A physical and genetic map of the Mycoplasma flocculare ATCC 27716 chromosome reveals large genomic inversions when compared with that of Mycoplasma hyopneumoniae strain J(T).

Pulsed-field gel electrophoresis and DNA hybridization data were used to construct a chromosomal map of Mycoplasma flocculare ATCC 27716, a non-pathogenic inhabitant of porcine respiratory tracts. Twenty-one genetic markers were placed on the map. Comparison of the genetic map with that of the closely related Mycoplasma hyopneumoniae strain J(T), the type strain of the causative agent of enzootic pneumonia in pigs, identified three chromosomal inversions that differentiate these genomes. One of these inversions involves genes that possibly may be involved in M. hyopneumoniae pathogenicity.

A physical and genetic map of the Mycoplasma hyopneumoniae strain J genome.

A macrorestriction map of the genome of Mycoplasma hyopneumoniae strain J, the type strain of the causative agent of enzootic pneumonia in pigs, was constructed using pulsed-field gel electrophoresis (PFGE) and DNA hybridization. The size of the genome as determined by PFGE was approximately 1070 kb. Assembly of the M. hyopneumoniae genomic map was facilitated and complimented by the simultaneous construction of an ordered cosmid library. Five contigs of overlapping cosmids were assembled, which together represent coverage of approximately 728 kb. Forty-two genetic markers (including three types of repeated elements) were placed on the M. hyopneumoniae map. Closer examination of an ApaI restriction fragment contained entirely within a single cosmid insert suggests that the genome size may be overestimated by PFGE.

Gene amplification (PCR) to detect and differentiate mycoplasmas in porcine mycoplasmal pneumonia.

The causative agent of porcine mycoplasmal pneumonia, Mycoplasma hyopneumoniae, is difficult and time-consuming to isolate. Serological identification using antibodies induced by the disease is confused by cross-reaction with a closely related organism, Myc. flocculare. From pig lungs obtained at slaughter for meat and deemed free of acute disease, it was possible to detect by culture both Myc. hyopneumoniae and Myc. flocculare. This study has improved on an earlier PCR detection of DNA from the former species by using a nested PCR capable of detecting the purified DNA equivalent to one mycoplasmal genome. With this PCR assay both mycoplasma species were detected and differentiated directly from lung tissue.

Phylogenetic relationships of the porcine mycoplasmas Mycoplasma hyosynoviae and Mycoplasma hyopharyngis.

The phylogenetic positions of the porcine mycoplasmas Mycoplasma hyosynoviae and Mycoplasma hyopharyngis were determined by using PCR-amplified 16S rRNA gene sequences. M. hyosynoviae is a member of the Mycoplasma hominis group, while M. hyopharyngis belongs to the Mycoplasma fermentans group of mollicutes. Neither species is closely related to previously characterized porcine mycoplasmas belonging to the Mycoplasma hyorhinis group.

Ureaplasma gallorale, an isolate from chickens, is most closely related to the human isolate, U. urealyticum.

Ureaplasma gallorale is a urease-containing mycoplasma (a member of the Mollicutes) which is pathogenic for chickens, from which it was originally isolated. We amplified the 16S rRNA gene of this bacterium and then cloned and sequenced the amplicon. A phylogenetic analysis based on an alignment of the 16S rRNA sequences of U. gallorale and several other Ureaplasma species revealed that U. gallorale is more closely related to Ureaplasma urealyticum than to other Ureaplasma species.

An unusual rRNA gene organization in Mycoplasma fermentans (incognitus strain).

The macro-restriction map of Mycoplasma fermentans (incognitus strain) was constructed and its rRNA genes were located on the map. It was found that this organism contains two sets of rRNA genes. The 16S and 23S rRNA genes were closely linked as two clusters. However, both 5S rRNA genes were separated from the 16S and 23S genes. The two 16S-23S rRNA gene clusters were arranged in an unusual tail to tail orientation.

Comparison of 16S rRNA genes within the T960 and parvo biovars of ureaplasmas isolated from humans.

Two-by-two sequence alignment revealed that the levels of homology between 16S rRNA gene sequences of strains belonging to the two biovars of Ureaplasma urealyticum (class Mollicutes) ranged from 98.5 to 98.9%. Within the biovars, three serovars of the T960 biovar exhibited levels of homology of > or = 99.7%, and the four serovars of the parvo biovar exhibited levels of homology of > or = 99.7%. A dendrogram of the Mycoplasma pneumoniae-Ureaplasma clade of the Mollicutes reflected the distinctiveness of the biovars.

Cloning the ribosomal RNA operons of Mycoplasma flocculare and comparison with those of Mycoplasma hyopneumoniae.

In contrast to other mycoplasma species the 16S/23S rRNA and 5S rRNA operons of Mycoplasma flocculare and Mycoplasma hyopneumoniae map at least 150 kb apart (20% of the genome). Both operons from M. flocculare have been cloned and sequenced. The 23S rRNA gene sequence showed 96.7% homology with the corresponding gene of M. Hyopneumoniae, equalling that found earlier for 16S rRNA and confirming the close phylogenetic relationships of these organisms. A possible upstream promoter was identified. Sequence elements upstream and downstream from each structural gene could form a stem needed for maturation of the immature rRNA transcript to mature 16S and 23S rRNA. We also identified two possible stem-and-loop sequences 3' to the 23S rRNA gene. The 5S rRNA gene itself also showed high homology with the corresponding structural gene of M. hyopneumoniae, although the upstream and downstream sequences were highly heterologous.

Differentiation of Mycoplasma hyopneumoniae, M flocculare, and M hyorhinis on the basis of amplification of a 16S rRNA gene sequence.

To differentiate Mycoplasma hyopneumoniae, the cause of mycoplasmal pneumonia in pigs, from M flocculare and M hyorhinis, an assay, using the polymerase chain reaction to amplify a segment of the 16S rRNA gene sequence, was developed. The assay was found to be useful for identification of field isolates, as well as for identification of laboratory-adapted strains. Amplification of DNA from M hyopneumoniae and M flocculare resulted in products of 200 and 400 base pairs, respectively. The DNA from M hyorhinis was not amplified. The assay was sensitive enough to detect as little as 1,000 genome equivalents of M hyopneumoniae and M flocculare DNA. Sensitivity was increased 100-fold by increasing the concentration of magnesium ion in the reaction buffer from 2 to 4 mM; however, DNA from M hyorhinis was also amplified under these conditions. The DNA from several walled bacteria and from other mycoplasmas was also tested, but none of these DNA samples was amplified, suggesting that the assay was specific for porcine mycoplasmas.

Polymerase chain reaction using 16S rRNA gene sequences distinguishes the two biovars of Ureaplasma urealyticum.

Several fundamental phenotypic and genotypic differences have separated strains of the genital mycoplasma Ureaplasma urealyticum into two clusters or biovars. However, the lack of an easily performed and unambiguous test to discriminate between them has hampered investigation of the relationship between these biovars and disease. We determined the 16S rRNA nucleotide sequence of U. urealyticum 27, the serovar 3 standard and representative of the parvo biovar (serovars 1, 3, 6, and 14). This sequence was compared with the published sequence of U. urealyticum T960, which is the type strain and the serovar 8 standard and is representative of the T960 biovar which is composed of the 10 intervening serovars. Homology between the two sequences was 98.8%; differences were exploited to provide primers for biovar-specific polymerase chain reactions (PCRs). The results of these reactions placed all 14 serovar standard strains into the correct biovar. The PCRs were also applied to 10 cloned and 8 noncloned isolates that had been serotyped earlier. For 16 of them, we deduced their biovars from the serotyping data and then confirmed them by PCR. One unpredictable isolate and one nonserotypeable isolate were also classified as to biovar. Thus, we have developed a method for biotyping U. urealyticum that is applicable to both laboratory-adapted strains and wild-type isolates and that is appropriate for testing large numbers of clinical isolates. The amplification by the T960 biovar PCR protocol of DNAs from ureaplasmas of animals and certain Mycoplasma species suggested that the parvo biovar has diverged from the mainstream of the evolution of this clade.

Construction of the physical maps of Mycoplasma hyopneumoniae and Mycoplasma flocculare and the location of rRNA genes on these maps.

The genomes of Mycoplasma flocculare and Mycoplasma hyopneumoniae, two mycoplasmas of the porcine respiratory system, were studied. Based upon antigenic cross-reactivity and DNA-DNA hybridization, these species have given indication of a close genetic relationship. By using field-inversion gel electrophoresis and employing the restriction digest fragments obtained from the gels as the probes, physical maps of the genomes of the two species were constructed. Mycoplasma hyopneumoniae is similar to M. flocculare in having a single set of rRNA genes and the 5S-rRNA gene is separated from the 16S and 23S rRNA genes. Based upon the location of the rRNA genes on the physical maps in both species, the distance between the 5S and the 16S and 23S rRNA genes is at least 150 kbp. Thus, there is further evidence for the close relationship between these organisms.

Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.

The growth response of Mycoplasma hyopneumoniae and Mycoplasma flocculare based upon ATP-dependent luminometry.

Cultures of Mycoplasma hyopneumoniae and M. flocculare in Friis' broth grew faster and to higher titers in air than in 8% CO2; cultures in air grew better when shaken than when stationary. Under the optimal conditions, both species have generation times of about 10 h and achieve maximum titers of at least 10(9) organisms per ml. Maximum growth was reached near pH 7.0, before the phenol red indicator had noticeably changed colour. Changes in growth were readily detected by an ATP assay based upon the luciferin-luciferase reaction. Concentrations of ATP fell rapidly after peak growth. Although the addition of 27 mM glucose to the medium did not change the pattern of growth and gas chromatography gave no evidence of the production of volatile or non-volatile end-products, washed harvested cells of both species metabolized [14C]-glucose. The addition of 29 mM arginine to the medium inhibited growth.

Human ureaplasmas show diverse genome sizes by pulsed-field electrophoresis.

Contour clamped homogeneous field (CHEF) agarose gel electrophoresis (AGE), ramped to give linear separation of DNA molecules of 600-1600 kilobase pairs (kbp), was used to determine mobilities for full-sized genomic DNA of the serotype standard strains of the human genital mollicutes, Ureaplasma urealyticum relative to yeast chromosomal DNA markers. Indicated genome sizes (in kbp) were 760 for the four biotype 1 strains and 840-1140 for eleven biotype 2 strains. Other estimates were: 720 for Mycoplasma hominis, 1070 for Mycoplasma hyopneumoniae, 890 for Mycoplasma flocculare, 1180 and 1350 for Mycoplasma mycoides subsp. mycoides Y and GC1176-2, respectively, and 1650 and 1580 for Acholeplasma laidlawii B and PG 8, respectively. These data supplement previous evidence from CHEF AGE that the genomes of the Mycoplasmataceae are diverse in size with some larger than previously estimated from DNA renaturation kinetics.

A gene probe to detect Mycoplasma hyopneumoniae, the etiological agent of enzootic porcine pneumonia.

Enzootic porcine pneumonia is caused by Mycoplasma hyopneumoniae. Since the disease is of world-wide importance it is important to detect and identify the causative agent. In experience laboratories this mycoplasma can usually be detected by culture but its identification still is difficult and time consuming. We have cloned random Eco R1 fragments of M. hyopneumoniae DNA to M13mp19 and used the resultant recombinant to produce a probe capable of detecting approximately 10 pg of the mycoplasma DNA (10(4) organisms). By using appropriate stringency the test was made specific for M. hyopneumoniae, although at lower stringency reaction was positive with Mycoplasma flocculare at 1000 x the concentration limit. The assay did not detect M. hyopneumoniae in DNA from lungs of chronically infected animals but it did react with DNA isolated from the organisms cultured from the infected lung material.

Characterization of tetracycline-resistant strains of Ureaplasma urealyticum.

Gel electrophoresis revealed no plasmids in cloned strains of Ureaplasma urealyticum for which minimal inhibitory concentration (MICs) of tetracycline were greater than 64 mg/l. However, DNA from these strains hybridized with the tetM sequence from Streptococcus agalactiae in dot blot hybridization, whilst DNA from more-susceptible strains did not do so. Our results confirm and extend previous work, in which the tetM sequence was associated with resistance to the tetracycline antibiotics in strains of U. urealyticum. The strains examined in this study were isolated primarily in western North America but included representatives from Europe and the United Kingdom. Serotyping showed an increase in strains with the serotype 9 determinant and a decrease in those with the serotype 14 determinant together with a shift to the biotype 2 cluster (P = less than 0.02). To characterize these strains and to identify alternative antibiotics for therapeutic and basic research applications, 26 tetracycline-resistant strains were tested against 25 diverse antimicrobial agents. All demonstrated in-vitro susceptibility to rosaramicin but were resistant to 2 mg/l erythromycin. Only two of the strains were resistant to 16 mg/l erythromycin. Most antibiotics were not active against these isolates, but low concentrations of filipin and unconventional agents such as flurofamide and hydroquinone inhibited the growth of some strains.

Septic arthritis due to dual infection with Mycoplasma hominis and Ureaplasma urealyticum.

Acute septic arthritis of a knee and shoulder developed in a 32-year-old renal transplant patient. Cultures yielded Mycoplasma hominis and at least 1, and possibly 2, strains of Ureaplasma urealyticum. Doxycycline therapy controlled the symptoms and signs, and the joints became culture negative. On stopping therapy after 7 months, the arthritis recurred and U. urealyticum was again isolated from the shoulder joint. Cessation of doxycycline almost 4 years after the initial episode resulted in another recurrence. To our knowledge, this is the 1st case in which both M. hominis and U. urealyticum have been isolated from a joint.

Purification of urease from Ureaplasma urealyticum.

We have purified urease from the Mollicutes, Ureaplasma urealyticum, using high performance liquid chromatography methods and DEAE-Sephadex chromatography. While only small amounts of material could be utilized in these methods, urease was purified at least 180-fold, yield a major band on SDS-PAGE of 66,000 daltons, a minor band of 64,000 daltons, and several faint bands of lower molecular mass. These results suggest that the 380,000 dalton intact urease is a pentamer or hexamer of these two larger subunits. The highly purified urease from DEAE-Sephadex retained full activity for at least 20 days at 4 degrees C in sodium phosphate buffer (pH 7.2) with 1% bovine serum albumin. The estimated specific activity of the DEAE peak fractions, 180 IU/micrograms, is at least 90-fold greater than that of jack bean urease.

ATP measurements obtained by luminometry provide rapid estimation of Ureaplasma urealyticum growth.

ATP content obtained by luciferin-luciferase luminometry with commercially available reagents provided rapid estimates of Ureaplasma urealyticum populations. Each cell contained about 4.7 X 10(-18) mol of ATP. We could detect 10(4) CCU50 (color change unit50) per 100 microliters. We correlated urease activity with growth and confirmed the differential response of ureaplasma strains to Mn2+.