SIR MYCOPLASMA 6278110 TESTSANTIBIOGRAM FOR UROGENITAL MYCOPLASMA

1- CLINICAL USESIR MYCOPLASMA is a reagent for the antibiogram analysis of urogenitalmycoplasma: Ureaplasma spp (Ureaplasma urealyticum, Ureaplasmaparvum) and Mycoplasma hominis (Mh). At present, the differentialidentification of Ureaplasma urealyticum and U. parvum is difficult toachieve.SIR MYCOPLASMA consists of a microplate containing 8 antibiotics activeagainst these microorganisms and commonly used for the treating ofgenital infections.The emergence of resistant strains, particularly to cyclines and macrolides(first–line treatment antibiotics) (10, 11, 18), renders antibiogram analysisabsolutely necessary in order to avoid therapeutic failures.The use of a standard inoculum makes this technique highly concordantwith the method of reference (9).

2- PRINCIPLESIR MYCOPLASMA is a liquid–medium antibiogram where each antibioticis present at two different concentrations (doxycycline, tetracycline,azithromycin, josamycin, erythromycin, ofloxacin) or a single concentration(clindamycin, pristinamycin).The test is based on metabolic inhibition.The growth of mycoplasma is objectively measured by their metabolicactivity : hydrolysis of urea in U9 broth by Ureaplasma spp, and hydrolysisof arginine in arginine broth by Mh, with release of ammonia, which makesthe medium turn alkaline and the phenol red pH indicator in the mediumturns from yellow to red.If the microorganism is sensitive to the tested antibiotic, its metabolism isinhibited and the medium remains yellow.If the microorganism is resistant, it is able to grow and the medium turns red.

3- PRESENTATIONSIR MYCOPLASMA comprises:• 10 SIR microplates (the diagram of the 16 wells is reproduced on the

label, thus allowing identification of the sample).• 10 adhesive cover seals.• 1 package insert.Each microplate is individually wrapped in aluminium with a cover seal anda desiccating sachet.

4- STORAGEWhen stored at + 2 – 8°C, SIR MYCOPLASMA can be used until theexpiration date printed on the packaging.

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5- EQUIPMENT REQUIRED (NOT SUPPLIED)• U9 broth: code 62762 (pack of 10 ampoules of lyophilised U9 broth

Q.S.P. 2 ml)• Arginine broth: code 62763 (pack of 10 ampoules of lyophilised arginine

broth Q.S.P. 2 ml)• MYCOPLASMA DUO (box of 20 tests – code 62740)• Sterile distilled water• 2 ml, 200 µl, 100 µl, 20 µl pipettes• Incubator at 37°C• Contaminated waste disposal container

6- MODE OF OPERATION

A) THE SIR MICROPLATEThe SIR microplate is a microstrip comprising two rows of 8 wells in whichthe various antibiotics are coated. The antibiotics are rehydrated when theinoculum is aliquoted in the wells to obtain concentrations that areapproximate to the critical concentrations, thus allowing analysis of thesensitivity profile of the strains tested and classification as sensitive,intermediate or resistant for each antibiotic.

Growth control in the absence of antibiotic

Doxycycline (4 mg/L and 8 mg/L)

Tetracycline (4 mg/L and 8 mg/L)

Azithromycin (2 mg/L and 4 mg/L)

Josamycin (2 mg/L and 8 mg/L)

Erythromycin (1 mg/L and 4 mg/L)

Clindamycin (2 mg/L)

Pristinamycin (2 mg/L)

Ofloxacin (1 mg/L and 4 mg/L)

B) CHOICE OF ANTIBIOTICSThe choice of antibiotics to be tested was made based on therapeuticprescriptions advised for neonatal and genital mycoplasma infections (17).There is to date no critical concentration specific for mycoplasma; as a result,the critical concentrations chosen in the SIR MYCOPLASMA microarray arebased on those published in a number of scientific journals and/orrecommended by medical societies such as the CA–SFM (AntibiogramCommittee – French Microbiological Society). The choice of azithromycinconcentrations is based on an evaluation carried out using clinical strains (19).

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Ureaplasma spp

Mycoplasma hominis

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Cyclines tested: tetracycline and doxycycline (at concentrations of 4 and8 mg/L).The Mh and Ureaplasma spp. strains described as being resistant totetracyclines are detected unambiguously since it has been published thatthe Minimum Inhibitory Concentrations (MICs) were ≥ 8 mg/L as opposedto ≤ 2 mg/L for sensitive strains (15, 18). For this class of antibiotics, thereare low–level resistances that require an incubation time of 48 hours (19).The frequency rate of resistance to cyclines among strains variesaccording to the country and the level of exposure to antibiotics (18).

Macrolides tested: erythromycin (at concentrations of 1 and 4 mg/L),azithromycin (at concentrations of 2 and 4 mg/L), josamycin (atconcentrations of 2 and 8 mg/L).Mh strains are naturally resistant to erythromycin and azithromycin.Josamycin remains active. Consequently, the results obtained with theseantibiotics should confirm the identification of the strain previouslyobtained. The frequency of acquired resistance to macrolides among Mhand Ureaplasma spp strains isolated in the clinic is not known butprobably is very low (18).

Lincosamide tested: clindamycin (at a concentration of 2 mg/L).Ureaplasma spp strains are naturally resistant to this antibiotic (18).

Streptogramine tested: pristinamycin (at a concentration of 2 mg/L).Mycoplasma strains are highly sensitive to this antibiotic.

Fluoroquinolone tested: ofloxacin (at concentrations of 1 and 4 mg/L).This molecule is active on Mh and Ureaplasma spp. The resistancefrequency is not well known but has been estimated at less than 1% forUreaplasma spp. (6, 18).

C) CARRYING OUT THE ANTIBIOGRAMThe antibiogram can be achieved starting from the content of well X ofMYCOPLASMA DUO (code 62740).For duration and temperature of biological sample storage, please refer torecommendations currently in use (13).

1) Standardisation of inoculumIn order to seed the antibiogram with an inoculum containing 103 to 105

CCU/ml (CCU = Colour Changing Units), it is necessary to carry out apre–culture of the medium seeded with the sample.Pre–culture (carried out following the protocols described below) leads togrowth of the mycoplasma to a maximum titre of 106 to 107 CCU/ml. A1/100 dilution of such a pre–culture in U9 or arginine broth, depending onthe species isolated, produces a standard inoculum.

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• Using MYCOPLASMA DUOWhen the antibiogram is carried out using MYCOPLASMA DUO, thecontent of well X at 24 or 48 hours of incubation for Mh, and 24 hours forUreaplasma spp, corresponds to the pre–culture; thereafter, the content ofthe well needs to be diluted in U9 or arginine broth following the protocoldescribed below in order to obtain the standard inoculum.

1. For Mycoplasma hominisStarting from well X (at 24 or 48 h of incubation), make a 1/100 dilutionin arginine broth (20 µl of the content of well X in 2 ml of arginine broth).

2. For Ureaplasma sppAt 24 h of incubation: make a 1/100 dilution of the content of well X inU9 broth (20 µl of the content of well X in 2ml of U9 broth).

• At 48 h of incubation: in this case, it is not possible to use the contentfrom well X because there is a risk that Ureaplasma might haveundergone autolysis. Make a 1/10 dilution of the Transport suspensionmedium stored at + 4°C (200 µl in 2 ml of U9 broth).

• Starting from mycoplasma–enriched culture brothThe urogenital sample can be placed in culture medium specific formycoplasma (15). If one is waiting for identification and numeration results,the antibiogram can be carried out later and the medium stored at +4°C(cf. the product insert of the culture medium being used). Thereafter,incubation of the culture medium at 37 °C for 16 hours is recommended inorder to obtain a mycoplasma–enriched inoculum. The usual titre obtained is 106–107 CCU/ml. According to the publishedrecommendations (15), the realization of the antibiogram suggests using astandard inoculum. Thus, 20 µL of the enriched culture medium are dilutedin 2 ml of U9 or arginine broth (1/100 dilution), depending upon theidentification of the mycoplasma strain.

2) Aliquoting of the standard inoculum100 µl of standard inoculum (1/100 dilution of the pre–culture, in U9 orarginine broth) is aliquoted in each well of the SIR microplate.Cover the microplate with sealing film and incubate at 37°C for 48 hours.

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D) SAMPLES CONTAINING Ureaplasma spp and MhIn this case, seed 2 SIR microplates: one with the standard inoculumobtained by diluting in U9 broth (Ureaplasma spp strain antibiogram), andthe other with the standard inoculum obtained by diluting in arginine broth(Mh strain antibiogram).

7- INTERPRETATION OF RESULTSThe antibiogram can be read as soon as the growth control wells haveturned from yellow to red.A reading is taken at 24 h and another at 48 h. The 48–h reading allowsthe detection of low–level resistances to cyclines (19) and confirmation ofresults when the starting inoculum turns out to be low (103 CCU/ml) orwhen the colour change was doubtful after 24 hours.

For clindamycin and pristinamycin (a single concentration), only 2 resultsare possible: Sensitive or Resistant.

Example:

yellow/yellow: S Sensitive strain

red/yellow: I Intermediate strain

red/red: R Resistant strain

Particular case: if an orange colour is observed.One may observe the beginning of a change from yellow to red, resultingin an orange colour in the low–concentration wells. This must beinterpreted as positive and a clear–cut colour change can be obtainedafter 48 hours.

8- QUALITY CONTROL OF MANUFACTURERAll products manufactured and marketed by the Bio–Rad Company aremonitored by a quality assurance system from reception of the rawmaterials through to marketing of the finished products.

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2 red wells:Growth in the presence of antibioticResistant strain

The well with a low concentration of antibiotic: redThe well with a high concentration of antibiotic: yellow

Intermediate strain

Every batch of finished product is subject to quality control and will onlybe marketed if it complies with acceptance criteria.The documents on the production and control of each batch are keptfor reference.

9- INTERNAL QUALITY CONTROLQuality control can be carried out using a strain from a lyophilisedcollection (Ureaplasma parvum ATCC 700970). Resuspend the lyophilisedstrain in 1 ml of freshly reconstituted U9 medium (reference 62762). Makea 1/10 dilution in freshly reconstituted U9 medium.Incubate the seeded U9 medium at 37°C for 18 hours under aerobicatmosphere.Starting from this Ureaplasma–enriched U9 medium, make a 1/100 dilution(take a 20 µl aliquot of medium and add to 2 ml of reconstituted U9 broth).Seeding of the SIR MYCOPLASMA microarray is carried out following theusual protocol, i.e. by aliquoting 100 µl in each well. Cover the microarraywith sealing film and incubate at 37°C for 48 hours. The microarray can beread if the growth control wells have turned from yellow to red.

The expected profile is as follows:

10- PERFORMANCESThe choice of antibiotics tested using SIR MYCOPLASMA has evolvedaccording to the therapeutic treatments, SIR MYCOPLASMA antibiogramhas been re–evaluated in view of the modified treatments.

In the case of the two evaluations described below, the reference methodused was that of Minimal Inhibitory Concentrations in liquid medium (MIC)(6, 15).

The first evaluation (without azithromycin wells) was carried out on 80strains including 60 strains of Ureaplasma spp and 20 strains ofMycoplasma hominis (9).

With Ureaplasma spp strains:

For tetracyclines, concordance percentages varied from 90 % [79.5–96.2%] a to 95 % [86.1–99 %] a depending on the antibiotic.

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For macrolides, concordance percentages varied from 92 % [81.6–97.3 %]a to 100 % [95.1–100 %] a depending on the antibiotic.

For clindamycin, the concordance percentage was 85 % [73.4–92.9 %]a

For pristinamycin, the concordance percentage was 100 % [95.1–100 %]a.

For f luoroquinolones, the concordance percentage was 93 %[83.8–98.2 %]a.

With Mycoplasma hominis strains:

The concordance percentage is 85 % [62.1–96.8 %]a for tetracycline and100 % [86.1–100 %]a for all other antibiotics.

Total concordance for all the antibiotics mentioned previously is 94.6 %[92.4–96.4 %]a with 2.6 % major discordances. No strain was foundsensitive with the SIR MYCOPLASMA microarray when it was resistantwith the reference method (no very major discordance).

A second evaluation was carried out following replacement of minocyclinby azithromycin at concentrations of 2 and 4 mg/L.

A comparative study was made between the method for determining MICsin liquid medium and the SIR MYCOPLASMA microarray for azithromycinwells. The strains tested are reference strains and wild–type clinicalstrains. Each inoculum was standardised and confirmed to be withinacceptable and recommended limits, i.e. 104 and 105 CCU/ml (15). Theresults of the two methods were interpreted as Sensitive (S), Intermediate(I) or Resistant (R) for the various microarray concentrations.

For 15 mycoplasma reference strains (13 ATCC Ureaplasma spp strainsand 2 ATCC Mycoplasma hominis strains), the concordance percentagefor the S or R clinical classification was 100 % [81.9–100 %]a. AllUreaplasma spp strains had a sensitive profile and the two Mycoplasmahominis strains had a resistant profile, which corresponds to the naturalresistance of this species.

For 15 clinical isolates of Ureaplasma spp, the concordance was 100 %[81.9–100 %]a between the clinical classification afforded by the SIRMYCOPLASMA microarray and that obtained with reference MICs.a: [CI 95%] = 95% confidence interval.

11- LIMITS OF THE TESTIf the medium turns to red and becomes cloudy, this indicates growth ofbacteria other than mycoplasma (the growth of the latter does not alter theclarity of the medium). This phenomenon can be observed in arginine andU9 culture broth if they have been incubated at 37 °C. The presence ofother bacteria may be confirmed on chocolate agar.

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The final interpretation of results, as for any biological result interpretation,cannot be made on the basis of a single test and needs to be based onclinical data and biochemical, cytological and immunological results.

If the inoculum used for seeding of the SIR MYCOPLASMA microarray islow (<103 CCU/ml), the colour change in the wells may be unreliable.Should the result appear abnormal, repeating the standard inoculum andantibiogram using a SIR MYCOPLASMA microarray is recommended.

If, for a given antibiotic, the well with the highest concentration is red andthat with the lowest concentration remains yellow, the result cannot beused. It is recommended that another antibiogram be carried out startingfrom a new standard inoculum.

Macrolides, especially erythromycin, are known to be pH sensitive (14).Indeed, colour change in the low–concentration well may be observed,thus leading to an intermediate result owing to a pH effect rather thanantibiotic activity.

This colour change phenomenon may also be observed with azithromycinwells.

12- BIBLIOGRAPHY1. BEBEAR C. Activité comparée de la minocycline et doxycycline sur les

mycoplasmes pathogènes pour l'homme. Pathol. Bio., 1985, 33,577–580.

2. CANTET P et BEBEAR C. Activité comparée in vitro de sept quinolonessur Ureaplasma urealyticum. Pathol. Bio. 1983, 31, 501–503.

3. DAVIS J.W. Antimicrobial Susceptiblity of Ureaplasma urealyticum. J.Clin. Microb., 1981, 13, 320–325.

4. EVANS R.T and TAYLOR ROBINSON D. The Incidence of TetracyclineResistant Strains of Ureaplasma urealyticum. J. Antimicrob. Chemother,1978 , 4, 57–63.

5. ROBERT M.C. Dissemination of the Tet–M Tetracycline ResistanceDeterminant to Ureaplasma urealyticum. Antimicrob. Agents Chemother,1986, 29, 350–352.

6. ROBERTSON J.A. Standardized Method for Determining AntimicrobialSusceptibility of Strains of Ureaplasma urealyticum and their Responseto Tetracycline, Erythromycin and Rosaramincin. Antimicrob. AgentsChemother, 1981, 20, 50–58.

7. TAYLOR–ROBINSON D. Clinical Antibiotic Resistances of Ureaplasmaurealyticum. Pediatr. Infec. Dis., 1986, 5, 335–337.

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8. SOBETZKO R., HARTMANN A.A., ELSNER P. Susceptibil ity ofUreaplasma urealyticum to Ten Chemotherapeutic Agents. Zbl.Bakt.Hyg. 1988, A269, 245–250.

9. RENAUDIN H., BEBEAR C. Evaluation des systèmes Mycoplasma Pluset SIR Mycoplasma pour la détection quantitative et l'étude de lasensibilité aux antibiotiques des mycoplasmes génitaux. Path.Biol.,1990, 38, n°5, 431–435.

10. CUMMINGS M.C., Mc CORMACK W.M. Increase in Resistance ofMycoplasma hominis to Tetracyclines. Antimicrob. Agents Chemother.1990, 34, 2297–2299.

11. BAURIAUD R., SEROR C.,LARENG M.B., LEFEVRE J.C. Sensibilité invitro aux antibiotiques des mycoplasmes génitaux isolés à Toulouse.Etude de nouvelles molécules (macrolides et quinolones).Path. Biol.1992, 40, n°5, 479–482.

12. WAITES K., DUFFY L., HAMRICK W., CROUSE D., CASSEL G.Microbiologic Efficacy of Intravenous erythromycin against Ureaplasmaurealyticum in the Lower Respiratory Tract of Preterm Neonates.10th International IOM congress – Bordeaux July 19–26th 1994 – Postern°163.

13. BEBEAR C.M., RENAUDIN H., CHARRON A., CLERC M., PEREYRE S.,BEBEAR C. 2003. DNA Gyrase and Topoisomerase IV Mutations inClinical Isolates of Ureaplasma spp. and Mycoplasma hominis Resistantto Fluoroquinolones. Antimicrob. Agents Chemother. 47:3323–3325.

14. KENNY G. E, CARTWRIGHT F.D. 1993. Effect of pH, Inoculum size, andIncubation time on the susceptibility of Ureaplasma urealyticum toEryhtromycin in vitro. Clin. Infect. Dis.; 17 (Suppl 1).

15. WAITES K.B., BEBEAR C.M., ROBERTSON J.A., TALKINGTON D. F.,KENNY G. E. Laboratory Diagnosis of Mycoplasmal Infections.Cumitech 34.

16. Basic laboratory procedures in clinical bacteriology. Geneva : WorldHealth Organization (WHO), 1991, 1st edition.

17. Sexually transmitted diseases treatment guidelines. MMWR. 2002. May10, 2002 / 51 / No-RR6.

18. BEBEAR C.M. 2006. L’antibiogramme. Chap. 43. Paris : Eska Editor,1st edition.

19. BEBEAR C.M., RENAUDIN H. 2007. Evaluation de la galerie SIRMycoplasma incluant l’azithromycine.

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