Multilocus Variable-Number Tandem-Repeat Analysis-Confirmed Emergence of a Macrolide Resistance-Associated Mutation in Mycoplasma pneumoniae during Macrolide Therapy for Interstitial Pneumonia in an Immunocompromised Child

A child with Job's syndrome was treated for pneumonia due to Mycoplasma pneumoniae. A mixed population of wild-type bacteria and an A2059G mutant was detected during josamycin treatment failure. The same multilocus variable-number tandem-repeat analysis (MLVA) type (MLVA type I) was isolated before and after treatment failure. The child recovered after ciprofloxacin treatment.     

Macrolide and fluoroquinolone resistance in Mycoplasma genitalium in two Swedish counties, 2011–2015

Mycoplasma genitalium, causing non‐gonococcal non‐chlamydial urethritis and associated with cervicitis, has developed antimicrobial resistance (AMR) to both the macrolide azithromycin (first‐line treatment) and the fluoroquinolone moxifloxacin (second‐line treatment). Our aim was to estimate the prevalence of resistance, based on genetic AMR determinants, to these antimicrobials in the M. genitalium population in two Swedish counties, Örebro and Halland, 2011–2015. In total, 672 M. genitalium positive urogenital samples were sequenced for 23S rRNA and parC gene mutations associated with macrolide and fluoroquinolone resistance, respectively. Of the samples, 18.6% and 3.2% in Örebro and 15.2% and 2.7% in Halland contained mutations associated with macrolide and fluoroquinolone resistance, respectively. The predominating resistance‐associated mutations in the 23S rRNA gene was A2059G (n = 39) in Örebro and A2058G (n = 13) and A2059G (n = 13) in Halland. The most prevalent possible resistance‐associated ParC amino acid alterations were S83I (n = 4) in Örebro and S83N (n = 2) in Halland. Resistance‐associated mutations to both macrolides and fluoroquinolones were found in 0.7% of samples. Our findings emphasize the need for routine AMR testing, at a minimum for macrolide resistance, of all M. genitalium‐positive samples and regular national and international surveillance of AMR in M. genitalium, to ensure effective patient management and rational antimicrobial use.     

Mycoplasma genitalium Prevalence,Coinfection, and Macrolide Antibiotic Resistance Frequency in a Multicenter Clinical Study Cohort in the United States

The prevalence rates of Mycoplasma genitalium infections and coinfections with other sexually transmitted organisms and the frequency of a macrolide antibiotic resistance phenotype were determined in urogenital specimens collected from female and male subjects enrolled in a multicenter clinical study in the United States. Specimens from 946 subjects seeking care from seven geographically diverse clinical sites were tested for M. genitalium and for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. Sequencing was used to assess macrolide antibiotic resistance among M. genitalium-positive subjects. M. genitalium prevalence rates were 16.1% for females and 17.2% for males. Significant risk factors for M. genitalium infections were black race, younger age, non-Hispanic ethnicity, and female symptomatic status. Female M. genitalium infections were significantly more prevalent than C. trachomatis and N. gonorrhoeae infections, while the M. genitalium infection rate in males was significantly higher than the N. gonorrhoeae and T. vaginalis infection rates. The macrolide-resistant phenotype was found in 50.8% of females and 42% of males. These results show a high prevalence of M. genitalium single infections, a lower prevalence of coinfections with other sexually transmitted organisms, and high rates of macrolide antibiotic resistance in a diverse sample of subjects seeking care across a wide geographic area of the United States.     

Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate.

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.     

Absence of Mycoplasma pneumoniae cytadsorption protein P1 in Mycoplasma genitalium and Mycoplasma gallisepticum

Polyclonal and monoclonal antibodies to Mycoplasma pneumoniae protein P1 were nonreactive with whole-cell or soluble preparations of M. genitalium and M. gallisepticum. However, radioimmunoprecipitation performed with hyperimmune rabbit sera raised against each mycoplasma species indicated antigenic cross-reactivity between M. pneumoniae and M. genitalium.     

Mycoplasma genitalium attaches to human spermatozoa

BACKGROUND: Mycoplasma genitalium causes urogenital diseases in men and women and is presumed to be sexually transmitted. We wanted to investigate whether spermatozoa could serve as vectors for M.genitalium in order to cause upper genital diseases in women. METHODS: By use of Nomarski light microscopy and transmission X-ray microscopy, the attachment of M.genitalium to spermatozoa was studied. Semen was incubated in vitro with M.genitalium. Purified, motile spermatozoa were examined for attachment of M.genitalium by immunofluorescence microscopy. RESULTS: Mycoplasma genitalium was shown to adhere to the head, midpiece and tail of the spermatozoa. The spermatozoa became immotile when many M.genitalium were attached. However, the motile spermatozoa were demonstrated to carry M.genitalium and in this case the mycoplasmas were seen to attach mostly to the midpiece or neck region. Occasionally, M.genitalium was seen at the head but not at the tail. By X-ray microscopy, it was possible to observe the diffentiated structure of M.genitalium, and the attachment seemed to be mediated by the tip. CONCLUSIONS: Mycoplasma genitalium can bind to human spermatozoa and thus could be carried by motile sperm. This ability may be important in the process of causing female genital diseases and infertility.     

The mature MgPa-adhesin of Mycoplasma genitalium

A high molecular weight protein of Mycoplasma genitalium (MgPa-protein) was isolated by fractionated solubilization with 1% CHAPS, followed by subsequent extraction with 2% octylglucoside and size exclusion chromatography. The comparison of the N-terminal sequence reported here with published nucleotide sequence data revealed the existence of a signal sequence; the molecular weight of the mature MgPa-protein was calculated to be 153, 134 dalton. The protein shares antigenic determinants with the adhesin of Mycoplasma pneumoniae (P1-protein). Therefore the amino acid sequence of the MgPa-protein was matched to the P1-protein sequence. Five of seven computer predicted hydrophobic regions of both amino acid sequences were located in corresponding regions.     

Serological investigation of Mycoplasma genitalium in infertile women

BACKGROUND: The role of Mycoplasma genitalium in the pathogenesis of pelvic inflammatory disease has not been characterized. METHODS: Sera from 308 infertile women were investigated for antibodies to M. genitalium by immunoblotting. Women with tubal factor infertility (TFI) made up 132 of the patients, 67 of the women had an infertile male partner and 109 were infertile for unknown reasons. RESULTS: Of the TFI patients 29 (22.0%) were seropositive to the major adhesin, MgPa, of M. genitalium versus 11 (6.3%) in the group of women with normal tubes. No cross-reactions between MgPa and P1 of the related Mycoplasma pneumoniae were found. Besides, MgPa positive sera were confirmed by immunoblotting using a cloned fragment of the C-terminal part of MgPa specific to M. genitalium. Chlamydia trachomatis is known to be able to cause infertility as a result of salpingitis. Therefore, the sera were tested against C. trachomatis using a commercial ELISA test. Seventy-five (56.8%) of the TFI patients were seropositive to C. trachomatis. Eight (27.6%) TFI patients seropositive to MgPa were negative to C. trachomatis. CONCLUSIONS: This study indicates that M. genitalium may be an independent risk factor in the development of an inflammatory process leading to scarring of the uterine tubes in women and thereby causing infertility.     

Direct Detection of Macrolide Resistance in Mycoplasma genitalium Isolates from Clinical Specimens from France by Use of Real-Time PCR and Melting Curve Analysis

Mycoplasma genitalium is a sexually transmitted organism commonly treated with azithromycin. However, macrolide resistance has been reported and is associated with point mutations in the 23S rRNA gene. To evaluate the prevalence of macrolide resistance in M. genitalium isolates from clinical specimens from France, we first used a previously reported high-resolution melting assay. Because susceptible and resistant M. genitalium isolates were hardly discriminated in M. genitalium-positive clinical specimens, we developed a new molecular assay for the rapid detection of macrolide resistance. An assay using real-time PCR based on fluorescence resonance energy transfer (FRET) coupled with melting curve analysis was designed. The assay was first validated on characterized macrolide-resistant M. genitalium isolates and then applied to 202 urogenital M. genitalium-positive specimens collected from 178 patients from France in 2011 and 2012. Resistant genotypes were confirmed by 23S rRNA gene sequencing. Among the 202 M. genitalium-positive specimens, 155 were amplified, demonstrating a sensitivity of 76.7%. A substitution in the 23S rRNA gene was found in 14.2% of the patient samples. Nine and six patients had M. genitalium isolates with a substitution at positions 2059 and 2058, respectively. In four cases, a mixed population of wild-type and mutated M. genitalium isolates was observed. The prevalence of M. genitalium macrolide resistance has been stable in France since its detection in 2006. Our FRET PCR assay is able to discriminate between wild-type and resistant genotypes directly from clinical specimens. This assay will allow clinicians to shorten the time to the initiation of effective disease treatment.     

Diagnostic assessment of Mycoplasma genitalium in culture-positive women

Detection of Mycoplasma genitalium-mediated, chlamydia-negative nongonococcal urethritis and other M. genitalium-linked infectious etiologies has been very challenging. Although M. genitalium is considered a leading cause of genitourinary symptoms in men and women, extreme difficulties in its cultivation due to its highly fastidious nature and the lack of routine and effective diagnostic tests have slowed the generation of clinical data which directly implicate the presence of M. genitalium in disease pathogenesis. In this study, we compared enzyme-linked immunosorbent assays (ELISAs) and immunoblot and PCR assays in M. genitalium culture-positive women over 1 to 3 years of clinical visits to determine the usefulness of independent diagnostic strategies. Furthermore, the value of combinatorial diagnostic assessments is described, which provides insights into the dynamics of M. genitalium-host interactions. Overall, we show that neither ELISA nor PCR, alone or in combination, provides the sensitivity required to confidently predict the existence of viable M. genitalium organisms in cervical and vaginal samples. Additionally, culture-positive women exhibited a range of antibody responsiveness to M. genitalium based upon ELISA and immunoblot assessments, indicating immune diversity among this high-risk population.     

Homologous regions shared by adhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium

A lambda gt11 library of Mycoplasma genitalium genomic DNA was generated, and clones were identified using a pool of monoclonal antibodies directed against different epitopes of the 140 kDa adhesin protein. Because the 140 kDa protein of M. genitalium and the 170 kDa P1 adhesin of M. pneumoniae share biological properties such as a tip-associated location, cytadherence function and immunologic crossreactivity, we performed Southern blot analysis using these cloned partial 140 kDa gene fragments and 14 subclones that span the P1 structural gene of M. pneumoniae. Homologous regions of the two genes were identified.     

DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium.

DNA probes specific for Mycoplasma pneumoniae and Mycoplasma genitalium were selected from genomic libraries prepared in pUC13. The 32P-labeled probes could detect, by dot blot hybridization, down to about 0.1 ng of the specific mycoplasma DNA or 10(5) CFU. Biotinylation of probe decreased the sensitivity of detection and produced nonspecific background reactions with nonhomologous DNAs. Sulfonation of probe yielded a similar level of sensitivity with less background.     

A Mycoplasma genitalium protein resembling the Mycoplasma pneumoniae attachment protein.

In previous studies with hyperimmune rabbit sera and monoclonal antibodies against the P1 protein of Mycoplasma pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of Mycoplasma genitalium. Because of biologic and morphologic similarities between these two human Mycoplasma species, attempts were made to characterize this cross-reacting protein of M. genitalium (designated MgPa). The protein was surface exposed and had an estimated molecular size of 140 kilodaltons. Electron microscopy with monoclonal antibodies produced against either MgPa or P1 demonstrated that MgPa is located over the surface of the terminal structure of M. genitalium which is covered by a nap layer. These immunologic and morphologic findings suggest that the MgPa protein of M. genitalium could be the counterpart of the P1 protein of M. pneumoniae.     

Cross-hybridization between the cytadhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium and genomic DNA of Mycoplasma gallisepticum

Immunological cross-reactivity was observed between the cytadhesin proteins of Mycoplasma pneumoniae and Mycoplasma genitalium and a 155 kDa protein of Mycoplasma gallisepticum. Furthermore, the cytadhesin genes of M. pneumoniae and M. genitalium were used to demonstrate homology with M. gallisepticum genomic DNA under low stringency conditions suggesting that a family of adhesin-related genes exists among these pathogenic mycoplasmas.     

Mycoplasma genitalium: from Chrysalis to multicolored butterfly

The history, replication, genetics, characteristics (both biological and physical), and factors involved in the pathogenesis of Mycoplasma genitalium are presented. The latter factors include adhesion, the influence of hormones, motility, possible toxin production, and immunological responses. The preferred site of colonization, together with current detection procedures, mainly by PCR technology, is discussed. The relationships between M. genitalium and various diseases are highlighted. These diseases include acute and chronic nongonococcal urethritis, balanoposthitis, chronic prostatitis, and acute epididymitis in men and urethritis, bacterial vaginosis, vaginitis, cervicitis, pelvic inflammatory disease, and reproductive disease in women. A causative relationship, or otherwise strong association, between several of these diseases and M. genitalium is apparent, and the extent of this, on a subjective basis, is presented; also provided is a comparison between M. genitalium and two other genital tract-orientated mollicutes, namely, Mycoplasma hominis, the first mycoplasma of human origin to be discovered, and Ureaplasma species. Also discussed is the relationship between M. genitalium and infertility and also arthritis in both men and women, as is infection in homosexual and immunodeficient patients. Decreased immunity, as in HIV infections, may enhance mycoplasmal detection and increase disease severity. Finally, aspects of the antimicrobial susceptibility and resistance of M. genitalium, together with the treatment and possible prevention of mycoplasmal disease, are discussed.     

Sequence-based typing of Mycoplasma genitalium reveals sexual transmission

Mycoplasma genitalium causes male nonchlamydial, nongonococcal urethritis and is associated with cervicitis and pelvic inflammatory disease in women. Epidemiological studies indicate that M. genitalium is sexually transmitted, and the aim of the present study was to further substantiate this by means of a DNA typing system. A typing assay based on a diagnostic mgpB gene PCR was developed, evaluated, and applied directly to urogenital specimens. The assay had a low limit of detection and hence a high typeability. Sequences of isolates from 52 unrelated patients were divided into 29 different sequence types, giving a discriminatory index of 0.95. Two to six M. genitalium-positive specimens were collected from each of 44 patients over a median interval of 56 days (range, 11 to 1,395). Forty had the same sequence type in consecutive specimens. Specimens collected from two men were repeatedly positive at intervals of 472 and 1,395 days, respectively, but the sequence types had changed. A new strain was introduced in one sexual dyad, and the sequence types changed subsequently. Seventy-nine M. genitalium-positive specimens from 19 couples were investigated, and all partners initially had concordant sequence types, but one couple had discordant types at one time point before a newly introduced strain took over. The present typing system is simple and reproducible and has an excellent discriminatory capacity which might prove useful in studies of sexual networks and for evaluation of treatment failures. In the laboratory, this system may document the uniqueness of newly isolated M. genitalium strains.     

Cervicitis and genitourinary symptoms in women culture positive for Mycoplasma genitalium

PROBLEM: Mycoplasma genitalium has been associated with male urethritis. We sought to relate M. genitalium to genitourinary signs and symptoms in women. METHOD OF STUDY: We compared 26 culture-positive women (group 1), 257 additional polymerase chain reaction-positive women (group 2), and 107 negative control women. We used logistic regression to evaluate signs and symptoms, controlling for co-infections, pregnancy, age, and intervention group assignment. RESULTS: Comparing group 1 with controls, we found significantly elevated odds ratios (ORs) for intermediate vaginal discharge (OR = 5.4; 95% confidence interval 1.01, 29.2) and action in response to discharge [3.9 (1.1, 13.5)]. Non-significant increases were observed for pathologic vaginal discharge [3.8 (0.78, 18.2)], pathologic dyspareunia [1.5 (0.25, 9.0)], vaginal odor [2.1 (0.75, 5.7)], and cervical mucopus [4.1 (0.74, 22.4)]. Group 2 results were similar, but showed no increase in cervical mucopus relative to controls. CONCLUSION: Infection with M. genitalium in women is independently related to increased genitourinary symptomatology.     

Amino Acid Changes in Elongation Factor Tu of Mycoplasma pneumoniae and Mycoplasma genitalium Influence Fibronectin Binding

Mycoplasma pneumoniae and Mycoplasma genitalium are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized M. pneumoniae elongation factor Tu (EF-Tu_Mp) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated M. genitalium EF-Tu (EF-Tu_Mg), in spite of sharing 96% identity with EF-Tu_Mp, does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu_Mp that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu_Mg. Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu_Mp. Synthetic peptides corresponding to this region of EF-Tu_Mp (EF-Tu_Mp 340-358) blocked both recombinant EF-Tu_Mp and radiolabeled M. pneumoniae cell binding to Fn. In contrast, EF-Tu_Mg 340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.Many pathogens express surface proteins that facilitate colonization and cellular invasion (12, 39, 44, 49, 55). The human mycoplasmas, Mycoplasma pneumoniae and Mycoplasma genitalium, have genome sizes of 816,394 bp (20) and 580,070 bp (12), respectively, with the latter considered the smallest self-replicating biological cell (14, 38). These bacterial pathogens possess terminal tip-like structures comprised of specific membrane adhesins and adherence-related accessory proteins that mediate surface parasitism of target cells (5) and are essential for virulence (4). While adherence of virulent M. pneumoniae is mediated primarily by tip organelle-associated adhesins (10, 24), the absence of these proteins in hemadsorption-negative mutants (HA⁻ class II mutants) (17) still permits detectable adherence (18), suggesting the involvement of alternative mechanisms by which mycoplasmas bind to host cells.Recently, we showed that M. pneumoniae surface-associated elongation factor Tu (EF-Tu_Mp; MPN665) and the pyruvate dehydrogenase E1 beta subunit (MPN392) interact with fibronectin (Fn) (11). In addition, we demonstrated that HA⁻ class II mutants also bind Fn through EF-Tu (11). Fn is an abundantly available pathogen target (22) that exists in soluble form in blood fluids and plasma and in fibrillar form in the extracellular matrix (56). M. pneumoniae could readily access the extracellular matrix through virulence-related determinants following epithelial cell damage (29) and could directly bind to subepithelial tissue targets through EF-Tu interactions with Fn. Furthermore, these distinct pathogenic pathways may also contribute to the ability of M. pneumoniae to invade and to establish intracellular and perinuclear residence (9, 57).Detailed analyses of EF-Tu_Mp-Fn interactions revealed the critical role of the carboxyl region of EF-Tu (amino acids 192 to 219 and 314 to 394) in Fn recognition (3). Other mycoplasmas with tip organelles, such as Mycoplasma penetrans and Mycoplasma gallisepticum, have been reported to bind Fn through a 65-kDa protein (13) and the PlpA and Hlp3 proteins (34).Following our initial findings of EF-Tu_Mp-Fn interactions, surface-associated EF-Tu proteins from other microorganisms, including Lactobacillus johnsonii, Listeria monocytogenes, and Pseudomonas aeruginosa, were reported to bind mucin (16), fibrinogen (43), plasminogen, and factor H (32). Since EF-Tu is one of the most highly conserved proteins in mycoplasmas, it has been used to create an EF-Tu sequence-based mycoplasma phylogeny tree. This allows the classification of the human pathogens, M. genitalium and M. pneumoniae, along with M. gallisepticum, a poultry pathogen, in the same group (28). M. pneumoniae is an established pathogen of the respiratory tract (54) but has also been isolated from the urogenital tract (15). M. genitalium, an emerging sexually transmitted disease pathogen (27, 51), has also been associated with respiratory (6) and joint (50) pathologies. It has been suggested that the tissue-specific tropisms and pathogenic mechanisms of these two mycoplasmas are determined by genetic distinctions between them (19). Most of the open reading frames proposed for M. genitalium are present in M. pneumoniae. Overall, M. pneumoniae and M. genitalium share 67.4% average identity at the amino acid level, while conserved housekeeping proteins exhibit 70 to 97% identity (19). Among the latter proteins, EF-Tu displays a high sequence identity (96%).In this study, we compared EF-Tu-Fn binding between M. pneumoniae and M. genitalium and discovered biological and biochemical differences that facilitated the identification of key amino acids responsible for these interactions. Such distinctions provide evidence of unique colonization capabilities of these bacteria.