JAC Advance Access published online on August 27, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn337
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Original research |
Differences in biofilm development and antibiotic susceptibility among clinical Ureaplasma urealyticum and Ureaplasma parvum isolates
Servicio de Microbiología, Hospital Universitario Ramón y Cajal y CIBER en Epidemiología y Salud Pública (CIBERESP), Ctra Colmenar Km 9.1, Madrid 28034, Spain
* Corresponding author. Tel: +34-91-3368542; Fax: +34-91-3368809; E-mail: rosacampo{at}yahoo.com
Received 15 April 2008; returned 7 July 2008; revised 23 July 2008; accepted 27 July 2008
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Abstract |
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Objectives: The aim of this work was to study the ability of clinical isolates of Ureaplasma spp. to form biofilms in vitro and to compare the antibiotic susceptibility of sessile cells and their planktonic counterparts.
Methods: A total of nine Ureaplasma spp. isolates recovered from unrelated male patients diagnosed with urethritis or chronic prostatitis and two isolates isolated from the urine of two healthy volunteers were included. Ureaplasma species identification was performed by 16S rDNA gene amplification and sequencing. Conventional antibiotic susceptibility tests were carried out by the broth microdilution method. Biofilm susceptibility assays were performed following the method proposed by Moskowitz using 10C urea broth medium and confirming bacterial growth by colour shift of the medium. The 
2 test was applied to analyse the statistical differences between the MIC and the minimal biofilm inhibitory concentration.
Results: Isolates were identified as Ureaplasma urealyticum serovar 7 (five isolates), U. urealyticum serovar 13 (four isolates) and Ureaplasma parvum serovar 3 (two isolates). Biofilm formation was observed in 9 out of the 11 strains studied (82%); two isolates of U. urealyticum serovar 13 were non-biofilm formers. Global resistance percentages of planktonic cells compared with sessile cells were different for erythromycin (0% versus 44%, P = 0.02), telithromycin (22% versus 77%, P = 0.02), ciprofloxacin (66% versus 100%), levofloxacin (0% versus 33%) and tetracycline (0% versus 33%). All nine biofilm-forming strains were fully susceptible to clarithromycin in both planktonic and biofilm types of growth.
Conclusions: These results indicate that biofilm formation can protect mycoplasma cells from antibiotics and host defences, favouring their persistence in chronically infected or colonized patients while increasing resistance to antimicrobial agents. Therefore, the capacity to form biofilms by Ureaplasma spp. isolates should be considered when antibiotic treatments are required.
Key Words: biofilm formation , U. parvum , U. urealyticum
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Introduction |
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About 17 different species of the class Mollicutes have been detected that colonize or infect humans; Mycoplasma pneumoniae, which causes respiratory tract infections, and Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma spp., which are responsible for genito-urinary tract infections, being the most important pathogens.1 Although these microorganisms can be intracellularly located, they usually colonize mucosal surfaces using bacterial structures that facilitate adherence to eukaryotic cells.2
Biofilms are complex aggregations of sessile bacterial cells enveloped by an extracellular matrix of biopolymeric substances. Clinically, biofilm infections are notoriously difficult to treat with antimicrobial agents and disinfectants, and the host immune system is generally ineffective at clearing infection due to limited access. The biofilm mode of life conveys a survival advantage that results in persistent infections.3 The reduced permeability of biofilms usually impairs the action of antibiotics. As an exception, azithromycin has demonstrated a specific ability to avoid biofilm formation by Pseudomonas aeruginosa inhibiting alginate production and blocking quorum sensing signals.4 Therapeutic options for Mycoplasma spp. infections are limited, and macrolides are the most active agents.
The ability to form biofilms by animal Mycoplasma species has already been examined, showing considerable intra-species differences.5 Conversely, biofilm formation by human mycoplasmas has not been reported, although their involvement in persistent infections and their recognized adherence to mucosal surfaces firmly suggest this possibility. The aim of this work was to study the ability of clinical isolates of Ureaplasma urealyticum and Ureaplasma parvum to form biofilms in vitro and to compare the antibiotic susceptibility of sessile cells and their planktonic counterparts.
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Methods |
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A total of seven U. urealyticum and two U. parvum isolates, recovered from urethral exudates and semen from unrelated male patients diagnosed with ureaplasmal urethritis or chronic prostatitis, were used in this study. Two U. urealyticum 7 (serovar 1) isolates cultured from the urine of two healthy volunteers were included as controls. All isolates were recovered during 2007 at the Microbiology Service of the Ramón y Cajal University Hospital, Madrid, Spain. Clinical samples were inoculated in 10C urea broth medium [70 mL of pleuropneumonia-like organism media (PPLO) pH 5.5, 20% horse serum, 10% yeast extract, 0.01% L-cystein hydrochloride, 0.04% urea, 0.5% FILDES (Difco), Phenol Red, ampicillin and GHL tripeptide]. A positive growth was considered when a colour shift due to urea hydrolysis resulting in concomitant alkalinization of the broth medium was observed and confirmed with the growth of typical brown colonies after subculture on A7 agar. Ureaplasma biovars were determined by 16S rDNA gene amplification and sequencing using primers and conditions previously described.6
Conventional MIC susceptibility testing with erythromycin, clarithromycin, telithromycin, tetracycline, ciprofloxacin, levofloxacin and linezolid was performed by the broth microdilution method following the Mycoplasmal Chemotherapy Working Team Guidelines.7 All antibiotics were supplied by their corresponding manufacturers or purchased from Sigma (Sigma Chemical Co., St Louis, MO, USA). Microdilution panels were aerobically incubated overnight at 37°C after inoculation. All tests were performed in duplicate and results expressed as the mean value of both experiments.
Biofilm susceptibility assays were performed as previously described with some modifications.8 Briefly, 96-well microtitre plates (Alpha Laboratories LTD, Hampshire, UK) were inoculated with 100 µL of a 1/100 dilution of a Ureaplasma overnight culture in 10C urea broth medium. Bacterial biofilms were formed by immersing the pegs of a modified polystyrene microtitre lid (catalogue no. 445497; Nunc TSP system Roskilde, Denmark) into this biofilm growth plate incubated at 37°C until the medium changed from orange to fuchsia (due to pH shift occurring between 24 and 36 h). Lids were then washed three times in sterile PBS to eliminate the planktonic organisms and placed on another microtitre plate containing serial dilutions of the corresponding antibiotic and incubated again until the medium colour changed again. The biofilms formed in the lids were transferred to the microtitre wells by centrifugation at 3000 rpm for 10 min. The lid was rejected and replaced by another clean lid and the plate was reincubated for another 24 h. An adequate biofilm growth of the positive growth control well (without antibiotic) was defined by the colour change of the broth medium (from orange to fuchsia). MIC and minimal biofilm inhibitory concentration (MBIC) are defined as the lowest concentration of antimicrobial that prevents a colour change of the planktonic and of the sessile cell cultures, respectively, at the time when the positive growth control shows its initial colour change. Comparison between MBIC50–MIC50 and MBIC90–MIC90 values was carried out only for biofilm-forming strains.
Statistical analysis of the results was performed using the 2 test.
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Results |
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Ureaplasma species identification by the 16S rDNA gene sequence is shown in Table 1. Biofilm formation was observed in 9 out of the 11 strains studied (82%). The two U. urealyticum serovar 13 strains recovered from urethral exudates were not able to form biofilms under our experimental conditions. Antibiotic susceptibility results are shown in Table 1. In general, MBICs were one or two dilutions higher than MICs, although some exceptions were observed. These exceptions comprise results found for telithromycin, levofloxacin and tetracycline in the case of four strains (numbers 1, 7, 66711 and 151302) in which one or more of these antimicrobials exhibited greater activity against sessile cells than against planktonic cells. Nevertheless, when comparing the global MIC with the MBIC values for all biofilm-former strains, MBICs were higher than MICs for all antibiotics.
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Considering antibiotic susceptibility and resistance concepts, most Ureaplasma spp. isolates were susceptible to all antibiotics tested when conventional MICs were determined. However, resistance rates considerably increased when antibiotic susceptibility tests were carried out for sessile cells. Thus, global resistance percentages in planktonic cells compared with sessile cells were lower for erythromycin (0% versus 44%, P = 0.02), telithromycin (22% versus 77%, P = 0.02), ciprofloxacin (66% versus 100%), levofloxacin (0% versus 33%) and tetracycline (0% versus 33%). All nine biofilm-forming strains were fully susceptible to clarithromycin, in both planktonic and biofilm types of growth. At present, no breakpoints are available for linezolid against Ureaplasma spp.; however, the results obtained appear to indicate that this antibiotic is not effective against these microorganisms. However, it is of note that strain number 1 has an MIC value of 0.5 mg/L and strain number 7 exhibited a significantly lower MBIC value (0.06 mg/L) than the MIC value (8 mg/L) of this compound (Table 1).
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Discussion |
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At least 17 well-documented Mycoplasma and Ureaplasma species either colonize or infect humans, 11 of them being considered as normal microbiota. Conversely, M. pneumoniae, M. hominis, Mycoplasma fermentans, M. genitalium, U. urealyticum and U. parvum are considered pathogenic species.1,2 In recent years, molecular techniques have improved the detection of these fastidious microorganisms in view of the fact that their in vitro culture remains difficult, mainly due to their slow growth and to their complex nutritional requirements.7 In order to identify the Ureaplasma isolates included in this work, the 16S rDNA coding gene was fully sequenced. At present, the two Ureaplasma biovars (urealyticum and parvum) include a total of 14 serovars. Most human isolates belong to biovar parvum and mainly comprise serovars 1, 3, 6 and 14. It is of note that in our work biovar urealyticum was more represented than biovar parvum.
The ability of Ureaplasma spp. to form biofilms has not yet been explored and it has been scarcely studied in the case of Mycoplasma spp. Both the biofilm-forming capacity of and biofilm antibiotic susceptibility determinations for Ureaplasma species were the objectives of the present study. With such a purpose, the previously described microtitre plate system was considered the most suitable method.5,8 However, several modifications were introduced due to the slow growing rate of these microorganisms. Following other authors' recommendations, a final pH value of 6.0 was stringently maintained in broth medium because differences in antibiotic susceptibilities, mainly in the case of macrolides, have been observed at higher pH values.9
In the present study, we confirmed the ability of U. urealyticum and U. parvum clinical isolates to form biofilms in vitro. This behaviour could reproduce in vivo situations, as recently reported for an M. hominis strain able to form biofilm (observed with scanning electron microscopy) within the amniotic cavity of a woman presenting acute necrotizing chorioamnionitis.10 In the case of chronic bacterial prostatitis, it has also been demonstrated by scanning electron microscopy that bacteria form biofilms that adhere to the epithelium of the ductal system. This situation leads to persistent prostate inflammation and is responsible, at least in part, for frequent antimicrobial treatment failures.11
Mycoplasmas posses both the smallest cell size and the smallest genome size of any bacteria known.1,2 Moreover, these bacteria apparently lack the two-component regulatory system responsible for the quorum sensing machinery directly involved in biofilm formation. Keeping this in mind, we tried to find the putative genes responsible for biofilm formation in the corresponding genome databases analysing all available Mollicutes genome sequences. A comparison with the previously biofilm-associated genes esp (from Enterococcus faecalis), bap (from Staphylococcus aureus), mus20 (from Pseudomonas putida) and sty2875 (from Salmonella Typhi) was performed but no homologous genes were found. Considering this approach, it can be speculated that mycoplasmas are able to form biofilms using a minimal and stochastically regulated genetic system not yet described.
As reported for other organisms, antibiotic susceptibility is reduced in Ureaplasma sessile cells forming biofilms when compared with planktonic cultures. In the present study, the major and statistically significant susceptibility differences between both types of cell growth were observed for erythromycin and telithromycin, these compounds being more active against planktonic cultures. The most active antibiotic was clarithromycin, with all strains being fully susceptible irrespective of the type of growth. As previously reported, linezolid was not active against Ureaplasma spp.
We conclude that the ability of Ureaplasma spp. isolates to form biofilms may represent a strategy to survive and persist in different clinical situations, thus contributing to the pathogenesis of chronic infections and increasing their recalcitrance to antimicrobial agents and host defence action.
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M. G.-C. and R. del C. were supported by a pre-doctoral fellowship and a contract from the Fondo de Investigaciones Sanitarias (Instituto de Salud Carlos III) FIS PI 061008 and CB05/137, respectively. This work was partially funded by a research grant from the CIBER en Epidemiología y Salud Pública (CIBER-ESP) and the Microbial Sciences Foundation, Madrid, Spain.
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None to declare.
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References |
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