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 ~  Abstract
 ~ Introduction
 ~ Clinical Features
 ~ Diagnosis
 ~ Conclusion
 ~  References
 ~  Article Figures

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REVIEW ARTICLE
Year : 2015  |  Volume : 33  |  Issue : 2  |  Page : 205-214
 

Ureaplasma: Current perspectives


Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India

Date of Submission19-Sep-2014
Date of Acceptance21-Feb-2015
Date of Web Publication10-Apr-2015

Correspondence Address:
B Dhawan
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.154850

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 ~ Abstract 

Ureaplasma species are the most prevalent genital Mycoplasma isolated from the urogenital tract of both men and women. Ureaplasma has 14 known serotypes and is divided into two biovars- Ureaplasma parvum and Ureaplasma urealyticum. The organism has several genes coding for surface proteins, the most important being the gene encoding the Multiple Banded Antigen (MBA). The C-terminal domain of MBA is antigenic and elicits a host antibody response. Other virulence factors include phospholipases A and C, IgA protease and urease. Besides genital tract infections and infertility, Ureaplasma is also associated with adverse pregnancy outcomes and diseases in the newborn (chronic lung disease and retinopathy of prematurity). Infection produces cytokines in the amniotic fluid which initiates preterm labour. They have also been reported from renal stone and suppurative arthritis. Genital infections have also been reported with an increasing frequency in HIV-infected patients. Ureaplasma may be a candidate 'co factor' in the pathogenesis of AIDS. Culture and polymerase chain reaction (PCR) are the mainstay of diagnosis. Commercial assays are available with improved turnaround time. Micro broth dilution is routinely used to test antimicrobial susceptibility of isolates. The organisms are tested against azithromycin, josamycin, ofloxacin and doxycycline. Resistance to macrolides, tetracyclines and fluoroquinolones have been reported. The susceptibility pattern also varies among the biovars with biovar 2 maintaining higher sensitivity rates. Prompt diagnosis and initiation of appropriate antibiotic therapy is essential to prevent long term complications of Ureaplasma infections. After surveying PubMed literature using the terms 'Ureaplasma', 'Ureaplasma urealyticum' and 'Ureaplasma parvum', relevant literature were selected to provide a concise review on the recent developments.


Keywords: Adverse pregnancy outcomes, antimicrobial susceptibility, genital tract infections, genome, Ureaplasma, Ureaplasma parvum, Ureaplasma urealyticum, virulence factors


How to cite this article:
Kokkayil P, Dhawan B. Ureaplasma: Current perspectives. Indian J Med Microbiol 2015;33:205-14

How to cite this URL:
Kokkayil P, Dhawan B. Ureaplasma: Current perspectives. Indian J Med Microbiol [serial online] 2015 [cited 2018 Dec 12];33:205-14. Available from: http://www.ijmm.org/text.asp?2015/33/2/205/154850



 ~ Introduction Top


Mycoplasmas are the smallest free-living, self replicating organisms. They are widespread in nature as parasites of humans, mammals, reptiles, fish, arthropods and plants. [1] Mycoplasmas are found mainly in the mouth, the upper respiratory tract, and the distal parts of the genitourinary tracts of humans. Among the genital Mycoplasmas, Ureaplasma species are the most prevalent, potentially pathogenic bacteria isolated from the urogenital tract of both men and women. [2] They are also frequently associated with preterm birth and other adverse pregnancy outcomes. The present review focuses on Ureaplasma and recent developments in its pathogenesis, diagnosis and treatment. We surveyed PubMed literature using the terms 'Ureaplasma', 'Ureaplasma urealyticum' and 'Ureaplasma parvum'. The relevant literatures were selected to provide a concise review on the recent developments.

Taxonomy

Ureaplasma belongs to the family Mycoplasmataceae, class Mollicutes order Mycoplasmatales. Ureaplasma was first discovered in 1954 by Shepard M et al., as a pathogen causing non-gonococcal urethritis in men. [3] Since the organisms produced small colonies (7-15 μm diameter), they were originally called T (tiny) strains, T-strain Mycoplasmas or T-Mycoplasmas. They were deemed unique among the Mycoplasmas of human origin in that they metabolized urea and not arginine or glucose. [4] It was thus proposed that there should be a new genus and species designation for these unique organisms within the order Mycoplasmatales. They were named Ureaplasma urealyticum by Shepard M et al., in 1974. [5]

Ureaplasma - General Characters

Ureaplasmas have evolved from Gram-positive bacteria by degenerative evolution to lose their peptidoglycan cell wall. [6]

They are spherical or coccobacillary-shaped bacteria with diameters between 0.2-0.3 μm. The lack of a cell wall renders these organisms insensitive to beta-lactams. This also prevents them from staining by Gram stain and is responsible for their pleomorphic form. Since they have limited biosynthetic capabilities due to their small genome, they require enriched growth medium with serum supplementation for their growth in vitro. Ureaplasma has a generation time of approximately one hour. [1]

Ureaplasma resides in the urogenital tract and respiratory tract and penetrates the sub mucosa only during immunosuppression or instrumentation. In humans, Ureaplasma is transmitted through sexual contact; it can also be transmitted from mother to offspring vertically in utero or through infected body fluids at the time of birth. [7]

Ureaplasma has 14 known serotypes and is divided into two groups-Ureaplasma parvum, (UPA, biovar 1, parvo) and Ureaplasma urealyticum, (UUR, biovar 2, T960). Biovar 1 includes serotypes 1, 3, 6 and 14, and the remaining 10 serotypes belong to biovar 2. Their separation into biovars is based on phylogenetic evidence through DNA-DNA hybridisation studies. The biovars also differ in their sensitivity to manganese salts, restriction fragment length polymorphisms and whole cell patterns on polyacrylamide gel electrophoresis (PAGE). [8] Studies of Ureaplasma published before its division into the two biovars refer to the organism as Ureaplasma sp. or Ureaplasma urealyticum.

U diversum, U gallorale, U felinum, U cati and U canigenitalium are Ureaplasma isolated from bovine, avian, feline, and canine species, respectively. [9]

Ureaplasma - genome

The genome of UPA serovars is between 0.75-0.78 Mbp and that of UUR serovars is between 0.84-0.95 Mbp. The genome of U. parvum is significantly smaller than that of U. urealyticum. UPA serovars have on an average 608 genes, of which 201 encode hypothetical proteins, and UUR serovars have on an average 664 genes, of which 230 encode hypothetical proteins. The Ureaplasma pan genome contains 1020 protein coding genes of which 515 genes are universally conserved among all serovars (i.e. core genome). [10] Paralanov V et al., performed a whole genome comparison on the nucleotide level using ATCC strains of all the 14 serovars. They found that the average intra-species difference was 0.62% with the least difference between UUR4 and UUR12 (0.06%), and the greatest difference between UUR9 and UUR13 (1.27%). On the inter-species level, the average difference was 9.5%, with the greatest difference between UPA1 and UUR9 (10.2%). [10]

Ureaplasma has several genes coding for surface proteins and lipoproteins. The gene encoding the Multiple Banded Antigen (MBA) has been frequently studied. The 5' region of the MBA encodes a conserved N-terminal anchor of the lipoprotein whereas the 3' region of the MBA encodes the C-terminal domain, consisting of multiple tandem repeat units, which are surface-exposed. The C-terminal domain is antigenic and elicits an antibody host response during Ureaplasma infection. Additions or deletions in the number of repeat units in the downstream region of the MBA are associated with antigenic variation. [11] Furthermore, MBA can phase vary with neighbouring genes, and UPA3 was recently shown to produce chimeric genes through phase variation. [10]

Ureaplasma contains one or more integrase recombinase genes. Some serovars contain transposases, or its remnants, and some phage related proteins. The tetM gene was identified as part of a Tn916 transposon, in serovar 9 which has acquired tetracycline resistance. Although tetracycline-resistant Ureaplasma were probably less frequent when serovar 9 was isolated, now they comprise 25-35% of all patient isolates. A report covering the years 2000-2004 from several states in the USA showed that 45% of unique clinical isolates of Ureaplasma spp. contain tetM and are tetracycline-resistant. [3]


 ~ Clinical Features Top


Ureaplasma and Genital tract infections

Ureaplasma is considered a part of normal genital flora with a colonization rate of 40-80%. [2] Despite this, the organism has been implicated in several conditions like non-gonococcal urethritis (NGU), prostatitis, urinary stones, gynecological diseases, infertility, and chronic lung disease in neonates. [12] They are considered opportunistic pathogens because they can be readily isolated from the lower urogenital tract of healthy humans as well as individuals with disease.

Several studies indicate that Ureaplasmas are a cause of non gonococcal, non chlamydial urethritis in men though its pathogenicity is still unclear. Experimental inoculation of Ureaplasma in human volunteers resulted in urethral symptoms, shedding of organisms and pus cells, and gradual amelioration and eradication of infection with minocyclin for 6 days. However, it is also a frequent finding in healthy men. Hence, it ranks behind Chlamydia trachomatis and Mycoplasma genitalium as a cause of NGU. [13] Studies from India by Gupta et al., and Deodhar et al., have shown a prevalence of 11% and 16.1% Ureaplasma infection respectively among patients with non gonococcal urethritis. [14],[15]

Ureaplasma has been isolated from the affected  Fallopian tube More Detailss in patients with Pelvic Inflammatory Disease (PID), but its occurrence at this site is rare and is usually in association with other known pathogens. [13] Though Ureaplasma has been isolated from a large proportion (62-97%) of patients with Bacterial Vaginosis (BV), its role as a pathogen remains unclear. [13]

Rates of vaginal colonization by Ureaplasma range from 8.5-77.5%. Rates of vaginal colonization are related to sexual activity with a higher likelihood seen in individuals with multiple partners. [13] In a study by Dhawan B et al., Ureaplasma was detected in 25.8% patients with genital tract infections and 20.8% in infertile women. [16] Previous studies have shown that U urealyticum biovars were associated with pathogenicity. A study by Chua KB et al., confirmed that biovar 2 was more associated with the loss of lactobacilli in women than biovar 1. They demonstrated that biovar 2 was associated with genital-urinary tract infections (58.18%), as compared to biovar 1 which was only a coloniser of the genitourinary tract. [17]

Ureaplasma and HIV

Ureaplasma
spp. genital infections have been reported with an increasing frequency in HIV-infected patients. In a study by Martinelli F et al., involving 187 human immunodeficiency virus type 1 (HIV-1)-infected male patients with no clinical signs of urethritis, the prevalence of U. urealyticum was higher in AIDS patients (12.3%) than in asymptomatic, HIV-1-infected patients (8.5%) and in healthy individuals (7%). [18] Ghosh A et al., demonstrated a 6% infection rate by Ureaplasma in HIV infected patients compared to 2% in healthy volunteers. [19] These studies indicate that infection with Ureaplasma sp., potentially increase the susceptibility of acquiring and transmitting HIV. Other genital Mycoplasmas like M. hominis and M. genitalium have been termed as candidate 'co factors' in the pathogenesis of AIDS since they act in synergy with HIV virus and exacerbate the retroviral disease. [19] Though not proven, Ureaplasma may have a similar role to play in HIV infected patients.

Ureaplasma and Infertility

Colonisation of lower genital tract by Ureaplasma has been evaluated as a cause of infertility. Gupta A et al., have demonstrated the presence of Ureaplasma urealyticum in 32% of infertile women enrolled in their study. [20] Ureaplasma has been isolated from fallopian tubes of patients with PID. Moreover, it has been shown to adhere to sperm cells thereby causing decreased sperm motility. [21] These may explain the association of Ureaplasma with infertility.

Ureaplasma and renal stones

Ureaplasma has been more frequently isolated from the urine of patients with infection stones than those with metabolic stones. Like Proteus, Ureaplasma can degrade urea due to their urease metabolism and crystallize struvite in animal models. [22] Infections with urease-producing bacteria significantly increase the risk of struvite formation due to increases in urine pH and direct damage of the uroepithelium by ammonia. These bacteria live within the interstices of the urinary stones where antimicrobial agents do not penetrate; thus, persistent infection results in rapid growth of stones and filling of all, or part of, the renal pelvis over a period of weeks or months. [23] In a study by Dewan B et al., extensive culture of stones was performed in 70 patients of nephrolithiasis. Ureaplasma urealyticum was cultured from the renal stones of only two patients. [24] Shafi H et al., conducted a study including samples of 45 renal stones along with samples of urine obtained by simultaneous bladder catheterisation. Though E. coli was the most common bacteria grown from the stones, Ureaplasma urealyticum was the most common organism causing UTI, grown in 62.5% of the urine samples. [25]

Ureaplasma and Adverse pregnancy outcomes

Ureaplasma has been isolated from amniotic fluid from women with chorioamnionitis leading to premature labour. Using PCR amplification of 16S ribosomal DNA, Ureaplasma species was the most common microbial genus identified in the amniotic fluid of women with preterm premature rupture of membranes. [26] Gerber S et al., sampled amniotic fluid by amniocentesis from 254 asymptomatic women at 15-17 weeks' gestation, and Ureaplasma species were identified in 11% subjects. Preterm births were also significantly higher in Ureaplasma-positive women compared to Ureaplasma-negative women. [27] A recent study of 150 women with premature rupture of membranes reported that 96% of the affected women were colonised with Ureaplasma urealyticum. [28] A study from Japan found that among 151 placentas from pregnancies that ended with spontaneous preterm birth, 63 were culture positive for Ureaplasma and 83% of these showed histological chorioamnionitis. [29]

Ureaplasma infection is also associated with other adverse pregnancy outcomes. Ureaplasma has been isolated more frequently from spontaneously aborted foetuses and from stillborn and premature infants than from foetuses delivered by induced abortion or from healthy full-term infants. The recovery of organisms from aborted foetuses is not entirely due to superficial contamination, since the organisms have been isolated from internal organs. [13]

Ureaplasma and Diseases in the newborn

Ureaplasma occasionally causes respiratory disease in newborns. Ureaplasma appears to be involved in respiratory disease in very-low-birth weight infants. Thus, infants weighing < 1,000 g who have had Ureaplasma isolated from tracheal aspirates within the first 24 hours of life have been found to be twice as likely to die or to develop chronic lung disease as are uninfected infants of similar birth weight or who weigh > 1,000 g. [30]

Isolation of Ureaplasma from endotracheal secretions of newborns shows that infection of the foetus can occur in utero or alternatively be acquired by vertical transmission at birth. Sethi S et al., isolated Ureaplasma urealyticum from 14% of tracheal aspirates taken from low birth weight infants with respiratory distress. [31] It is hypothesized that intrauterine inflammation and Ureaplasma colonization induce foetal lung maturation prematurely, predisposing the infant to the future likelihood of broncho-pulmonary dysplasia (BPD) and chronic lung disease (CLD). However, there are several reports which accept or refute the association of Ureaplasma in the respiratory secretions of newborns with the development of BPD. Pandey A et al., had reported in their study that colonization of the airways with U. urealyticum had no significant role in development of CLD in Indian preterm infants. [32] Schelonka RL et al., performed a meta-analysis of published studies and found that the relative risk for BPD in Ureaplasma positive infants was 1.6 (C.I. 1.1-2.3) for BPD at 36 weeks, or 2.8 (C.I. 2.3-3.5) for BPD at 28 weeks compared to Ureaplasma negative group. [33] Heterogeneity in studies maybe due to different patient populations, divergent ventilator management practices, non-uniform Ureaplasma culture techniques and publication bias.

Ureaplasma infection may also cause retinopathy of prematurity (ROP) in preterm infants by interfering with normal retinal vascularisation. Ozdemir R et al., demonstrated that Ureaplasma urealyticum colonization is associated with severe ROP and the need for laser ablation surgery. [34]

Ureaplasma and other infections

Ureaplasma has been isolated from the joints of patients with hypogammaglobulinemia presenting with suppurative arthritis. The arthritis in these patients is seen to persist for months even after treatment with antibiotics and anti-inflammatory agents. Sethi S et al., reported a case of septic arthritis in a young girl of 10 years. Ureaplasma urealyticum was isolated from the synovial fluid aspirated from her knee joint. It is hypothesized that the spread to the target joint may be haematogenous. [35]

Pathogenesis

Ureaplasma attaches to mucosal surfaces with the help of cytadherence proteins. These are expressed on the surface of the bacterial cell. Ureaplasma is known to adhere to a variety of human cells including erythrocytes, spermatozoa, and urethral epithelial cells.

The key virulence factor of Ureaplasma is the MBA, a surface-exposed lipoprotein. TLR1, TLR2 and TLR6 are the pattern recognition receptors through which the MBA activates nuclear factor kappa B. [36] The MBA is known to demonstrate size variation. Robinson et al., have shown that the severity of chorioamnionitis correlated inversely with the number of MBA size variants that existed within infected amniotic fluid (AF), suggesting that variation of the MBA was associated with Ureaplasma pathogenicity. [37]

Zimmerman CR et al., demonstrated that the MBA of U. parvum underwent alternate phase variation with an adjacent gene, UU376. Furthermore, phase variation of MBA N-terminal paralogs (UU171 and UU172) was recently described in both U. parvum and U. urealyticum. [38] A primary function of antigenic variation is to evade the adaptive immune response. Dando SJ et al., have demonstrated using a sheep model that MBA size variability did not prevent recognition by host pattern recognition receptors. However, it may prevent the host immune response from eradicating Ureaplasma from the amniotic cavity and thus play a role in the virulence of these microorganisms. [39]

Ureaplasma expresses Phospholipase A and C. These generate prostaglandins-a known trigger of labour. [3] Kacerovsky et al. evaluated the amniotic fluid protein profiles and the intensity of intra amniotic inflammatory response to Ureaplasma spp., using the multiplex xMAP technology. The presence of Ureaplasma spp. in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinase-9. This generalized inflammation of the amnion and the chorio-decidua resulting in the production of pro-inflammatory mediators such as interleukin-1beta (IL-1β), IL-6 and prostaglandins ultimately initiate preterm labour. [40]

Ureaplasma has also shown an IgA protease activity, which could destroy mucosal IgA. Since IgA is the predominant immunoglobulin secreted at mucosal surfaces, IgA proteases may facilitate colonization by microorganisms by degrading this important component of the mucosal immune system. [3] However, genome analysis of all serovars has not shown the presence of this gene. It is speculated that the enzyme may have diverged so far from orthologues in other bacteria they were unrecognizable, or they may have convergently evolved an enzyme with no recognizable similarity to other enzymes. [10]

The urease activity of Ureaplasma generates ammonia from the cleavage of urea, which can cause toxicity to host tissues due to change in pH. Chronic lung disease after exposure to ammonia has been reported in adults. [3] However, Robinson et al., showed that an elevated pH in either the amniotic fluid or foetal lung fluid did not correlate with increased inflammatory cell counts in the chorioamnion or foetal lung tissue. But the ammonia liberated by Ureaplasma may contribute to the chronic tissue damage and pathology observed within the chorioamnion and the foetal lung in utero. [37]

Virulence and persistence of Ureaplasma is also influenced by the ability of microorganisms to form biofilms. A study conducted by Pandelidis K et al., confirmed that most clinical Ureaplasma isolates form biofilms in vitro. These biofilms may contribute to organism persistence and chronic inflammation but biofilm-formation did not impact MICs for azithromycin or erythromycin. [41]


 ~ Diagnosis Top


Specimen collection

Appropriate specimens for detection of Ureaplasma include urethral swab, urine, endocervical swab and endometrial biopsy. Dacron swabs, calcium alginate and polyester swabs on plastic shafts are ideal since cotton swabs on wooden shafts have an inhibitory effect on the organisms. Since these organisms are extremely delicate and sensitive to drying and heat, transport medium such as SP4, 2SP, Shepard's 10 B and PPLO broths should be used. Other media include Stuart's medium, Amie's medium and trypticase soy broth with 5% bovine serum albumin. Specimens should be refrigerated if immediate transportation to the laboratory is not possible. Since Ureaplasma is considered to be category 2 pathogen, laboratory bench and/or class 2 safety cabinet is sufficient for work on this organism. [1]

In the laboratory, PPLO broths containing swabs are vortexed and the swabs are discarded. The samples are then concentrated to 10-fold by centrifugation at 500 g for 30 minutes and processed through 0.45 μm membrane filter. [15]

Microscopy

Lack of a rigid cell wall makes it nearly impossible to directly visualize Ureaplasma by light microscopy. Though Gram stain precludes visualisation, it is useful to rule out other causative bacteria. DNA fluorochrome stains like acridine orange and Hoechst 33258 may be useful in centrifuged samples like amniotic fluid. [1]

Culture

Culture is considered the gold standard in the detection of Ureaplasma but it is difficult since these fastidious organisms require the presence of serum, metabolic substrate and growth factors like yeast extract for isolation. The recommended culture media include SP4 broth and agar, Shepard's 10 B broth and agar and PPLO broth and agar. Since the organisms do not produce any turbidity on growth, pH indicators like phenol red are added. The growth of organisms leads to a change in pH of the media which is visualised as a change in colour of the indicator. Antibiotics like Penicillin G and antifungals like Nystatin are also incorporated to prevent growth of contaminants.

The commonly used medium for isolating Ureaplasma is the PPLO broth containing urea. The processed samples are serially diluted 10-fold from 1:10 to 1:10 5 for inoculation. Dilution is essential since it overcomes interference by antibiotics, antibodies or other inhibitors, overcomes rapid decline in culture viability and indicates the number of organisms present. The inoculated broths are incubated at 37°C under 5% CO 2 and inspected twice daily. A rise in pH visualized by change in colour without any turbidity is indicative of growth. The growths are then sub cultured into broth again and onto agar. The highest dilution which shows change in colour represents the number of organisms present in the sample in colour changing units per ml (CCU/ml) [Figure 1]. A concentration of > 10 4 CCU/ml is the cut off for Ureaplasma. On PPLO agar, they show growth with a characteristic fried egg appearance enhanced by Dienes stain [Figure 2]. [16]
Figure 1: Culture of Ureaplasma spp. in PPLO broth containing urea Tube 1: Culture Positive, Tube 2: Culture Negative

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Figure 2: Characteristic Ureaplasma colonies on PPLO Agar (10×)

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Mycofast Revolution (ELiTech Diagnostic, France) assay is a new commercial assay which provides easy identification and enumeration of Ureaplasma spp. and M. hominis within 24 h to 48 h. The assay is a liquid method based on the ability of Ureaplasma spp. and Mycoplasma hominis to metabolize urea and arginine, respectively and consists of 20 wells that are pre-coated with a dehydrated culture medium (foal serum, yeast extract, cysteine, arginine, urea, phenol red and antibiotics) and contains a single broth with antimicrobials for transport and preservation of genital Mycoplasmas (UMMt) (ELiTech Diagnostic, France). [42]

Other commercially available diagnostic assays with similar methods of identification, antimicrobial susceptibility testing, turnaround time and ease of use include the Mycoplasma Duo kit (Sanofi Diagnostics Pasteur, France), the Mycoview (Ivagen) and MycoIST2 (BioMérieux) test kits. [43] The advantage of the Mycofast Revolution assay is that antimicrobial susceptibility testing is performed against different antimicrobial agents with specific minimum inhibitory concentrations (MICs) as defined by the 2011 Clinical and Laboratory Standards Institute (CLSI) guidelines. Antimicrobial susceptibility testing is performed against five antimicrobial agents that include levofloxacin, moxifloxacin, erythromycin, clindamycin and tetracycline. [42]

In a study by Redelinghuyhs MJ et al., Mycofast Revolution assay was compared with PCR for the detection of genital Mycoplasmas in pregnant women from self collected swabs. The Mycofast Revolution kit had a sensitivity and specificity of 77.3% and 80% respectively, against the PCR assay. [42]

Serology

Serological test methods for Ureaplasma include microimmunofluorescence, metabolism inhibition, and enzyme immunoassay, but the ubiquity of Ureaplasma in healthy people makes interpretation of antibody titres against these organisms difficult. No serological assays for the genital Mycoplasmas have been standardized, and they are not available for diagnostic purposes. [3]

Nucleic Acid detection tests

Gel based conventional PCR assays target sequences of 16S rRNA and 16S rRNA to 23S rRNA intergenic spacer regions, urease gene, and MBA, whereas real time PCR assays mainly target the urease genes and their subunits or MBA. A study conducted by Dhawan B et al., determined the prevalence of U. urealyticum in patients with genital discharge by both culture and PCR. The PCR targeted a 429 bp region in the urease structural gene of U. urealyticum [Figure 3]. The prevalence of U. urealyticum as determined by culture was 32% and by PCR was 45% with an agreement of 93.75%. [44] In yet another study, a multiplex PCR targeting the urease gene and the 16S rRNA was used to detect Ureaplasma and M. hominis respectively. The positive Ureaplasma isolates were further subjected to a second PCR targeting the multiple banded antigen gene for biotyping [Figure 4]. The majority of the Ureaplasma isolates belonged to biovar 1 (U. parvum). Primers like UMS83/UMA269, UMS125/UMA269 and UMS54/UMA269 were further used for identification of serovars [Figure 5] and serovars 3/14 was found to be the commonest. [16]
Figure 3: PCR for urease gene, Lane 1: 100 bp ladder, Lane 2: U. urealyticum positive control (NCTC 10177; urease positive), Lane 3-8: Clinical sample (positive), Lane 9: Negative control

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Figure 4: PCR for MBA gene, Lane 1: Positive control (biovar 2) - NCTC 10177, Lane 2: Positive control (biovar 1) - Serovar 3, Lane 3: Clinical sample positive for biovar 1, Lane 4: Clinical sample positive for biovar 2, Lane 5: Clinical sample positive for biovar 2, Lane 6: Negative control, Lane 7: 100 bp ladder

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Figure 5: PCR amplification for identification of serovars of U. parvum Lane 1: 100bp Marker, Lane 2: Serovar 1 positive control, Lane 3: Serovar 6 positive control, Lane 4: 3/14 positive control, Lane 5: Negative control, Lane 6: Positive clinical sample for Serovar 3/14

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The UAB Diagnostic Mycoplasma Laboratory performs a real-time PCR assay for detection and differentiation of Ureaplasma species based on UU063 (NP_077893), which encodes a conserved hypothetical protein that is identical in all four U. parvum serovars and a 15,072 bp open reading frame, UUR10_0680 (NC_011374.1), that is conserved (≥99.97%) in all 10 U. urealyticum serovars. [45]

STD6 and STD6B ACE Detection (Seegene, Inc), is a molecular assay commercially available in various European countries only. The assay simultaneously detects Trichomonas vaginalis, M. hominis, M. genitalium, C. trachomatis, N. gonorrhoeae, and Ureaplasma species in endocervical/urethral swabs. The novel feature of this assay technology is a dual-priming oligonucleotide system that contains two separate priming regions linked by a polydeoxyinosine spacer. The kit works with any thermocycler, and the post-PCR assay is designed for either manual or automated gel electrophoresis. The STD6 Ureaplasma assay amplifies a 130-bp region of the ureD cassette. Their new version, STD6B, differentiates U. urealyticum from U. parvum ureC genes. [45]

Ureaplasma species are common commensals in the lower urogenital tracts of healthy people. Therefore, a positive PCR assay from specimens from these sites is usually expected. An increased bacterial load determined by real-time PCR is more valuable as a clinical indication of infection. [45] Positive PCR results for Ureaplasma species from the urethra in men with urethritis, from tracheal aspirates of neonates with respiratory distress, from the bloodstream or cerebrospinal fluid in neonates with pleocytosis, and from normally sterile extragenital sites should be considered diagnostic of clinically significant infection.

Antibiotic susceptibility testing

Several antibiotic susceptibility testing methods have been used on genital Mycoplasmas. Agar dilution method, Broth micro dilution method and E test are some of them.

Broth micro - dilution method

The most practical, economic and widely used method is the broth micro dilution test. The method uses a 96 well microtitre plate in which decreasing concentrations of antibiotics are mixed with a standard concentration of organisms (usually 10 4 /ml) in broth medium and the mixtures are incubated. The medium used is 10B broth or PPLO broth containing urea. The multiplying organisms metabolize urea in the medium resulting in change in pH visible as a colour change to red due to the presence of phenol red in the medium. This colour change is usually seen 16-18 hours after incubation. If the organism is susceptible to the concentration of antibiotic in the well, growth is inhibited and there is no colour change. The MIC is the highest dilution of antibiotic that inhibits the colour change at the time when the change in the control without antibiotic has just developed [Figure 6]. The method allows several antibiotics to be tested in the same micro titre plate but it requires standardisation since time of reading and inoculum size affects the test. Moreover, heterogeneous population of organisms with resistant and sensitive strains can multiply in the medium and obscure the sensitive ones. It is important to read the MIC endpoint at the first appearance of colour change in the growth control well since longer incubation period will shift the MIC and thus give a falsely elevated MIC. [46]
Figure 6: Microbroth dilution in 96 micro-titre plate for reference strain U. urealyticum NCTC 10177 and clinical isolate. Doxy- doxycycline; Azithro - azithromycin; Josa - josamycin; Oflox - ofloxacin; Cipro-ciprofloxacin and Levo-levofloxacin

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Agar dilution method

The agar dilution method for the determination of MICs is based on the incorporation of doubling dilutions of the antimicrobial agents into molten agar plates, with each plate containing a different concentration. A8 agar or PPLO agar with urea can be used.

The appropriate dilutions of the drugs are mixed with the molten agar. After the agar plates solidify, 10 μl of a defined organism inoculum of 10 4 to 10 5 CFU/ml prepared in the appropriate broth is added to the agar plates. The plates are incubated in ambient air plus 5% CO 2 at 37°C. The MIC is read as the lowest concentration of the antimicrobial agent that prevents colony formation when examined under a stereomicroscope at the same time that the antimicrobial-free control plate demonstrates growth of approximately 30-300 CFU per spot of inoculum. The length of time until MICs could be read is similar to what is typically observed with the broth microdilution assay. [46]

Though laborious and time consuming, the method has advantages that the end point is relatively stable over time and it allows detection of mixed cultures. To simplify the technique, two modifications have been frequently used. The E test uses a strip which contains a specific antibiotic in a concentration gradient. MICs are defined as the antibiotic concentration on the strip at the point of intersection with the zone of colony inhibition. The second method involves using filter paper discs which contain serial two-fold decreasing concentrations of antibiotic. The lowest concentration of antibiotic causing a zone of inhibition is the MIC.

There are no universal guidelines from any regulatory agency for the performance of these tests as well as for the interpretation of MIC breakpoints. The micro broth dilution method is routinely used to test antimicrobial susceptibility of isolates of M. hominis and Ureaplasma sp. The organisms are tested against azithromycin, josamycin, ofloxacin and doxycycline. Cut off MICs for susceptibility, intermediate and resistance for azithromycin, josamycin and ofloxacin are ≤ 2, 4 and ≥ 8μg/ml respectively and those for doxycycline are ≤ 4, 8 and ≥ 16 μg/ml respectively. [15] Tetracycline resistant strains have MIC of ≥ 8μg/ml and susceptible strains usually have MICs of ≤ 2 μg/ml. [46]

Susceptibility profiles and antimicrobial resistance

Genital Mycoplasmas, like any other Mollicutes, are innately resistant to antibiotics which act on cell wall components (the beta lactams). Ureaplasma species have natural resistance to lincosamides (e.g. clindamycin). [47] Observed resistance to macrolides is associated with mutations in the 23S rRNA gene, while resistance to tetracycline is associated with the presence of the moveable tet M transposon. [48] The tet M gene encodes a protein which binds to ribosomes and in the case of U. urealyticum it has been demonstrated to be associated, on the chromosome, with Tn916, a conjugative transposon. [3] Previous study suggested that Ureaplasma resistance to quinolone is mainly due to the mutants of target enzyme-DAN helicase; the residues 68-107 areas are the quinolone-resistant areas (quinolones regions of drug-resistance, QRDR). [49]

Besides beta lactams, Mycoplasmas also show resistance to sulphonamides, trimethoprim and rifampicin. Resistance to rifampicin is attributed to the presence of a single amino acid at position 526 of the beta sub unit of RNA polymerase. [50]

In a recent study conducted by Dhawan B et al., involving patients with infertility and genital discharge, all isolates of M. hominis were resistant to erythromycin but sensitive to the doxycycline, josamycin and ofloxacin. The Ureaplasma sp. isolates were susceptible to doxycycline and josamycin. 77% of the isolates were susceptible to ofloxacin and 71% to azithromycin. [16] Though most studies report lower resistance rates for tetracyclines (<5%), a recent study by Redelinghuyhs MJ et al., demonstrated only 27% susceptibility of Ureaplasma isolates to tetracycline. [51] In a study by Chiang-tai Z et al., in Shanghai, biovar 1 showed high sensitivity rates (above 90%) to all antimicrobial agents; but biovar 2, maintained higher sensitivities (above 95%), only to doxycycline and minocycline. In fact, only a small number of biovar 2 strains were sensitive to roxithromycin and quinolones. [12]

Treatment

Since syndromes characteristic of genitourinary infection is not caused only by genital Mycoplasmas but also by various other organisms, antibiotic susceptibility to all of them must be taken into account while prescribing empirical therapy. Taking this into consideration, the preferred treatment is azithromycin 1 g orally as single dose or doxycycline 100 mg orally twice a day for 7 days. However, since resistance to tetracyclines is on the rise, a patient who fails to respond to doxycycline may also be treated with erythromycin 500 mg orally for 7 days. Fluoroquinolones also demonstrate efficacy equal to doxycycline in the treatment of non gonococcal urethritis. [50] The efficacy and safety of a 7-day course of sparfloxacin were comparable to those of doxycycline in a double-blinded randomized multicenter study of 725 men with clinically diagnosed NGU from chlamydial infection and 5 PMNs/hpf on urethral microscopy who were tested by culture for N. gonorrhoeae, C. trachomatis, U. urealyticum, and M. hominis.[52] However, Ureaplasma sp. is known to cause persistent infection in which case a prolonged course of antibiotics may be required.

The administration of antimicrobial agents to pregnant women with preterm rupture of the membranes (PROM) may extend the gestation period and decrease the risks of associated complications and neonatal infections. Macrolides are often used empirically since tetracyclines and fluoroquinolones are contraindicated in pregnancy. However, the amniotic sac is not effectively penetrated by erythromycin and Ureaplasmas are not eradicated from the vagina or cervix by this agent. Treatment with azithromycin is equally successful compared to erythromycin but with fewer side effects. [53]

Neonates clinically ill with pneumonitis due to Ureaplasma sp. are treated with erythromycin. Waites KB et al., studied the pharmacokinetics and microbiological efficacy of intravenous erythromycin in preterm neonates colonized in the lower respiratory tract with Ureaplasma sp. The study provided data supporting the use of 40 mg/kg/day of erythromycin for intravenous treatment of preterm neonates. [54] A limited number of studies report the use of macrolides for eradication of Ureaplasma in neonates at risk for developing BPD. Ballard et al., randomized 220 mechanically ventilated preterm infants ≤ 1250 g and treated them with azithromycin or placebo for up to 6 weeks. In the subgroup of infants who had Ureaplasma isolated in tracheal aspirates, azithromycin decreased BPD from 94% in the placebo arm to 73% in the antibiotic arm. [55] Recently, a randomized trial from Turkey evaluated clarithromycin in 74 preterm infants and found that treatment for 10 days decreased the incidence of BPD. [56] Although no significant side-effects were reported in these studies, antibiotics must be used with caution in preterm neonates since prolonged antibiotic exposure has been associated with increased rates of necrotizing enterocolitis or late onset sepsis. For invasive mycoplasmal infections like infection of CSF, tetracyclines are the best treatment. [50]


 ~ Conclusion Top


Ureaplasma are associated with a wide spectrum of diseases including non-gonococcal urethritis, urinary stones, gynaecological diseases, infertility, neonatal broncho pulmonary dysplasia, chronic lung disease and retinopathy of prematurity. Since they are smaller than conventional bacteria in cellular and genomic dimensions and have specific nutritional requirements, their identification, isolation and characterization require molecular techniques to complement culture. Prompt initiation of appropriate antibiotic therapy is important to prevent long term complications and sequel of these infections.

 
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2004 - Indian Journal of Medical Microbiology
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