Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks every 6 years, in older children and young adults. Diagnosis of M. pneumoniae is challenging because of the fastidious nature of the pathogen and the possibility of asymptomatic carriage. Laboratory diagnosis of M. pneumoniae infection based on antibody titration in the serum samples of patients remains the most practiced method. Because of the potential problem of immunological cross-reactivity with the use of polyclonal serum for M. pneumoniae, an antigen-capture enzyme-linked immunosorbent assay (ELISA) has been developed to improve the specificity of serological diagnosis. ELISA plates are coated with M. pneumoniae polyclonal antibodies, raised in rabbits and rendered specific after adsorption against a panel of heterologous bacteria that share antigens with M. pneumoniae species and/or are known to colonize the respiratory tract. The reacted M. pneumoniae homologous antigens are then specifically recognized by their corresponding antibodies in the serum samples. Further optimization of the physicochemical parameters to which the antigen-capture ELISA is subjected led to a highly specific, sensitive, and reproducible ELISA.

The Relationship between Mycoplasmas and Cancer: Is It Fact or Fiction ? Narrative Review and Update on the Situation.

More than one million new cancer cases occur worldwide every year. Although many clinical trials are applied and recent diagnostic tools are employed, curing cancer disease is still a great challenge for mankind. Heredity and epigenetics are the main risk factors often related to cancer. Although, the infectious etiological role in carcinogenesis was also theorized. By establishing chronic infection and inflammation in their hosts, several microorganisms were suggested to cause cell transformation. Of these suspicious microorganisms, mycoplasmas were well regarded because of their intimate parasitism with host cells, as well as their silent and insidious role during infections. This assumption has opened many questions about the real role played by mycoplasmas in oncogenesis. Herein, we presented a sum up of many studies among the hundreds which had addressed the Mycoplasma-cancer topic over the past 50 years. Research studies in this field have first started by approving the mycoplasmas malignancy potential. Indeed, using animal models and in vitro experiments in various cell lines from human and other mammalians, many mycoplasmas were proven to cause varied modifications leading to cell transformation. Moreover, many studies have looked upon the Mycoplasma-cancer subject from an epidemiological point of view. Diverse techniques were used to assess the mycoplasmas prevalence in patients with cancer from different countries. Not less than 10 Mycoplasma species were detected in the context of at least 15 cancer types affecting the brain, the breast, the lymphatic system, and different organs in the genitourinary, respiratory, gastrointestinal, and urinary tracts. Based on these revelations, one should concede that detection of mycoplasmas often linked to ''wolf in sheep's clothing" is not a coincidence and might have a role in cancer. Thorough investigations are needed to better elucidate this role. This would have a substantial impact on the improvement of cancer diagnosis and its prevention.

Clonal dissemination of antibiotic resistance among Tunisian Mycoplasma gallisepticum isolates as revealed by gene-targeted sequencing analysis.

SummaryTo date, very little is known about avian mycoplasma infections in Tunisia. Mycoplasma gallisepticum is one of the most economically significant pathogen for poultry in Tunisia and worldwide. Based on the paucity of data regarding the genetic profiles and antibacterial behavior of M. gallisepticum strains in Tunisia, the present study was conducted. Genetic typing and phylogenetic relationships of 40 M. gallisepticum strains (20 Tunisian isolates, 19 international strains collection, and S6 reference strain) were investigated by gene-targeted sequencing (GTS) using 4 loci ( pvpA , mgc2 , vlhA and the InterGenic Spacer Region (IGSR) between the 16S and the 23S rRNA genes). GTS reveals 12 STs that were found to spread over 2 clonal complexes (CC) and 5 singletons.Emergence of enrofloxacin and spiramycin resistance among M. gallisepticum local isolates have been revealed using the broth microdilution method. Causal mutations have been identified by sequencing the quinolone-resistance determining region (QRDR) and domain II and V of 23S rRNA as well as the rplD and rplV genes for enrofloxacine- and macrolide-resistant isolates, respectively. The emersion of antibiotic resistance to enrofloxacin and spiramycin has been identified as being related to a distinctive clonal complex formed by 4 different STs (ST2, ST3, ST4 and ST5) which would suggest that this phenotype was clonally disseminated.

Clonal Spread of Tetracycline Resistance Among Mycoplasma hominis Clinical Strains, Tunisia.

Antimicrobial resistance in a number of bacterial pathogens has been shown to spread clonally. To our knowledge, data about the phylodistribution of drug resistance in Mycoplasma hominis are very scarce. The aims of this study were to assess the antimicrobial susceptibility of Mycoplasma hominis clinical strains in Tunisia, to identify the molecular basis of antibiotic resistance, and to investigate the phylogenetic relationships of resistant strains. This study included 65 molecularly typed Mycoplasma hominis clinical strains recovered from Tunisian patients over 18 years (2000-2018). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Minimum spanning tree was constructed to establish the phylogenetic relationships among resistant isolates. Fluoroquinolones, doxycycline, and josamycine were found to be the most effective antibacterial agents. However, 22 strains belonging to 11 expanded multilocus sequence types (eSTs) proved resistant to tetracycline. The majority of these eSTs were genetically related, indicative of clonal expansion of tetracycline resistance. The present study provides relevant information on the antibiotic susceptibility of Tunisian M. hominis clinical strains, lending support to a clonal transmission of tetracycline resistance. This is likely to have an important implication in monitoring the spread of drug resistance among M. hominis.

Spread of multidrug resistance among Ureaplasma serovars, Tunisia.

Background: Ureaplasma spp. have been implicated in a variety of clinical conditions and certain serovars are likely to be disease-associated. Hence, the ascending trend of Ureaplasma spp. resistance to antimicrobials should deserve more attention. Here we assessed the extent of antimicrobial resistance of Ureaplasma serovars in Tunisia, and investigated the underlying molecular basis. Methods: This study included 101 molecularly typed Ureaplasma spp. clinical strains isolated over a 12-year time period (2005-2017). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Neighbor-joining tree was constructed to establish the phylogenetic relationships among isolates. Results: We found that all ureaplasma isolates were resistant to ciprofloxacin and erythromycin, intermediately resistant to azithromycin, and susceptible to doxycycline, moxifloxacin and josamycin. Ofloxacin and levofloxacin resistance was found in 73.27 and 17.82%, respectively, while 37.62% of isolates proved resistant to tetracycline. Consequently, we detected an elevated multidrug resistance rate among ureaplasma isolates (37.62%), particularly among serovars 2, 5, 8, and 9 (77.77% overall), as well as serovars 4, 10, 12, and 13 (52.63% overall). In most cases, drug resistance was found to be associated with known molecular mechanisms, yet we have identified two novel mutations in the L22 protein, which might be associated with macrolide-resistance. Conclusion: To our knowledge, this is the first study that reports the widespread expansion of multidrug resistance among Ureaplasma serovars, a finding of importance in terms of both surveillance and antimicrobial usage.

Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme.

To our knowledge, the phylodistribution of M. hominis clinical strains associated with various pathological conditions of the urogenital tract has not been explored hitherto. Here we analyzed the genetic diversity and phylogenetic relationships among 59 M. hominis Tunisian clinical isolates, categorized as gynecological infections- or infertility-associated pathotypes. For this purpose, we developed an expanded multilocus sequence typing (eMLST) scheme, combining the previously reported multilocus sequence typing (MLST) loci (gyrB, tuf, ftsY, uvrA, gap) with a new selected set of putative virulence genes (p120', vaa, lmp1, lmp3, p60), referred herein to as multi-virulence-locus sequence typing (MVLST) loci. In doing so, M. hominis population was segregated into two distinct genetic lineages, which were differentially associated with each pathotype. Such a clear dichotomy was supported by several phylogenetic and population genetic analysis tools. Recombination was found to take place, but not sufficient enough to break down the overall clonal population structure of M. hominis, most likely as a result of purifying selection, which accommodated the most fit clones. In sum, and owing to the eMLST scheme described herein, we provide insightful data on the phylogenetics of M. hominis, arguing for the existence of genetically differentiable urogenital pathotypes.

Genome Sequences of Two Tunisian Field Strains of Avian Mycoplasma, M. meleagridis and M. gallinarum.

Mycoplasma meleagridis and Mycoplasma gallinarum are bacteria that affect birds, but little is known about the genetic basis of their interaction with chickens and other poultry. Here, we sequenced the genomes of M. meleagridis strain MM_26B8_IPT and M. gallinarum strain Mgn_IPT, both isolated from chickens showing respiratory symptoms, poor growth, reduction in hatchability, and loss of production.

Mm19, a Mycoplasma meleagridis Major Surface Nuclease that Is Related to the RE_AlwI Superfamily of Endonucleases.

Mycoplasma meleagridis infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, M. meleagridis is characterized by its inability to synthesize purine and pyrimidine nucleotides de novo. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a M. meleagridis gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941-bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_AlwI superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact M. meleagridis cells, the E. coli-expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg2+ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with M. meleagridis intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that M. meleagridis expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_AlwI superfamily of endonucleases.

Genome Sequence of Mycoplasma meleagridis Type Strain 17529.

Mycoplasma meleagridis is a prominent turkey bacterial pathogen associated with airsacculitis and reproductive disorders. Notwithstanding the economic losses caused by M. meleagridis, its genome has still not been sequenced. For a better understanding of its genetic background and pathogenicity mechanisms, we sequenced the genome of M. meleagridis type strain ATCC 25294.