Flow Cytometric Determination of the Effects of Antibacterial Agents on Mycoplasma agalactiae , Mycoplasma putrefaciens , Mycoplasma capricolum subsp. capricolum , and Mycoplasma mycoides subsp. mycoides Large Colony Type

Abstract

Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin-and ciprofloxacin-treated cells.Contagious agalactia of small ruminants is a serious disease responsible for causing severe economic losses in goat and sheep farms throughout the world (3,14). It has four causal agents, Mycoplasma agalactiae, M. putrefaciens, M. capricolum subsp. capricolum, and M. mycoides subsps. mycoides large colony type (LC) (3), which cause a variety of clinical syndromes like mastitis, arthritis, keratoconjunctivitis, and, occasionally, abortion and respiratory disease (3,14). Contagious agalactia is currently controlled by vaccination and antimicrobial treatment. However, uncontrolled antimicrobial treatment is very common and can lead to the development of antimicrobial agent-resistant strains.The efficacies of all antimicrobials are described in terms of MICs. Despite reservations about their clinical relevance, MICs are still generally considered the reference point for comparison and evaluation of the sensitivities of other tests (8). Mycoplasmas are slowly growing and highly fastidious; and they do not produce turbidity in broth, nor do they grow on agar surfaces at levels sufficient for conventional antibacterial testing (8).Flow cytometry is a very powerful technique that makes it possible to study the morphological and physiological characteristics of individual cells and their distributions within large cell populations in a short period of time (1,4,6,17). It has wide-ranging clinical and experimental applications in investigations of eukaryotic cells and also looks very promising in the case of bacteria (4). On the basis of these considerations, it is appropriate to attempt to apply this methodology to the assessment of the antibiotic susceptibilities of these four mycoplasma species because of their importance in disease and because of their increasing resistance to previously active antibiotics (7,12,18,19).The reference strains of M. mycoides subsp. mycoides LC, M. agalactiae, M. putrefaciens, and M. capricolum subsp. capricolum were obtained from the National Collection of Type Cultures (United Kingdom). Enrofloxacin and ciprofloxacin were obtained from Sigma (St. Louis, MO); and gentamicin, chloramphenicol, oxytetracycline, and tylosin were obtained from Serva (Heidelberg, Germany). Stock solutions of the antibacterial agents were made by standard protocols (8). The MIC was defined as the lowest concentration of agents at which no growth occurred after 1 day in pH broth medium (11) plus 1% glucose for M. mycoides subsp. mycoides LC, M. putrefaciens, and M. capricolum subsp. capricolum and in pH broth medium plus 1% glucose and 0.5% pyruvate for M. agalactiae and was determined by the standard method (8). The MICs of ...

In vitro antimicrobial inhibition of Mycoplasma bovis isolates submitted to the Pennsylvania Animal Diagnostic Laboratory using flow cytometry and a broth microdilution method

“…These methods have included broth dilutions, solid agar testing, E-strip testing, and most recently, flow cytometry. 2,6,9,10,15,16,18 The MIC values have been determined for Mycoplasma agalactiae, Mycoplasma hyopneumoniae, Mycoplasma putrefaciens, Mycoplasma capricolum, and Mycoplasma mycoides using a selection of antimicrobials with flow cytometry for one isolate of each. …”

“…Under a light microscope, a small section of the agar was identified that contained less than 5 Mycoplasma colonies and no gross contamination from other bacteria. The section was excised from the agar and placed in PPLO broth a for 48 hr at 37°C and 5% CO 2 . The resulting broth suspension was plated again and the process repeated.…”

“…These levels were selected as starting concentrations based upon previous re search. 2,4,6,9,10,15,16,18 All plates were sealed to prevent gas exchange between wells. Each isolate was run in duplicate on a sterile 96-well round-bottom plate composed of 9 dilutions of the 7 antimicrobials, 1 negative control (no antimicrobial and no culture), and 2 positive controls (no antimicrobial present with culture).…”

Genome‐wide analysis of longevity in nutrient‐deprived Saccharomyces cerevisiae reveals importance of recycling in maintaining cell viability

“…Propidium iodide has been widely used for viability determination on the basis that it is excluded by the intact membrane of viable animal cells (Jones and Senft, 1985), bacteria (Assunção et al ., 2006) and fungi including S. cerevisiae (Deere et al ., 1998; Achilles et al ., 2006). On entering the cell, PI binds to nucleic acids and its fluorescence is enhanced and thus cells with damaged membranes (dead) fluoresce red while those with intact membranes (alive) do not.…”

Antimycoplasmal Activities of Compounds from Solanum aculeastrum and Piliostigma thonningii against Strains from the Mycoplasma mycoides Cluster

“…Growth of Mycoplasma has been determined by color changing units (CCU), measuring a decrease in pH after sufficient growth by a change in color (red to yellow) of the pH indicator ( Stemke and Robertson, 1982 ). In this paper, we have used both CCU and a flow cytometric (FCM) method ( Assunção et al, 2005 , 2006 ) to follow growth of Mycoplasma and its inhibition by plant compounds.…”