examined for infection with NSU corynebacteria, mycoplasmas, and gonococci. No classic and T-mycoplasmas or gonococci were cultured, but one species of NSU corynebacteria was isolated not only from the patient's urethral discharge during three episodes of NSU but also from his consort. It was not isolated after successful treatment of the patient with tetracycline and the use of condoms prevented reoccurrence of urethritis. This NSU corynebacterium was isolated previously from one epididymitis patient and two NSU patients but not from any of the normal male and female subjects examined. Therefore, this strain is considered to be one of the etiologic agents of NSU and female subjects are asymptomatic carriers. In consequence, it is suggested that NSU corynebacteria which are commensals and pathogens of the male and female urogenital tracts should be incorporated in a new species, of the Coryneform group, and that this strain should be the type species, Corynebacterium genitalium n. sp.

Evaluation of differential media for the identification of Corynebacterium genitalium and Corynebacterium pseudogenitalium (group JK corynebacteria).

Tween purple agar containing 1% fructose (TFP agar) differentiated Corynebacterium genitalium from C. pseudogenitalium, which respectively formed colorless and yellow colonies after 72 h incubation at 37 degrees C aerobically or in 5-10% CO2 in air. Thus TFP agar is a differential medium. Corynebacteria-like colonies grown on nonspecific urethritis (NSU) chocolate agar from urogenital material were identified as C. genitalium, C. pseudogenitalium, or commensals when subcultured on TPF agar. TFP agar was unsuitable for their primary isolation since the commensals turned the medium yellow with 24 h incubation. Gentamicin cannot be employed as a selective agent in medium for the isolation of these corynebacteria. TFP agar containing 10 micrograms/mL entamicin inhibited most strains of C. pseudogenitalium and C. genitalium isolated from urogenital infections. It did not inhibit isolates of these corynebacteria from cancer patients or suppress the normal bacterial flora of the urogenital tract. Evidence that gentamicin-resistant strains are characteristic of nosocomial infections is presented.

Patient-controlled analgesia with and without background infusion. Analgesia assessed using the demand: delivery ratio.

Sixty adult patients following general surgical operation were treated with patient-controlled analgesia using morphine. Patients were allocated into three groups to receive: no background infusion, a 1 mg.h-1 or a 2 mg.h-1 background infusion. The other controls on the patient-controlled analgesia machine were set to allow a maximum dose of morphine of 6 mg.h-1 to each group. Analgesia was assessed after 4 and 24 h using a 100 mm horizontal visual analogue scale. The number of analgesic requests made by the patient and the number of those requests which resulted in successful deliveries was recorded. Patients who received a regimen including a background infusion had improved pain relief, particularly in the first 4 h of treatment (p < 0.05). Patients who received a background infusion of 2 mg.h-1 had an increased incidence of nausea (p < 0.05). A background infusion of 1 mg.h-1, with a 1 mg bolus dose and a 12 min lockout interval provided acceptable pain relief without excessive nausea. In all three groups the ratio of analgesic requests to successful deliveries correlated with the degree of pain reported by visual analogue score (p = 0.0001).

An evaluation of ultrasound imaging for identification of lumbar intervertebral level.

The accuracy of ultrasound imaging to identify lumbar intervertebral level was assessed in 50 patients undergoing X-ray of the lumbar spine. Using an ultraviolet marker, an anaesthetist attempted to mark the L2/3, L3/4 and L4/5 intervertebral spaces. A radiologist unaware of these marks attempted to mark the same spaces with the aid of ultrasound imaging. X-ray-visible pellets were taped to the back at the various marks prior to lateral lumbar X-ray. Ultrasound imaging identified the correct level in up to 71% of cases, but palpation was successful in only 30% (p < 0.001). Up to 27% of marks using the palpation method were more than one spinal level above or below the assumed level using palpation, but none were more than one level high or low using ultrasound guidance.