One Test Microbial Diagnostic Microarray for Identification of Mycoplasma mycoides subsp. mycoides and Other Mycoplasma Species

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.     

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Heterogeneity of Mycoplasma Dispar

Seventy bovine mycoplasma strains recovered from cases of calf pneumonia, and all displaying the cultural characteristics of Mycoplasma dispar, were compared to the type strain of this species by the disc growth inhibition test, the metabolism inhibition test and indirect epi-immunofluorescence test applied to colonies on agar. Sixty-seven strains were found to be identical with M. dispar. The remaining three strains formed a distinct serogroup partially separate from the type strain of M. dispar, but the difference from the type strain was not considered great enough to warrant the establishment of a subspecies.     

Array-based genomic comparative hybridization analysis of field strains of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist, although it is clear that M. hyopneumoniae adheres to porcine ciliated epithelium by action of a protein called P97. Previous studies have shown variation in the gene encoding the P97 cilium adhesin in different strains of M. hyopneumoniae, but the extent of genetic variation among field strains across the genome is not known. Since M. hyopneumoniae is a worldwide problem, it is reasonable to expect that a wide range of genetic variability may exist given all of the different breeds and housing conditions. This variation may impact the overall virulence of a single strain. Using microarray technology, this study examined the potential variation of 14 field strains compared to strain 232, on which the array was based. Genomic DNA was obtained, amplified with TempliPhi, and labeled indirectly with Alexa dyes. After genomic hybridization, the arrays were scanned and data were analyzed using a linear statistical model. The results indicated that genetic variation could be detected in all 14 field strains but across different loci, suggesting that variation occurs throughout the genome. Fifty-nine percent of the variable loci were hypothetical genes. Twenty-two percent of the lipoprotein genes showed variation in at least one field strain. A permutation test identified a location in the M. hyopneumoniae genome where there is spatial clustering of variability between the field strains and strain 232.     

A Serologic Variant of Mycoplasma Hyorhinis Recovered from the Conjunctiva of Swine

In an examination of conjunctival samples from 40 piglets for mycoplasmas, 17 isolates were obtained. Eight could be identified as Mycoplasma hyorhinis, three as Mycoplasma flocculare, and one as Acholenlasma sp. Five strains were not readily identifiable, but together with two previously recovered strains they were found to represent a distinct serogroup. All seven strains were glucose and phosphatase positive. Incubation in a CO2-enriched atmosphere led to enhancement of the growth on solid medium. The serogroup was serologically related to M. hyorhinis, but not to a number of other glucose fermenting species of mycoplasma, and it may therefore be regarded as a new subspecies of M. hyorhinis.     

Characterization of Mycoplasma pulmonis Variants Isolated from Rabbits I. Identification and Properties of Isolates

Mycoplasma showing at least two colony types were isolated from the nares and oropharynx of New Zealand white rabbits. Two strains were purified by single-colony passages and characterized. Morphology by phase-contrast and electron microscopy was typical of Mycoplasmataceae. Both grew anaerobically as well as aerobically, caused hemolysis of guinea pig, sheep, and horse red blood cells, and fermented glucose. These characteristics are shared by members of the species M. pulmonis, commonly isolated from the respiratory tracts of laboratory rats and mice. By use of the growth-inhibition test and agar-gel double-diffusion tests, the two strains were found to be serologically related to each other and to M. pulmonis ATCC 14267 but not to other representative Mycoplasma species from man and animals.     

Localization of Enzymes in Mycoplasma.

Pollack, J. D. (University of Connecticut, Storrs), Shmuel Razin, and Robert C. Cleverdon. Localization of enzymes in Mycoplasma. J. Bacteriol. 90:617-622. 1965.-Cells of eight parasitic and two saprophytic Mycoplasma strains were lysed by use of osmotic shock, and the membranes were separated from the soluble fraction by use of differential centrifugation. Cell fractions were tested for reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase, reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) oxidase, glucose-6-phosphate dehydrogenase, adenosine triphosphatase, ribonuclease, and deoxyribonuclease activities. Adenosine triphosphatase was confined to the membrane fraction of all Mycoplasma strains. The NADH(2) oxidase activity was associated with the membranes of the saprophytic M. laidlawii and with the soluble fraction of the parasitic Mycoplasma strains. NADPH(2) oxidase activity was detected only in the soluble fraction of the parasitic strains. Glusose-6-phosphate dehydrogenase was demonstrated only in the soluble fraction of M. laidlawii. Ribonuclease activity was found usually in both membrane and soluble fractions, but was generally higher in the membrane fraction. In the human and bovine Mycoplasma strains, deoxyribonuclease activity could not be demonstrated in the soluble fraction; in the remaining strains, activity was highest in the soluble fraction. Dissolution of M. laidlawii strain B membranes by sodium deoxycholate significantly increased membrane-NADH(2) oxidase and adenosine triphosphatase activities.     

Evaluation of Some Serological Techniques for The Identification of Mycoplasma Hyorhinis and Mycoplasma Suipneumoniae

Eighteen strains of Mycoplasma hyorhinis and a strain of Mycoplasma suipneumoniae were tested in 4 serological tests, i. e., disc growth inhibition, metabolic inhibition, indirect haemagglutination and indirect epi-immunofluorescence. Only with immunofluorescence could all tested strains of M. hyorhinis be shown; no cross-reactions between M. hyorhinis and M. suipneumoniae could be detected. The other tests failed in many cases to identify strains of the same species, and they gave cross-reactions between M. hyorhinis and M. suipneumoniae.     

Occurrence of urease in T strains of Mycoplasma

A previously unknown metabolite necessary for growth of T strains of Mycoplasma in artificial culture media has been identified as urea. The source of this metabolite was the mammalian plasma or serum enrichment of the culture medium. Normal horse serum was the most satisfactory native protein enrichment for cultivation of T strains of mycoplasma, and it is believed that its superior performance in agar and fluid culture media is associated with its relatively high urea content (approximately 40 mg/100 ml). T-strain urease activity was maximal at pH 6.0 +/- 0.5. This is also the optimal pH for growth of T strains. Substrate concentrations greater than 1.0% urea were inhibitory to growth and urease activity of T-strain organisms, and optimal urea concentrations in fluid media appeared to lie within the range of 0.008 to 0.01 m. This range of urea concentration permitted maximal growth of T-strain organisms without rapid loss of viability due to excessive ammonia accumulation and rise in pH to lethal levels. T strains of Mycoplasma were cultivated in a serum-free fluid medium containing urea as the only added metabolite and nitrogen source. T strains are the only known human mycoplasmas which exhibit urease activity, and this biochemical marker can be employed as an aid in the detection and identification of T strains of Mycoplasma (urease color test) and in distinguishing T strains from other members of the human Mycoplasma group.     

Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains.

Restriction enzyme digestion and field inversion gel electrophoresis were used to analyze the chromosomes of strains of Mycoplasma hyopneumoniae and the related organism Mycoplasma flocculare. The chromosome size for the M. hyopneumoniae type strain was calculated from individual fragments to be 1,011.3 +/- 32.9 kbp. The chromosomes of M. hyopneumoniae field strains were approximately the same size. The restriction patterns obtained for the chromosomes of phenotypically similar M. hyopneumoniae strains were quite different. Therefore, the species M. hyopneumoniae seems to be very heterogeneous. A field inversion gel electrophoresis analysis of the entire chromosomes allowed us to distinguish M. hyopneumoniae strains easily and hence to characterize further the species M. hyopneumoniae. The chromosome size for M. flocculare was calculated to be 988.3 +/- 39.5 kbp. Restriction enzyme XhoI, which statistically should cut the M. hyopneumoniae chromosome frequently, did not cut the DNA of any of the M. hyopneumoniae strains but did digest M. flocculare DNA, indicating that there is a site-specific modification at CTCGAG which probably belongs to a restriction modification system in M. hyopneumoniae and is absent in M. flocculare.     

Characterization of gentamicin-resistant Mycoplasma hyorhinis.

A study was conducted to determine if a gentamicin-resistant strain of mycoplasma could be developed for use in validating current mycoplasma detection methods for biologic product harvest cell culture fluid (CCF) containing gentamicin. A strain of gentamicin-resistant Mycoplasma hyorhinis was isolated and characterized. The study showed that this organism was similar to the wild-type strain in all ways examined except gentamicin resistance. Both strains of mycoplasma (the gentamicin resistant and the wild-type) exhibited comparable growth patterns and showed 100% homology based on DNA sequencing and analysis of a 464-bp PCR product. Also, analysis using species-specific antisera identified both strains as M. hyorhinis. Two commonly used lot release mycoplasma detection methods (culture and DNAF) consistently detected mycoplasmas in spiked biologic product harvest CCF containing gentamicin but not in unspiked samples. This study demonstrates the first isolation and characterization of a gentamicin-resistant M. hyorhinis that can be used to validate mycoplasma detection methods for biologic product harvest CCF containing gentamicin.     

Attachment of Mycoplasma salivarium and Mycoplasma orale to glass surfaces

Mycoplasma salivarium ATCC 23064 and 24 other oral strains, and Mycoplasma orale ATCC 15539 and 22 other oral strains were examined quantitatively for attachment to glass surfaces by using ELISA method. Although all of the tested strains attached to glass surfaces, M. salivarium attached far more readily than M. orale. The results suggested that electrostatic bonds were involved in the attachment and that bivalent metal ions also played a role.     

Distribution of ecto 5'-nucleotidase on Mycoplasma species associated with arthritis

The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of Mycoplasma pulmonis, K(m) 0.63 microM 5'-AMP. The six M. fermentans strains lacking 5'N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5'-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5'N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma.     

Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains.

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.     

Comparison of various atmospheric conditions for isolation and subcultivation of Mycoplasma hyorhinis from cell cultures

The efficiency of aerobic incubation was compared with incubation under various oxygen and carbon dioxide conditions for the isolation and subcultivation of three strains of Mycoplasma hyorhinis from VERO-cell cultures and subcultivation of three laboratory strains. Under anaerobic conditions with a low oxidation-reduction potential (at or below -115 mV) as obtained in jars, with catalysts, containing mixtures of 5%-10% CO2 in H2, very poor or no growth of any of the six M. hyorhinis strains was observed. When traces of oxygen were present (that is, under conditions with higher oxidation-reduction potentials, e.g. when omitting the catalyst in the above gas mixtures or in 5% CO2 + 95% N2) isolation from cell cultures was successful in most tests, but subcultivation of these primary isolates was seldom possible under these semi-anaerobic conditions. However, in most cases these primary isolates could be subcultivated aerobically, although aerobic conditions were unsatisfactory for isolation in about half of the experiments. Isolation of M. hyorhinis was optimal in 5% O2 + 95% N2, under which condition the isolates could also always be subcultivated. Isolation failed occasionally when 5% O2 + 5% CO2 + 90% N2 was used, thus indicating that 5% CO2 was slightly inhibitory. 5% CO2 in air and 10% CO2 either in air, H2 or N2 were also inadequate for isolation from cell cultures. In contrast to the findings with these cell culture-adapted M. hyorhinis strains, the laboratory strains could be subcultivated easily under all conditions tested except those with an oxidation-reduction potential at or below -115 mV; 100% CO2 was inhibitory for all 6 strains. Our findings may partly explain why M. hyorhinis is often considered "non-cultivable" on artificial media once adapted to cell cultures. The findings emphasize the need to employ also a micro-aerophilic condition (5% O2 in 95% N2) in the examination of cell cultures for mycoplasma.     

An evaluation of PCR methods to detect strains of Mycoplasma fermentans.

A panel of 30 putative Mycoplasma fermentans strains, isolated from various sources including human, ovine and cell lines, were tested by a previously described polymerase chain reaction (PCR) to confirm their identity by amplification of a conserved 206 bp region of the insertion sequence IS1550. In addition, the application of another PCR based on the major part of the IS1550 element showed one or two products of different length (1144 and 1341 bp) enabling M. fermentans strains to be divided into two types designated as Type A and Type B. A PCR, which amplifies the macrophage activating lipopeptide gene (malp), supported the identification of all the strains as M. fermentans. Thirteen other species of Mycoplasma from human sources gave negative results in these tests, with the exception of Mycoplasma orale, which was detected by both IS1550-PCRs based on the major part and the conserved 206 bp region of the IS1550 element. This study suggests that all M. fermentans isolates possess both the IS1550 element and the malp gene. In contrast to the IS1550, the malp gene is shown to be species-specific and the use of a malp PCR described here could prove to be a useful adjunct to IS1550 detection as confirmation of the presence of M. fermentans in clinical material.     

Mycoplasma simbae sp. nov., Mycoplasma leopharyngis sp. nov., and Mycoplasma leocaptivus sp. nov., isolated from lions.

Mycoplasmas isolated from the throats of lions were shown to belong to three serotypes, all of which were serologically distinct from the previously recognized Mycoplasma and Acholeplasma spp. Eight mycoplasma colonies were cloned, including one from a leopard (strain LP), and were examined in detail for morphology, growth, and biochemical characteristics. The strains had the following properties: guanine-plus-cytosine contents of 37 mol% (strain LXT [T = type strain]), 28 mol% (strain LL2T), and 27 mol% (strain 3L2T) and a requirement for sterol. Strain 3L2T metabolized glucose, which was not metabolized by strains LXT and LL2T. Arginine and urea were not hydrolyzed. Strain LX (= NCTC 11724) is the type strain of a new species, Mycoplasma simbae; strain LL2 (= NCTC 11725) is the type strain of a second new species, Mycoplasma leopharyngis; and strain 3L2 (= NCTC 11726) is the type strain of a third new species, Mycoplasma leocaptivus.     

Mycoplasma Dispar As a Causative Agent in Pneumonia of Calves

Three field strains of Mycoplasma dispar were inoculated, by aerosol inhalation, into a total of eight naturally-born, colostrum-deprived calves. All three strains produced macroscopic pneumonia, each in one calf. Histopaithologically an exudative bronchitis accompanied by moderate interstitial cell proliferations was found. Reisolation studies indicated that the entire respiratory tract is the natural habitat of Mycoplasma dispar, which apparently does not spread via the blood stream.