Latest Advances in Laboratory Detection of Mycoplasma genitalium.

Mycoplasma genitalium is an important sexually transmitted pathogen affecting both men and women. Its extremely slow growth in vitro and very demanding culture requirements necessitate the use of molecular-based diagnostic tests for its detection in clinical specimens. The recent availability of U.S. Food and Drug Administration (FDA)-cleared commercial molecular-based assays has enabled diagnostic testing to become more widely available in the United States and no longer limited to specialized reference laboratories. Advances in the knowledge of the epidemiology and clinical significance of M. genitalium as a human pathogen made possible by the availability of molecular-based testing have led to updated guidelines for diagnostic testing and treatment that have been published in various countries. This review summarizes the importance of M. genitalium as an agent of human disease, explains the necessity of obtaining a microbiological diagnosis, describes currently available diagnostic methods, and discusses how the emergence of antimicrobial resistance has complicated treatment alternatives and influenced the development of diagnostic tests for resistance detection, with an emphasis on developments over the past few years.

In Vitro Activities of Eravacycline and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas.

We performed in vitro susceptibility testing for eravacycline in comparison to 4 other antimicrobials against 10 Mycoplasma genitalium, 40 Mycoplasma hominis, 44 Mycoplasma pneumoniae, 20 Ureaplasma parvum, and 20 Ureaplasma urealyticum isolates. All eravacycline MICs were ≤0.25 µg/ml, except that for one isolate of M. genitalium, for which the MIC was 2 µg/ml. Eravacycline was markedly more potent than tetracycline, azithromycin, moxifloxacin, and clindamycin against all isolates tested, which included 37 macrolide, tetracycline, and/or fluoroquinolone-resistant organisms.

Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae.

We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, the BioFire FilmArray respiratory panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex respiratory pathogen panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB research use only (RUO) PCR, and the SpeeDx Resistance Plus MP assays. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed positives for M. pneumoniae The highest clinical sensitivities were found with the InGenius PCR (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6, 64.6, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (P < 0.05). Specificities of all assays were 99.5 to 100%. The Resistance Plus MP assay detected macrolide resistance in 27/33 specimens, resulting in a sensitivity of 81.8%. This study provides the first large-scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of various test performances on patient outcome.

Macrolide-resistant Mycoplasma pneumoniae pneumonia in transplantation: Increasingly typical?

Mycoplasma pneumoniae is one of the most common bacterial causes of pneumonia. Macrolide-resistant M pneumoniae (MRMP) was documented in 7.5% of isolates in the United States. Resistance portends poor outcomes to macrolide therapy, yet patients respond well to fluoroquinolones or tetracyclines such as minocycline. However, MRMP may be under-appreciated because M pneumoniae generally causes relatively mild infections in non-immunosuppressed adults that may resolve without effective therapy and because microbiological confirmation and susceptibility are not routinely performed. We report two cases of pneumonia due to MRMP in kidney transplant recipients. Both patients required hospital admission, worsened on macrolide therapy, and rapidly defervesced on doxycycline or levofloxacin. In one case, M pneumoniae was only identified by multiplex respiratory pathogen panel analysis of BAL fluid. Macrolide resistance was confirmed in both cases by real-time PCR and point mutations associated with macrolide resistance were identified. M pneumoniae was isolated from both cases, and molecular genotyping revealed the same genotype. In conclusion, clinicians should be aware of the potential for macrolide resistance in M pneumoniae, and may consider non-macrolide-based therapy for confirmed or non-responding infections in patients who are immunocompromised or hospitalized.

Allergic airway sensitization impairs antibacterial IgG antibody responses during bacterial respiratory tract infections.

BACKGROUND: Mycoplasma pneumoniae, an atypical human pathogen, has been associated with asthma initiation and exacerbation. Asthmatic patients have been reported to have higher carriage rates of M pneumoniae compared with nonasthmatic subjects and are at greater risk for invasive respiratory infections. OBJECTIVE: We sought to study whether prior allergen sensitization affects the host response to chronic bacterial infection. METHODS: BALB/cJ and IL-4 receptor α-/- mice were sensitized with ovalbumin (OVA) and then infected with M pneumoniae or Streptococcus pneumoniae. Immune parameters were analyzed at 30 days postinfection and included cellular profiles in bronchoalveolar lavage fluid (BALF) and serum IgG and IgE antibody levels to whole bacterial lysate, recombinant P1 adhesin, and OVA. Total lung RNA was examined for transcript levels, and BALF was examined for cytokine protein profiles. RESULTS: Anti-M pneumoniae antibody responses were decreased in allergen-sensitized, M pneumoniae-infected animals compared with control animals, but OVA-specific IgG responses were unaffected. Similar decreases in anti-S pneumoniae antibody levels were found in OVA-sensitized animals. However, M pneumoniae, but not S pneumoniae, infection augmented anti-OVA IgE antibody responses. Loss of IL-4 receptor signaling partially restored anti-M pneumoniae antibody responses in IgG2a and IgG2b subclasses. Inflammatory cytokine levels in BALF from OVA-sensitized, M pneumoniae-infected or S pneumoniae-infected animals were reduced compared with those in uninfected OVA-sensitized control animals. Unexpectedly, airway hyperreactivity to methacholine was essentially ablated in M pneumoniae-infected, OVA-sensitized animals. CONCLUSIONS: An established type 2-biased host immune response impairs the host immune response to respiratory bacterial infection in a largely pathogen-independent manner. Some pathogens, such as M pneumoniae, can augment ongoing allergic responses and inhibit pulmonary type 2 cytokine responses and allergic airway hyperreactivity.

Evaluation of the ELITe InGenius PCR Platform for Detection of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia in persons of all ages. Due to the fastidious nature of this bacterium and the necessary specialized growth media, nucleic acid amplification testing is currently the most reliable means for patient diagnostics. Analytical sensitivity, specificity, reproducibility, and clinical performance of the ELITe InGenius automated PCR platform with its MGB Alert M. pneumoniae real-time PCR research use only reagents (ELITechGroup, Inc., Bothell, WA) were compared with those of a laboratory-developed real-time PCR assay targeting repMp1 for detection of M. pneumoniae The ELITe InGenius PCR assay successfully detected 31 distinct M. pneumoniae clinical isolates and reference strains, and there was no cross-reactivity with other mollicutes, Gram-positive bacteria, or Gram-negative bacteria. In testing 223 clinical samples, the ELITe InGenius PCR showed 95.79% and 99.22% positive and negative agreement with the repMp1 assay, respectively. Additionally, the ELITech platform showed 98.91% positive and 96.95% negative predictive values, and there was no significant difference detected between the two assays (McNemar's test, P = 0.375). The ELITe InGenius PCR assay limit of detection was 0.16 CFU/PCR test or 4.16 genome copies (GCs)/test. Accuracy, instrument ease-of-use, and decreased hands-on time make the ELITe InGenius platform suitable for detection of M. pneumoniae directly from clinical specimens.

Mycoplasma hominis Infections Transmitted Through Amniotic Tissue Product.

Background: Mycoplasma hominis is a commensal genitourinary tract organism that can cause infections outside the genitourinary tract. We investigated a cluster of M. hominis surgical site infections in patients who underwent spine surgery, all associated with amniotic tissue linked to a common donor. Methods: Laboratory tests of tissue product from the donor, including culture, quantitative real-time polymerase chain reaction (qPCR), and whole-genome sequencing were performed. Use of this amniotic tissue product was reviewed. A multistate investigation to identify additional cases and locate any unused products was conducted. Results: Twenty-seven tissue product vials from a donor were distributed to facilities in 7 states; at least 20 vials from this donor were used in 14 patients. Of these, 4 of 14 (29%) developed surgical site infections, including 2 M. hominis infections. Mycoplasma hominis was detected by culture and qPCR in 2 unused vials from the donor. Sequencing indicated >99% similarity between patient and unopened vial isolates. For 5 of 27 (19%) vials, the final disposition could not be confirmed. Conclusions: Mycoplasma hominis was transmitted through amniotic tissue from a single donor to 2 recipients. Current routine donor screening and product testing does not detect all potential pathogens. Clinicians should be aware that M. hominis can cause surgical site infections, and may not be detected by routine clinical cultures. The lack of a standardized system to track tissue products in healthcare facilities limits the ability of public health agencies to respond to outbreaks and investigate other adverse events associated with these products.

In Vitro Activities of Gepotidacin (GSK2140944) and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas.

Gepotidacin, a novel first-in-class triazaacenaphthylene topoisomerase II inhibitor, was tested against 85 type strains and clinical isolates of Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum in comparison to levofloxacin, moxifloxacin, azithromycin or clindamycin, and tetracycline. Gepotidacin MIC90s (µg/ml) were 0.125 (M. pneumoniae), 0.032 (M. genitalium), 2 (M. hominis), and 8 (Ureaplasma species). Gepotidacin activity was not affected by resistance to fluoroquinolones, tetracyclines, or macrolides in the strains tested. Gepotidacin merits further study for treating infections caused by these organisms.

In Vitro Activities of Lefamulin and Other Antimicrobial Agents against Macrolide-Susceptible and Macrolide-Resistant Mycoplasma pneumoniae from the United States, Europe, and China.

Lefamulin, an investigational pleuromutilin, was tested against a collection of 18 macrolide-susceptible and 42 macrolide-resistant Mycoplasma pneumoniae strains, and the results were compared with those of azithromycin, erythromycin, tetracycline, doxycycline, and moxifloxacin testing. Lefamulin was highly active against all strains tested, with all MICs at ≤0.008 µg/ml. The lefamulin MIC90 (0.002 µg/ml) for macrolide-resistant strains was the lowest among all drugs tested. Minimum bactericidal concentrations were within 2 dilutions of the MIC values, indicating a bactericidal effect.

In Vitro Activities of Omadacycline (PTK 0796) and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas.

In vitro activities of omadacycline, a new aminomethylcycline, were determined for Mycoplasma and Ureaplasma spp. and compared with those of azithromycin, clindamycin, moxifloxacin, tetracycline, and doxycycline. All omadacycline MICs were <2 µg/ml. MIC90s were 0.063 µg/ml for Mycoplasma hominis, 0.25 µg/ml for Mycoplasma pneumoniae, and 2 µg/ml for Ureaplasma spp. Omadacycline had the lowest MIC90 among all drugs tested against M. hominis Omadacycline activity was not affected by macrolide, tetracycline, or fluoroquinolone resistance.

The clinical presentation of Fusobacterium-positive and streptococcal-positive pharyngitis in a university health clinic: a cross-sectional study.

BACKGROUND: Pharyngitis guidelines focus solely on group A ß-hemolytic streptococcal infection. European data suggest that in patients aged 15 to 30 years, Fusobacterium necrophorum causes at least 10% of cases of pharyngitis; however, few U.S. data exist. OBJECTIVE: To estimate the prevalence of F. necrophorum; Mycoplasma pneumoniae; and group A and C/G ß-hemolytic streptococcal pharyngitis and to determine whether F. necrophorum pharyngitis clinically resembles group A ß-hemolytic streptococcal pharyngitis. DESIGN: Cross-sectional. SETTING: University student health clinic. PATIENTS: 312 students aged 15 to 30 years presenting to a student health clinic with an acute sore throat and 180 asymptomatic students. MEASUREMENTS: Polymerase chain reaction testing from throat swabs to detect 4 species of bacteria and signs and symptoms used to calculate the Centor score. RESULTS: Fusobacterium necrophorum was detected in 20.5% of patients and 9.4% of asymptomatic students. Group A ß-hemolytic streptococcus was detected in 10.3% of patients and 1.1% of asymptomatic students. Group C/G ß-hemolytic streptococcus was detected in 9.0% of patients and 3.9% of asymptomatic students. Mycoplasma pneumoniae was detected in 1.9% of patients and 0 asymptomatic students. Infection rates with F. necrophorum, group A streptococcus, and group C/G streptococcus increased with higher Centor scores (P < 0.001). LIMITATIONS: The study focused on a limited age group and took place at a single institution. Asymptomatic students-rather than seasonal control participants-and a convenience sample were used. CONCLUSION: Fusobacterium necrophorum-positive pharyngitis occurs more frequently than group A ß-hemolytic streptococcal-positive pharyngitis in a student population, and F. necrophorum-positive pharyngitis clinically resembles streptococcal pharyngitis. PRIMARY FUNDING SOURCE: University of Alabama at Birmingham and the Justin E. Rodgers Foundation.

Comparative genome analysis of Mycoplasma pneumoniae.

BACKGROUND: Mycoplasma pneumoniae is a common pathogen that causes upper and lower respiratory tract infections in people of all ages, responsible for up to 40% of community-acquired pneumonias. It also causes a wide array of extrapulmonary infections and autoimmune phenomena. Phylogenetic studies of the organism have been generally restricted to specific genes or regions of the genome, because whole genome sequencing has been completed for only 4 strains. To better understand the physiology and pathogenicity of this important human pathogen, we performed comparative genomic analysis of 15 strains of M. pneumoniae that were isolated between the 1940s to 2009 from respiratory specimens and cerebrospinal fluid originating from the USA, China and England. RESULTS: Illumina MiSeq whole genome sequencing was performed on the 15 strains and all genome sequences were completed. Results from the comparative genomic analysis indicate that although about 1500 SNP and indel variants exist between type1 and type 2 strains, there is an overall high degree of sequence similarity among the strains (>99% identical to each other). Within the two subtypes, conservation of most genes, including the CARDS toxin gene and arginine deiminase genes, was observed. The major variation occurs in the P1 and ORF6 genes associated with the adhesin complex. Multiple hsdS genes (encodes S subunit of type I restriction enzyme) with variable tandem repeat copy numbers were found in all 15 genomes. CONCLUSIONS: These data indicate that despite conclusions drawn from 16S rRNA sequences suggesting rapid evolution, the M. pneumoniae genome is extraordinarily stable over time and geographic distance across the globe with a striking lack of evidence of horizontal gene transfer.

Macrolide-Resistant Mycoplasma pneumoniae, United States.

Macrolide-resistant Mycoplasma pneumoniae (MRMP) is highly prevalent in Asia and is now being reported from Europe. Few data on MRMP are available in the United States. Using genotypic and phenotypic methods, we detected high-level MRMP in 13.2% of 91 M. pneumoniae--positive specimens from 6 US locations.

In vitro antibacterial activity of AZD0914 against human Mycoplasmas and Ureaplasmas.

In this study, susceptibilities were determined for AZD0914, a spiropyrimidinetrione DNA gyrase inhibitor, azithromycin, doxycycline, and levofloxacin against Mycoplasma and Ureaplasma species. The activity of AZD0914 was comparable to that of levofloxacin and doxycycline against Mycoplasma genitalium and Mycoplasma pneumoniae. The AZD0914 MIC90 against Mycoplasma hominis was 8-fold greater than that for levofloxacin. The AZD0914 MIC90 against Ureaplasma species was 4-fold less than that for azithromycin and 8-fold less than that for levofloxacin and doxycycline.

Suppression of antimicrobial peptide expression by ureaplasma species.

Ureaplasma species commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome of Ureaplasma infections. THP-1 cells, a human monocytoid tumor line, were cocultured with Ureaplasma parvum and U. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR. In vitro antimicrobial activities of synthetic AMPs against Ureaplasma spp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation with U. parvum and U. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1, DEFA5, DEFA6, and CAMP), was significantly downregulated compared to that of TNFA and IL-8, which were upregulated. In vitro flow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity against Ureaplasma spp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation with U. parvum infection. No DNA methylation status changes were detected upon Ureaplasma infection. In conclusion, AMPs have in vitro activity against Ureaplasma spp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization.

Chromosomal mutations responsible for fluoroquinolone resistance in Ureaplasma species in the United States.

We sequenced the full lengths of the gyrA, gyrB, parC, and parE genes in 13 fluoroquinolone-resistant Ureaplasma isolates (levofloxacin MICs, 4 to 32 µg/ml) and 10 susceptible isolates (MICs ≤ 2 µg/ml). Mutations were detected in all resistant isolates but in none of the susceptible isolates. The most prevalent mutation was the S83L substitution in the ParC protein. No plasmid-mediated fluoroquinolone resistance genes were detected.

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains.

BACKGROUND: Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. RESULTS: We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.75-0.78 Mbp genomes and UUR serovars were 0.84-0.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. CONCLUSIONS: Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that ureaplasmas exist as quasi-species rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.

Antimicrobial susceptibilities and mechanisms of resistance of commensal and invasive Mycoplasma salivarium isolates.

Mycoplasma salivarium, an oral commensal organism, can cause severe invasive infections in immunocompromised individuals. Currently there is no treatment guidance for such infections. We performed antimicrobial susceptibility tests on 39 commensal and invasive M. salivarium isolates and investigated the mechanisms of antimicrobial resistance. Clindamycin was the most active agent [minimum inhibition concentration (MIC) range: 0.004-128 mg/L, MIC50 = 0.031 mg/L, MIC90 = 0.125 mg/ml], followed by tetracycline and levofloxacin. All isolates were resistant to erythromycin (MIC ≥4 mg/L) due to the presence of 2057A (Escherichia coli numbering) in 23S rRNA. Three isolates with elevated clindamycin MICs (≥8 mg/L) harbored A2058T/G mutations in 23S rRNA gene; four sequential isolates from one patient developed C2611T and A2059G mutations accompanying the increase of clindamycin MICs. Five isolates with elevated tetracycline MICs (≥4 mg/L) had mutations in 16S rRNA gene (A965G/T, G966T, or A967C/T) and one of them harbored TetM. Nine isolates with elevated levofloxacin MICs (≥4 mg/L) had one or more mutations in gyrA, gyrB, parC, or parE. Susceptibility breakpoints for clindamycin, tetracycline and levofloxacin were suggested to be ≤0.125, ≤2, and ≤2 mg/L, respectively. Antimicrobial resistance to any of the three agents (clindamycin, tetracycline, or levofloxacin) was documented in 12 (34.3%) non-duplicate isolates, of which 10 were invasive. Levofloxacin resistance was most frequent (25.7%). Multi-drug resistance was also observed (14.3%). This study demonstrates the frequent occurrence of antimicrobial resistance in M. salivarium, emphasizing the need for culture and susceptibility testing to guide antimicrobial therapy.

Omadacycline Is Highly Active In Vitro against Mycoplasma genitalium.

Here, we performed in vitro susceptibility testing on 10 Mycoplasma genitalium isolates against omadacycline, minocycline, tetracycline, doxycycline, moxifloxacin, levofloxacin, and azithromycin. Omadacycline was the most potent agent, with all MICs of ≤0.5 µg/mL. MICs were not affected by resistance to other agents, including resistance to other tetracycline class drugs. Omadacycline may be a potential treatment option for M. genitalium infection. IMPORTANCE There are very few clinical isolates of Mycoplasma genitalium available for in vitro susceptibility testing. We studied 10 isolates and determined that the new semisynthetic aminomethylcycline omadacycline is active against isolates that are resistant to tetracyclines, macrolides, and quinolones. These data suggest that clinical studies should be performed in order to see if omadacycline may be useful to treat urogenital infections caused by M. genitalium.