Short paperIdentification of contaminating mollicutes in mammalian cell culture as spiroplasma species
Introduction
A number of mammalian cell culture samples were submitted to Mycoplasma Experience from a single source in Ireland during 1993. The samples contained contaminating isolates that grew at 36°C ±1°C, produced acid in mixed substrate broth and grew on agar. However they had a colony morphology characteristic of motile mollicutes, such as Mycoplasma mobile and Spiroplasma citri [Fig. 1], which grow at the lower temperatures of ~19.5°C and 30–32°C respectively but not at 36°C.
The originator decided to destroy infected cell stocks, but remainders of the samples and their isolates were stored at −70°C at Mycoplasma Experience. In 2000, the first sample, BL7, had been identified as being in the S.citri phylogenetic group by sequencing [1]. The second, CH1, had not had identification attempted. These isolates were resurrected and DNA extracts subjected to 16S rDNA sequencing in an attempt to identify them further.
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Materials and methods
Original 1993 cell culture samples BL7 and CH1 were thawed, inoculated onto commercially available Mycoplasma Experience agar and incubated at 36°C ±1°C in an atmosphere of 95% Nitrogen/5% Carbon dioxide until growth was observed. Single colonies were transferred to 2 ml ME liquid and cultured to provide material for DNA extraction.
DNA from the samples was extracted using the MagNA pure compact NA isolation kit I (Roche). PCR of the 16S rDNA genes was carried out using the MicroSEQ® full
Results
Comparing sequences with the EZbiocloud 16S rDNA database, BL7 aligned most closely with Spiroplasma melliferum BC-3 with 99.22% similarity on 1444bp of sequence. CH1 aligned most closely with S.melliferum BC-3 with 99.41% similarity on 1059bp of sequence. The relationship of BL7 with other similar spiroplasmas is shown in Table 1 and Fig. 2.
Similar results were given for CH1 (not shown).
Discussion
Insects are a globally distributed, rich source of spiroplasma species, which are most commonly found in their guts. S.melliferum, a honeybee pathogen, was first isolated from the hemolymph and gut of honeybees (Apis mellifera) in Maryland in 1976 [3]. It has been recovered from the hemolymph of bumble bees, leafcutter bees and a robber fly; the intestinal contents of sweat bees, digger bees, bumblebees and a butterfly. It was also found on a variety of plant surfaces (flowers). Spiroplasmas on
Declaration of competing interest
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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