Analysis of mutations in DNA gyrase and topoisomerase IV of Ureaplasma urealyticum and Ureaplasma parvum serovars resistant to fluoroquinolones.

This study aims to determine the prevalence of fluoroquinolone resistance of Ureaplasma biovars and serovars isolated from urogenital clinical samples and determine the underlying molecular mechanism for quinolone resistance for all resistant isolates. Of 105 samples confirmed as positive for U. urealyticum/U. parvum, 85 were resistant to quinolones by the Mycoplasma-IST2 kit. However, only 43 out of 85 quinolone resistant isolates had amino acid substitutions in GyrA, GyrB, ParC and ParE proteins underlining that this assay have mis-identified as fluoroquinolone resistant 42 isolates. The known ParC E87K and ParC S83L mutations were found in 1 and 10 isolates, respectively. An original mutation of ureaplasmal ParC (E87Q, 1 isolate) was found. Furthermore, we found a ParE R448K mutation in one isolate, already described. Among the additional alterations detected, the most prevalent mutation found was L176F in GyrA protein in 18 isolates with single infection and in 3 isolates with mixed ureaplasma infections. Mutations in GyrB (E502Q, 4 isolates), ParE (Q412K, Q412P, Q412T, 3 independent isolates), whose role is unknown, were also found. Other sporadic mutations in the four genes were identified. This investigation is the result of monitoring the data for molecular fluoroquinone resistance in Ureaplasma spp. in Italy. Resulting that this acquired resistance is high and that continued local epidemiological studies are essential to monitor and document their antimicrobial resistance trends.

Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.

Trends in the rates of resistance of Ureaplasma urealyticum to antibiotics and identification of the mutation site in the quinolone resistance-determining region in Chinese patients.

The resistance of Ureaplasma urealyticum to antibacterials, isolated from 804 patients from the outpatient clinic of the Sir Run Run Shaw Hospital Hangzhou, China, from March to June over six consecutive years (1999-2004) was reviewed. The quinolone resistance-determining region of six strains of U. urealyticum was analyzed. The level of resistance to doxycycline, josamycin, tetracycline, azithromycin, clarithromycin and pristinamycin was below 20% and did not change during the study period. The rate of resistance to fluoroquinolones (ofloxacin, ciprofloxacin) was much greater than these (>50%) and has increased since 1999. The rate of resistance to Erythromycin decreased from 63.9% in 1999 to about 20% from 2000 onwards. The widespread use of fluoroquinolones had led to high resistance rates in U. urealyticum and the emergence of quinolone resistance. Analysis of the gene sequence of topoisomerase IV and DNA gyrase suggested a role for the topoisomerase IV ParE subunit in fluoroquinolone-resistant U. urealyticum.

Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme.

Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, demonstrating that a lasso-like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5'-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2'-Ara position, but larger substitutions at the 3' position of the deoxyribose.

Development of real-time PCR for the differential detection and quantification of Ureaplasma urealyticum and Ureaplasma parvum.

Ureaplasma parvum and Ureaplasma urealyticum are recently recognized species of the genus Ureaplasma. In humans, Ureaplasma spp. can be found on mucosal surfaces, primarily in the respiratory and urogenital tracts. They have been implicated in various human diseases such as nongonococcal urethritis, intrauterine infections in association with adverse pregnancy outcome and fetal morbidity, and pneumonitis in immunocompromised hosts. We have developed two quantitative real-time PCR assays to differentially detect U. parvum and U. urealyticum. Based upon the sequence information of the urease gene (ureB), we designed two TaqMan primer and probe combinations specific for U. parvum and U. urealyticum, respectively. The assays did not react with nucleic acid preparations from 16 bacterial species commonly encountered in relevant clinical specimens, including seven urease-producing species. Each assay had a detection limit of approximately five copies per reaction of the respective gene target. The results suggest that these assays are both sensitive and specific for U. parvum and U. urealyticum. Further investigation of both assays using clinical specimens is appropriate.

Structures of thymidine kinase 1 of human and mycoplasmic origin.

Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3'-azido-3'-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an alpha/beta-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects beta-structures at the root of a beta-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin.

Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth.

Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu-TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu-TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu-TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.

Uropathogens and urinary tract concretion formation and catheter encrustations.

Infection stones (ammonium magnesium phosphate) and catheter encrustations have a common cause-urease producing microorganisms. With their rapid growth and frequent recurrences, infection stones are among the most troublesome of urinary system stones. For many patients with a long-term indwelling catheter, encrustations can be a severe problem. Urine composition is important, because, urine calcium enhances the crystallization process and urine citrate inhibits it. The role of non-urease producing microorganisms in stone forming processes is not well understood. Stones can now be successfully treated with a low morbidity index by percutaneous stone surgery or extracorporeal shock wave lithotripsy (ESWL) and recurrence of stone formation is then avoided by prolonged antibiotic treatment and oral citrate. Catheter encrustations and damage caused by ammonia released during urease activity can, however, be a serious problem in patients with indwelling catheters and our remedies are unsatisfactory.

Ureaplasma urealyticum: an opportunity for combinatorial genomics.

Examination of genomic or enzymatic activity data alone neither provides a complete picture of metabolic function or potential nor confidently reveals sites amenable to inhibition. Furthermore, in some cases, gene annotation and in aqua assays disagree by describing gene annotation without enzyme activity and enzyme activity without homologous annotation. The newly sequenced genome of Ureaplasma urealyticum (parvum) is another prokaryote example of the class Mollicutes where such confounding differences are observed. The little-considered role of some proteins as multifunctional enzymes - substitutes for 'missing' genes - could both partially explain the apparent anomalies and relate to any inaccurate deductions of inhibitor function. A combinatorial analysis involving available evidence of genomic sequence, transcription, translational phenomena, structure and enzymatic activity gives the best picture of the organism's vital metabolic alternatives.

Characterization of phospholipase A1, A2, C activity in Ureaplasma urealyticum membranes.

The presence of endogenous phospholipase A (PL-A) activity of U. urealyticum hydrolyzing the acyl ester bond and phospholipase C (PL-C) activity hydrolyzing the phosphodiester bond is primarily localized in the membranes of ureaplasmas. Characterization of the membrane PL-A and PL-C activity in exponential growing cells of serovars 3, 4, and 8 was investigated. The pH optimum was about 8.5-9 for phospholipase A1 (PL-A1) in the three serovars. A more acidic pH optimum of 6 was observed for phospholipase A2 (PL-A2) enzymes in serovars 3 and 4. However, a very significant stimulation of PL-A2 activity in serovar 8 occurred around pH 7. The specific activity of PL-A2 was always 50-100 fold higher than PL-A1 activity in the pH range studied. Ca2+ ions only slightly stimulated PL-A1 activity in all 3 serovars. PL-A1 activity was stimulated about 6-fold from 0.5-0.8 mM Ca2+ ion concentrations for serovar 3 and 12-fold for serovar 8. Only lower concentrations (0.2-0.4 mM) of calcium stimulated PL-A2 activity in serovar 4. EDTA inhibition corresponded to Ca2+ stimulation for PL-A1 activity for serovars 3 and 8. A general stimulation of PL-A1 activity by diethyl ether was evident but the degree of stimulation varied with the serovar. Sodium deoxycholate enhanced PL-A activity of serovars 4 and 3, but partially inhibited that of serovar 8. PL-A activity in the three serovars were not significantly affected by p-hydroxymercuribenzoate, a marker of -SH groups in the enzyme. All 3 serovars were inactivated by heat. A broad pH optimum for PL-C activity was evident around 7-8. Diethyl ether enhanced PL-C activity of serovar 8. Sodium deoxycholate and heat were inhibitory to PL-C activity. The results demonstrate that the major characteristics of ureaplasma membrane bound PL-A and PL-C are basically similar to those of other mollicutes and bacteria. However, the major differences in the specific characteristics of specially PL-A1 and PL-A2 suggest that the ureaplasma phospholipases are unique enzymes different from the phospholipases of bacteria. Both the PL-A and PL-C enzymes function over the broad range at which ureaplasma can grow, pH 5-9 essential for survival. The ureaplasma PL-As are also markedly different from one serovar to another. This variation in specific activity could contribute significantly to differences in virulence among serovars in specific host milieus. There is significant variation from acidic pH of the vagina and alveolar surface of the lung to a more neutral pH of the endometrium and placenta. There are marked differences in calcium concentrations under specific circumstances in various host tissues. Thus the differences in specific activity among the phospholipases of the serovars of U. urealyticum may be of physiological importance in interactions with host tissues and pathogenesis of disease.

Upper urinary tract stones & Ureaplasma urealyticum.

Extensive culture of stones and of pre-operative and renal pelvic urine for isolation of bacteria and Ureaplasma urealyticum were performed in 70 patients of nephrolithiasis. Stones were subjected to biochemical analysis and scanning electron microscopy. Micro-organisms were isolated from 33 (47%) of 70 renal stones. Of the 38 species of micro-organisms isolated, 14 were urea-splitting (U. urealyticum 2; Klebsiella pneumoniae 8; Morganella morganii 1; Acinetobacter spp 3) and 24 were nonurea splitting. U. urealyticum was cultured from the renal stones of two patients. Pelvic urine, unlike voided urine did reflect the bacteriology of the stone. Biochemically, 55 stones (79%) were calcium oxalate phosphate stones, 10 (14%) were calcium oxalate stones and 5 (7%) were uric acid stones. None of the stones were found to be of struvite composition. These data suggest that infection stones are uncommon in this part of the country. Further, infection of renal stones with fastidious organisms like U. urealyticum and multi-drug resistant bacteria necessitate their removal to ensure complete cure.

Biovar diversity is reflected by variations of genes encoding urease of Ureaplasma urealyticum.

Five oligonucleotide primers derived from the gene encoding urease of Ureaplasma urealyticum were designed to evaluate the relationship between the urease gene and biovar diversity of this organism. Five combinations of these primers were tested by PCR and the result revealed that there were variations in urease genes among different serovars of U. urealyticum. This result, in agreement with other PCRs based on other functionally unrelated (rRNA and MB antigen) genes, may reflect the phylogenetic relationship among organisms taxonomically classified as U. urealyticum.

Biovar-specific epitopes of the urease enzyme of Ureaplasma urealyticum.

The importance of Ureaplasma urealyticum as a pathogen in premature neonates and patients with a profound defect in humoral immunity has, over the last few years, become well recognised. U. urealyticum is unique amongst the Mycoplasmataceae for its use of urea metabolism as an essential source of energy. The urease enzyme responsible for this is, therefore, of prime importance and any variability in expression of this enzyme may play a role in virulence of the organism. U. urealyticum is divided into 14 serovars comprising two biovars -- the parvo-biovar and T960-biovar. In this study monoclonal antibodies (MAbs) were produced against the urease enzyme. Two distinct epitopes of the 72-kDa alpha-subunit were recognised by three different MAbs. Under denaturing conditions both epitopes were shown to be specific for the parvo-biovar.

Organization of Ureaplasma urealyticum urease gene cluster and expression in a suppressor strain of Escherichia coli.

Ureaplasma urealyticum is a pathogenic ureolytic mollicute which colonizes the urogenital tracts of humans. A genetic polymorphism between the two biotypes of U. urealyticum at the level of the urease genes was found. The urease gene cluster from a biotype 1 representative of U. urealyticum (serotype I) was cloned and sequenced. Seven genes were found, with ureA, ureB, and ureC encoding the structural subunits and ureE, ureF, ureG, and a truncated ureI) gene encoding accessory proteins. Urease expression was not obtained when the plasmid containing these genes was incorporated into an opal suppressor strain of Escherichia coli, although this enzymatic activity was found in the same E. coli strain transformed with pC6b, a plasmid with previously cloned urease genes from the U. urealyticum T960 strain of biotype 2 (serotype 8). Although there are 12 TGA triplets encoding tryptophan within urease genes, the level of expression obtained was comparable to the levels reported for other bacterial genes expressed in E. coli. Nested deletion experiments allowed us to demonstrate that ureD is necessary for urease activity whereas another open reading frame located downstream is not. The promoter for ureA and possibly other urease genes was identified for both serotypes.

Catalase activity as a predictor of amniotic fluid culture results in preterm labor or premature rupture of membranes.

OBJECTIVE: To evaluate catalase activity as a rapid predictor of microbial invasion of amniotic fluid (AF). METHODS: The study population consisted of 74 patients before 36 weeks' gestation with preterm labor or premature rupture of membranes (PROM). Subjects were excluded if there was evidence of clinical chorioamnionitis or fetal distress at admission. Amniocentesis was done within 24 hours of admission, and the AF was cultured for aerobic and anaerobic bacteria and for Mycoplasma species. All AF samples were Gram stained, and slides were examined by microbiology technologists. Amniotic fluid catalase activity was measured immediately after amniocentesis using a commercially available kit. The sensitivity of the Gram stain and catalase activity were compared using McNemar exact test. RESULTS: Amniotic fluid cultures were positive in 12 of 37 (32%) patients presenting with preterm labor and in 21 of 37 (56%) patients with PROM. Catalase activity was significantly more sensitive than Gram stain in detecting positive AF cultures in cases of PROM (P < .001) and preterm labor (P < .04). CONCLUSION: Catalase activity is a simple, rapid test that is useful in identifying subclinical intra-amniotic infection in patients with preterm labor or PROM.

Hydrolysis of urea by Ureaplasma urealyticum generates a transmembrane potential with resultant ATP synthesis.

When urea is added to Ureaplasma urealyticum, it is hydrolysed internally by a cytosolic urease. Under our measuring conditions, and at an external pH of 6.0, urea hydrolysis caused an ammonia chemical potential equivalent to almost 80 mV and, simultaneously, an increase in proton electrochemical potential (delta p) of about 24 mV with resultant de novo ATP synthesis. Inhibition of the urease with the potent inhibitor flurofamide abolished both the chemical potential and the increase of delta p such that ATP synthesis was reduced to approximately 5% of normally obtained levels. Uncouplers of electrochemical gradients had little or no effect on these systems. The electrochemical parameters and ATP synthesis were measured similarly at three other external pH values. Any change in delta p was primarily via membrane potential (delta psi), and the level of de novo ATP synthesis was related to the increase in delta p generated upon addition of urea and more closely to the ammonia chemical potential. Although the organisms lack an effective mechanism for internal pH homeostasis, they maintained a constant delta pH. The data reported are consistent with, and give evidence for, the direct involvement of a chemiosmotic mechanism in the generation of around 95% of the ATP by this organism. Furthermore, the data suggest that the ATP-generating system is coupled to urea hydrolysis by the cytosolic urease via an ammonia chemical potential.

Characterization of the immunoglobulin A protease of Ureaplasma urealyticum.

Ureaplasma urealyticum strains of all serotypes express a specific human immunoglobulin A1 protease that cleaves immunoglobulin A1 to produce intact Fab and Fc fragments. The use of a variety of inhibitors suggests that the enzyme is a serine protease. N-terminal sequencing of the Fc digestion product showed that the enzyme cleaves between the proline and threonine residues 235 and 236 in the hinge region of the heavy chain of immunoglobulin A1.