Use of PCR to detect mycoplasma DNA in respiratory tract specimens from adult HIV-positive patients.

The polymerase chain reaction (PCR) was evaluated retrospectively for its ability to detect Ureaplasma urealyticum and Mycoplasma spp. in respiratory tract specimens obtained from adult patients with AIDS. Mycoplasma DNA was detected in specimens from 12 of 84 patients. Of the 107 specimens tested, 13 and seven positive PCR results were obtained with the genus- and species-specific oligonucleotide primers used, respectively, in two different steps. With the latter, one sample was positive for U. urealyticum plus M. hominis, another for M. fermentans plus M. salivarium, and five others were positive for M. salivarium. The unexpected detection of U. urealyticum DNA in respiratory secretions from an adult AIDS patient suggested that this urogenital mycoplasma could have a role in determining or exacerbating respiratory tract infections in the HIV-positive population, but that its low rate of isolation could be related to the frequent failure of methods used currently to detect mycoplasmas.

Phylogenetic position of rare human mycoplasmas, Mycoplasma faucium, M. buccale, M. primatum and M. spermatophilum, based on 16S rRNA gene sequences.

The nucleotide sequence of the 16S rRNA genes of four rare human mycoplasma species, Mycoplasma faucium, M. buccale, M. primatum and M. spermatophilum, were partially sequenced and compared to published rRNA genes of mycoplasmas to determine their position in the Mollicutes phylogenetic tree. Nucleotide sequence motif and overall similarities allowed positioning of these mycoplasmas in the hominis phylogenetic group, as defined by Weisburg et al. [Weisburg, W. G., Tully, J. G., Rose, D. L. & 9 other authors (1989). J Bacteriol 171, 6455-6467]. Furthermore, these mycoplasmas could be clustered into two different subdivisions of the hominis group: (i) M. faucium and M. buccale were found to be included in the M. fermentans subdivision, and (ii) M. primatum and M. spermatophilum were included in the M. hominis one. Variable regions of the 16S rRNA genes were used to determine specific PCR primers to detect and identify M. faucium.

Neuropathy in diffuse infiltrative lymphocytosis syndrome: an HIV neuropathy, not a lymphoma.

OBJECTIVE: To determine whether CD8 lymphoid infiltrates in nerves of patients with HIV-associated diffuse infiltrative lymphocytosis syndrome (DILS) corresponds to a lymphomatous neoplastic process or to a proliferation of T cells reactional to HIV. BACKGROUND: DILS is characterized by persistent CD8 hyperlymphocytosis and multivisceral CD8 T-cell infiltration, which may affect peripheral nerves. METHODS: Presence of monoclonal T cells and HIV-1 proviral load were evaluated by polymerase chain reaction (PCR) techniques in frozen peripheral nerve samples from six patients with DILS neuropathy and 22 patients with other HIV-associated peripheral neuropathies, including mononeuritis multiplex (MM:6), inflammatory demyelinating polyneuropathies (IDP:6), distal sensory polyneuropathy (DSP:5), and toxic distal sensory polyneuropathy (TDSP:5). RESULTS: Five of six patients with DILS showed no detectable monoclonal T-cell clones in their nerves. Nerve proviral load in DILS (6.8 +/- 0.2 log/10(5) cells) was much higher than in MM (p < 0.008), IDP (p < 0.001), DSP (p < 0.001), and TDSP (p < 0.005). CONCLUSIONS: DILS neuropathy represents a separate entity among HIV-associated neuropathies. It is associated with massive HIV proviral load in nerve and must not be confused with a peripheral nerve T-cell lymphoma.

Application of an arbitrarily-primed polymerase chain reaction to mycoplasma identification and typing within the Mycoplasma mycoides cluster.

An arbitrarily-primed polymerase chain reaction (AP-PCR) was developed using a primer pair, Mlip1 and Mlip4, for members of the Mycoplasma mycoides cluster, a group containing important pathogens of small and large ruminants. Parameters that influence the reproducibility of this assay were optimized: magnesium, primer and template concentrations, and pH. AP-PCR fingerprinting, carried out on a number of strains of each of the six species or subspecies belonging to the mycoides cluster, allowed the typing of strains within each group. The AP-PCR assay showed that the cluster can be divided into two groups: (i) high and (ii) no genomic polymorphism variation. In addition, specific polymorphic bands for members of species or subspecies included in this cluster were amplified by this AP-PCR method, thus allowing their identification.

Cloning and characterization of the lipase operon from Mycoplasma mycoides subspecies mycoides LC.

Lipases, serine esterase enzymes, play an essential role in the mycoplasmal nutritional requirement for long-chain fatty acids. Although the lipase(s) activity in different mycoplasma species has been investigated, the molecular biology of the corresponding genes has not been studied. Using a single-primer PCR technique combined to more classical cloning systems, an operon containing three open reading frames (ORF), each of which could encode a lipase protein of 264, 264 or 269 amino acids (aa), was identified from Mycoplasma mycoides subsp. mycoides LC. Analysis of aa sequences of the encoded polypeptides showed that they display high aa similarity between each other (65-79%) and 28-31% identity to other prokaryotic lipases. Moreover, a lipase-esterase activity could be detected when the mycoplasmal lipase-encoding genes were expressed in a strong opal-suppressor-bearing Escherichia coli strain.

Rapid, sensitive PCR-based detection of mycoplasmas in simulated samples of animal sera.

A fast and simple method to detect mycoplasmal contamination in simulated samples of animal sera by using a PCR was developed. The following five mycoplasma species that are major cell culture contaminants belonging to the class Mollicutes were investigated: Mycoplasma arginini, Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, and Mycoplasma fermentans. After a concentration step involving seeded sera, genus-specific primers were used to amplify a 717-bp DNA fragment within the 16S rRNA gene of mycoplasmas. In a second step, the universal PCR was followed by amplification of variable regions of the 16S rRNA gene by using species-specific primers, which allowed identification of contaminant mycoplasmas. With this method, 10 fg of purified DNA and 1 to 10 color-changing units of mycoplasmas could be detected. Since the sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal mycoplasma-specific 32P-labelled oligonucleotide probe, a detection limit of 1 to 10 genome copies per PCR sample was obtained. This highly sensitive, specific, and simple assay may be a useful alternative to methods currently used to detect mycoplasmas in animal sera.

Conjugative transfer of a shuttle plasmid from Escherichia coli to Mycobacterium smegmatis [corrected].

The chimeric plasmid pMY10 containing the origin of replication of pAL5000, the origin of replication of pBR322, the origin of transfer of pRK2 and a kanamycin resistance gene was constructed and successfully transferred by conjugation from Escherichia coli harbouring the helper plasmid pRK4.24 into Mycobacterium smegmatis. This is the first report of conjugtive transfer of plasmid between E. coli and an acid fast organism.

Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum plasmid pAL5000.

A restriction map of Mycobacterium fortuitum var. fortuitum plasmid pAL5000 was established. The unique sites for ApaI, BamHI, BglII, BstEII, ClaI, EcoRI, EcoRV, HpaI, KpnI and NarI were located on the 5.0-Kb plasmid. The plasmid had no sites for AhaIII, BclI, HindIII, PstI, SphI and XbaI. pAL5000 was cloned into pBR322 and propagated in Escherichia coli. Three hybrid pAL5000-pBR322 plasmids carrying the complete pAL5000 sequence were constructed by joining the plasmids at their BamHI, EcoRI or EcoRV sites. We also cloned into these plasmids a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The construction of these plasmids will facilitate the analysis and manipulation of pAL5000, and may allow the development of a vector system for genetic analysis in mycobacteria.

Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B.

The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.

R factor-controlled restriction and modification of deoxyribonucleic acid: restriction mutants.

Restriction mutants of two different R factor-controlled host specificities (RI and RII) were isolated. All of the restriction mutants examined had a normal modification phenotype. No complementation was observed between the RI and RII host specificities. It is concluded that for each host specificity no protein subunit is shared by the restriction endonuclease and modification methylase.