JAC Advance Access originally published online on April 19, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):122-125; doi:10.1093/jac/dkn158
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Original research |
Incidence and antimicrobial susceptibilities of genital mycoplasmas in outpatient women with clinical vaginitis in Athens, Greece
1 Alimos Medifirst, Medical Group Practise, Department of Biopathology, Athens, Greece 2 Department of Informatics with Applications in Biomedicine, University of Central Greece, Lamia, Greece 3 Athens Medical School, Aeginition Hospital, Department of Biopathology and Clinical Microbiology, Athens, Greece
* Corresponding author. Tel: +30-210-7289192; Fax: +30-210-6004608; E-mail: chatlouk{at}hotmail.com/schatzi{at}med.uoa.gr
Received 24 January 2008; returned 6 March 2008; revised 12 March 2008; accepted 14 March 2008
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Abstract |
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Objectives: The incidence and antimicrobial susceptibilities of Ureaplasma urealyticum and Mycoplasma hominis, isolated from vaginal and endocervical swabs collected from 369 outpatient women, were determined.
Methods: Isolation, identification and typing of the pathogens were performed by means of conventional methods. The antimicrobial susceptibilities of the genital mycoplasmas were determined with commercially available kits and evaluated according to the CLSI.
Results and conclusions: In 65 (47.44%) out of the 137 positive specimens, U. urealyticum was grown as a single pathogen, in 0.72% M. hominis was grown as a single pathogen and in 2.92% both urogenital mycoplasmas were grown. In the remaining specimens (48.90%), there was a mixed growth with other microbes. Of the isolated U. urealyticum strains, 87.4% and 98.2% were susceptible to tetracycline and doxycycline, respectively, 79.2% were susceptible to josamycin, 48.6% were susceptible to clarithromycin and 91.8% were susceptible to pristinamycin, while erythromycin, azithromycin, ciprofloxacin and ofloxacin proved to be inactive against most of the strains. M. hominis isolates were 100% susceptible to tetracycline, doxycycline and pristinamycin, while susceptibilities to the other antimicrobial agents varied mainly in the range of ‘intermediate’ or ‘resistant’. As results originating from similar studies from various countries are very controversial, the simplest way to avoid therapeutic failures would be the implementation of rational treatment regimens based on culture isolation and the in vitro determination of the antimicrobial susceptibility of genital mycoplasmas in each clinical case.
Keywords: Mycoplasma hominis , Ureaplasma urealyticum , susceptibility
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Introduction |
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Mycoplasma hominis and Ureaplasma urealyticum belong to the normal commensal flora of the genital tract of sexually active healthy women, with colonization rates reaching up to 80% in certain areas of the world.1 The incidence of infection is affected by the menstrual cycle, pregnancy and the use of vaginal contraceptives.2 Poverty and increasing number of sexual partners, as well as bacterial and protozoan infections (co-infections), favour the colonization of the genital tract by mycoplasmas and ureaplasmas, thus increasing the risk of serious medical complications for the mother during gestation, as well as for the fetus and the neonate.3–6 These aggravating conditions require the therapeutic use of antimicrobials. Agents like β-lactams are completely inactive, since mycoplasmas and ureaplasmas do not have a cell wall. Tetracyclines and quinolones are the drugs of choice,7–9 while the antimicrobial susceptibilities to macrolides vary between the two species. Strains of M. hominis are naturally resistant to C14 macrolides (erythromycin, clarithromycin and roxithromycin), while those of U. urealyticum are moderately susceptible.9–12
The increase in resistance of many pathogens to antimicrobial agents has prompted the implementation of ongoing surveillance studies. As far as we know, there are no such studies from Greece hitherto, with respect to the prevalence, as well as to the antimicrobial susceptibilities, of M. hominis and U. urealyticum. The aim of the present study was to assess the incidence and antimicrobial susceptibilities of these two sexually transmitted pathogens, isolated from vaginal swabs collected from outpatient women with clinical vaginitis in Athens, Greece.
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Patients and methods |
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Patients
A total of 369 women, aged 18–62, were examined in the laboratory of the Medical Center of Alimos (Medifirst) of Eureko Group (Interamerican) during a 7 month period (from 23 December 2005 to 22 July 2006).
Ethics Committee approval and informed patient consent were not required for this study.
Specimen collection, processing, culture and antimicrobial susceptibility testing
For each patient, two vaginal swabs from the posterior vaginal fornix and three endocervical swabs were collected and processed as follows.
Two vaginal swabs and one endocervical swab were used for the detection of Trichomonas vaginalis, Neisseria gonorrhoeae, Gardnerella vaginalis, Streptococcus agalactiae and Candida spp. Isolation, identification and typing of the above pathogens, where needed, were performed by means of conventional microbiological procedures. Moreover, the presence or absence of natural vaginal flora (Lactobacillus spp.) and the number of leucocytes per low power field (l.p.f.) were registered.
One endocervical specimen was collected by means of the special swab of the commercial ‘Clearview Chlamydia Female Specimen Collection Kit’ combined and provided with the commercial direct qualitative immunoassay kit (Clearview Chlamydia Female Specimen Collection Kit-Clearview Chlamydia MF, Unipath Ltd, Bedford, UK) and investigated for the presence of Chlamydia trachomatis antigen, according to the manufacturer’s instructions.
The presence of M. hominis and U. urealyticum, as well as their antimicrobial susceptibilities, were investigated with the commercially available Mycoplasma IST 2 Kit (bioMérieux, Marcy-l’Étoile, France) as indicated by the manufacturer. Briefly, the endocervical cotton swab included in the kit was inoculated in R1 transport medium, inhibiting most of the Gram-negative and Gram-positive bacteria. The inoculated R1 medium was vortexed rapidly and 3 mL was added to the growth R2 medium, which contained 1 mL of lyophilized urea/arginine broth. After reconstitution and shaking, 55 µL was dispensed into each of the 22 test wells on the strip. Two drops of mineral oil were added to each well. The remainder of the R2 medium and the inoculated strip were then incubated at 37°C and observed for colour changes at 24 and 48 h. The antimicrobial susceptibility testing included tetracycline, doxycycline, erythromycin, azithromycin, clarithromycin, josamycin, ofloxacin, ciprofloxacin and pristinamycin. The development or absence of red colour on the relevant part of the strip provided an index of resistance or susceptibility to each antimicrobial agent, respectively, according to the guidelines of the CLSI. The breakpoints for the antimicrobials tested are given in Table 2.
Before incubation of the R2 medium, 100 µL was inoculated on an A7 agar plate (bioMérieux), left to dry and incubated at 37°C under anaerobic conditions (Genbox-anaer, bioMérieux) for 48 h. In samples with mixed bacterial flora, there was no growth of bacterial species other than Mycoplasma or Ureaplasma on A7 agar, while in specimens containing yeasts, there was a mixed growth and typical Mycoplasma or Ureaplasma colonies were picked up and subcultured in R1 medium for further antimicrobial susceptibility testing. A7 agar was used for the quantitative assessment of M. hominis and U. urealyticum in cfu/mL by direct microscopy (l.p.f), which was performed in accordance with the manufacturer’s instructions. Quantitative determination carried out by means of the colour change of the culture medium could only give a rough estimate of the titre. The exact titre was determined after plating on A7 Mycoplasma agar. Colonies resembling a fried-egg indicated the presence of M. hominis, while those colonies that were tiny and brown indicated the presence of U. urealyticum. Only cultures with a growth of 
104 cfu/mL were considered positive and were included in the present study.
Statistical analysis of the results was performed using the standard binomial test.
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Results |
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Detection of pathogenic microorganisms
Of the 369 specimens tested, 137 (37.1%) were positive for M. hominis or U. urealyticum or both, either alone or in combination with other microorganisms (Table 1). In 65 (47.44%) out of the 137 positive specimens, U. urealyticum was grown as a single pathogen, in 1 (0.72%) M. hominis was grown as a single pathogen and in 4 (2.92%) both urogenital mycoplasmas were grown. In the remaining 67 specimens (48.90%), there was a mixed growth including genital mycoplasmas and other microbes (Table 1). None of the mycoplasma-positive specimens was positive for C. trachomatis.
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Statistical analysis of the isolation rates showed: (i) a significant association between mixed cultures with U. urealyticum and Candida spp. with the presence of >5 leucocytes per l.p.f. (P < 0.05); and (ii) a significant association between U. urealyticum as a single isolated pathogen with the presence of Lactobacillus spp. (normal vaginal flora; P < 0.001). No other statistically significant association could be observed regarding the isolated microorganisms, the presence of normal vaginal flora and the number of leucocytes in the clinical samples.
Antimicrobial susceptibility patterns
The antimicrobial susceptibilities of M. hominis and U. urealyticum are shown in Table 2. The majority of U. urealyticum isolates were susceptible to tetracycline and doxycycline (87.4% and 98.2%, respectively), 79.2% were susceptible to josamycin and about half of the isolates (48.6%) were susceptible to clarithromycin, while erythromycin, azithromycin, ciprofloxacin and ofloxacin proved to be inactive against most of the isolates. Most of the U. urealyticum isolates were susceptible to pristinamycin (91.8%).
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The small number (only five) of M. hominis isolates were completely (100%) susceptible to tetracycline, doxycycline and pristinamycin, while susceptibilities to the other antimicrobial agents varied mainly in the range of ‘intermediate’ or ‘resistant’ (Table 2).
The susceptibilities of the mixed isolates including M. hominis and U. urealyticum resembled those of M. hominis (Table 2).
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Discussion |
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Genital mycoplasmas can be found as commensal colonizers in healthy populations.13 In the present study, the clinical symptoms of vaginitis together with a colony number
104 cfu/mL were strong evidence of pure or mixed mycoplasmal infection. In addition, a high proportion of the specimens (75 out of 137: 54.74%) contained numbers of leucocytes (>5 per l.p.f.) consistent with inflammation and infection. In about half of these specimens (29.93%), only genital mycoplamas were grown, while the rest (24.81%) comprised mixed cultures either with Candida spp. or other bacteria (Table 1). The normal vaginal flora (Lactobacillus spp.) was absent in all specimens positive for genital mycoplasmas and G. vaginalis. However, it is impossible to correlate these two points with each other, as there were no data available with respect to the menopausal status of the patients. Regarding antimicrobial susceptibility, tetracycline and doxycycline were active against 87.4% and 98.2%, respectively, of the U. urealyticum isolates and 100% of the M. hominis isolates. Among the macrolides, the highest activity against U. urealyticum was shown by josamycin (79.2%) followed by clarithromycin (48.6%), while the response to erythromycin and azithromycin was very weak (17.1% and 14.4%, respectively). M. hominis is known to be intrinsically resistant to erythromycin and that was fully in agreement with our results. Pristinamycin proved to be very active against the great majority of both mycoplasmal species included in the study (91.8% for U. urealyticum and 100% for M. hominis). Ofloxacin and ciprofloxacin proved to be ineffective against the majority of U. urealyticum and a great number of the strains were intermediately susceptible (81 out of 111 for ofloxacin: 72.9%). The same was observed with the few strains (only five) of M. hominis, where only 20% was susceptible to both quinolones and the rest were distributed in the intermediate and resistant categories.
We have to point out that antimicrobial susceptibilities of mixed isolates (M. hominis and U. urealyticum), as shown in Table 2, are only indicative for clinical therapeutic purposes (performed according to the manufacturer’s instructions included in the kit) and are not further discussed or evaluated in the present report. In that case, it would be time-consuming and cost-ineffective to isolate pure cultures and test the two species separately.
Results from previous reports, regarding the antimicrobial susceptibilities of genital mycoplasmas, originating from various countries, are very controversial.14–21 The discrepancies must be due to the different antimicrobial-use policies, which lead to the emergence of resistance to one or other antimicrobial group. Thus, it is very uncertain to establish common guidelines for the empirical treatment of genital mycoplasmal infections, especially when a great many of them are mixed with other pathogens.
Many different alternatives are available for the empirical treatment of genital mycoplasmas, including mainly tetracyclines and quinolones, while recent reports introduce newer drugs such as josamycin and pristinamycin as well. However, the empirical treatment can be ineffective for the reasons mentioned above, and it might be that drugs used in the past and considered effective can prove to be inactive in many cases.
The simplest way to avoid therapeutic failures would be the implementation of rational treatment regimens. This requires the in vitro determination of the antimicrobial susceptibility of the isolated genital mycoplasmas in each clinical case, which has now become a simple routine laboratory procedure, through the use of commercially available systems.
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Funding |
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This work was supported by Dr George Veliotes on behalf of Eureko Companies and the European Alliance Partners Company AG (EURAPCO), Nikolaos Mitsakakis on behalf of the Medical Center of Alimos (Medifirst) of Eureko Group (Interamerican) and Department of Biopathology and Clinical Microbiology, Aeginition Hospital, Athens Medical School.
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None to declare.
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Acknowledgements |
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We would like to thank Dr George Veliotes, the Chairperson of the Health Business Group of Eureko Companies and the European Alliance Partners Company AG (EURAPCO), and Nikolaos Mitsakakis, the Financial Manager of the Medical Center of Alimos (Medifirst) of Eureko Group (Interamerican), for their financial and moral support, without which this study would not have been possible.
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References |
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