Phenotypic and genotypic antimicrobial susceptibility patterns of the emerging human respiratory pathogen Mycoplasma amphoriforme isolated from the UK and Denmark.

OBJECTIVES: To determine the phenotypic and genotypic antibiotic susceptibility of Mycoplasma amphoriforme isolates recovered from patients in the UK and Denmark. METHODS: Seven isolates of M. amphoriforme were examined for antimicrobial susceptibility to seven antibiotics using the microbroth dilution assay in line with the CLSI guidelines for mycoplasmas. Each isolate was additionally subjected to WGS to identify resistance-associated mutations. Based on the consensus sequences from the genomic data, PCR primers were designed, and tested, for the amplification of the QRDR within the parC gene. RESULTS: Of the seven isolates investigated, four (57%) were resistant to moxifloxacin (0.5-1 mg/L) and levofloxacin (1-2 mg/L), compared with those that were susceptible (0.03-0.06 and 0.006 mg/L, respectively). Isolate H29 was resistant to five of the seven antibiotics tested: moxifloxacin, 0.5 mg/L; levofloxacin, 2 mg/L; azithromycin, 64 mg/L; erythromycin, 128 mg/L; and clindamycin, 64 mg/L. All isolates were susceptible to tetracycline (0.06 mg/L) and lefamulin (0.001-0.004 mg/L). Mutations from genomic data confirmed the presence of an S89F mutation within the ParC protein among all fluoroquinolone-resistant isolates and an A2059G mutation in the 23S rRNA gene in the macrolide- and lincosamide-resistant isolate H29. CONCLUSIONS: To the best of our knowledge, this is the first time where phenotypic and genotypic resistance data have been paired for M. amphoriforme confirming a correlation between the two. These data suggest the need for focused testing and resistance determination of isolates from high-risk patients given the backdrop of a high prevalence of antimicrobial resistance.

Identification of contaminating mollicutes in mammalian cell culture as spiroplasma species.

Cell cultures have provided an ideal habitat for a wide variety of Mycoplasma and Acholeplasma species since the earliest days of in-vitro culture. The possibility of contamination with Spiroplasma species was addressed by Regulatory Authorities due to the increased commercial use of insect cells, recognising that Spiroplasmas have been isolated from many types of arthropod and also that insect cell cultures support Spiroplasma growth as they have been used for cultivation of fastidious species. In this study we re-examined two cell culture samples previously confirmed as contaminated with mollicutes by cultural methods. One isolate had undergone sequencing which had placed it in the S. citri phylogenetic group, whilst the other had not been identified. Using modern sequencing methods we were able to further identify both isolates to species level.

Mycoplasma gallisepticum in pheasants and the efficacy of tylvalosin to treat the disease.

Infectious sinusitis, a common condition seen in adult pheasants, is primarily caused by Mycoplasma gallisepticum. The aims of the present study were to investigate the pathogenicity of M. gallisepticum in 14-day-old pheasants and evaluate the macrolide antibiotic tylvalosin (TVN) as a treatment for infectious sinusitis. The minimum inhibitory concentration of TVN for five isolates of M. gallisepticum taken from pheasants confirmed their susceptibility to TVN (range: 0.002 to 0.008 µg/ml). One of the isolates (G87/02) was inoculated intranasally into 72 pheasants (two groups of 36) at 14 days of age. Eight days later, when 18/72 (25%) of the pheasants showed clinical signs, one group was treated with 25 mg TVN/kg bodyweight daily in drinking water for three consecutive days. An uninfected, unmedicated control group (n=12) was also included. In contrast to the uninfected control group, a range of clinical signs typical of infectious sinusitis with varying severity was observed in challenged birds and M. gallisepticum was re-isolated from the infraorbital sinus and the eye/conjunctiva at necropsy, 22 days post challenge. In comparison with untreated birds, medication with TVN significantly reduced clinical signs and the re-isolation/detection of M. gallisepticum (P≤0.0021). The daily liveweight gain of treated birds was significantly increased in comparison with untreated birds (P=0.0002), and similar to daily liveweight gains observed in the uninfected control group. In conclusion, TVN at 25 mg/kg bodyweight daily for three consecutive days in drinking water was efficacious in the treatment of M. gallisepticum infection induced by challenging 14-day-old pheasants.

The growth and long term survival of Acholeplasma laidlawii in media products used in biopharmaceutical manufacturing.

Acholeplasma laidlawii is a potential contaminant of bovine serum and has also been found as a contaminant in serum free cell culture media products. Anecdotal evidence of A. laidlawii contamination of tryptone soya broth circulated for a number of years before it was acknowledged that the organism could contaminate microbiological broth powders. The occasional occurrence of A. laidlawii in broth powders and possibly in powdered components of cell culture media as part of the normal bioburden poses a serious threat to routine pharmaceutical and biopharmaceutical operations where filtration is the sterilisation method of choice. Absence of visual evidence of contamination cannot be relied upon as there is variation with both organism strain and media product in the ability to produce turbidity. Strains of A. laidlawii which have been isolated from broth powders are not significantly different in temperature or media preferences from other strains. A. laidlawii is capable of growing to high titre at refrigeration and ambient temperatures in unsupplemented bacteriological sterility media or serum free cell culture media and can survive for prolonged periods in these products.

The Development of a Microbial Challenge Test with Acholeplasma laidlawii To Rate Mycoplasma-Retentive Filters by Filter Manufacturers.

Mycoplasma are bacteria that can penetrate 0.2 and 0.22 µm rated sterilizing-grade filters and even some 0.1 µm rated filters. Primary applications for mycoplasma filtration include large scale mammalian and bacterial cell culture media and serum filtration. The Parenteral Drug Association recognized the absence of standard industry test parameters for testing and classifying 0.1 µm rated filters for mycoplasma clearance and formed a task force to formulate consensus test parameters. The task force established some test parameters by common agreement, based upon general industry practices, without the need for additional testing. However, the culture medium and incubation conditions, for generating test mycoplasma cells, varied from filter company to filter company and was recognized as a serious gap by the task force. Standardization of the culture medium and incubation conditions required collaborative testing in both commercial filter company laboratories and in an Independent laboratory (Table I). The use of consensus test parameters will facilitate the ultimate cross-industry goal of standardization of 0.1 µm filter claims for mycoplasma clearance. However, it is still important to recognize filter performance will depend on the actual conditions of use. Therefore end users should consider, using a risk-based approach, whether process-specific evaluation of filter performance may be warranted for their application. LAY ABSTRACT: Mycoplasma are small bacteria that have the ability to penetrate sterilizing-grade filters. Filtration of large-scale mammalian and bacterial cell culture media is an example of an industry process where effective filtration of mycoplasma is required. The Parenteral Drug Association recognized the absence of industry standard test parameters for evaluating mycoplasma clearance filters by filter manufacturers and formed a task force to formulate such a consensus among manufacturers. The use of standardized test parameters by filter manufacturers, including the preparation of the culture broth, will facilitate the end user's evaluation of the mycoplasma clearance claims provided by filter vendors. However, it is still important to recognize filter performance will depend on the actual conditions of use; therefore end users should consider, using a risk-based approach, whether process-specific evaluation of filter performance may be warranted for their application.