Evaluating the presence of Mycoplasma hyorhinis, Fusobacterium nucleatum, and Helicobacter pylori in biopsies of patients with gastric cancer

Background Gastric cancer is the third leading cause of cancer-related deaths worldwide and has been associated with infections that may promote tumour progression. Accordingly, we analysed the presence of Mollicutes, Mycoplasma hyorhinis, Fusobacterium nucleatum and Helicobacter pylori in gastric cancer tissues and evaluated their correlation with clinicopathological factors. Methods Using a commercial kit, DNA were extracted from 120 gastric samples embedded in paraffin: 80 from patients with gastric cancer and 40 from cancer free patients, dating from 2006 to 2016. Mollicutes and H. pylori were detected by PCR; F. nucleatum and M. hyorhinis were detected by qPCR, together with immunohistochemistry for the latter bacteria. Results Mollicutes were detected in the case and control groups (12% and 2.5%) and correlated with the papillary histologic pattern (P = 0.003), likely due to cell transformation promoted by Mollicutes. M. hyorhinis was detected in the case and control group but was not considered a cancer risk factor. H. pylori was detected at higher loads in the case compared to the control group (8% and 22%, P = 0.008) and correlated with metastasis (P = 0.024), lymphatic invasion (P = 0.033), tumour of diffused type (P = 0.028), and histopathological grading G1/G2 (P = 0.008). F. nucleatum was the most abundant bacteria in the case group, but was also detected in the control group (26% and 2.5%). It increased the cancer risk factor (P = 0.045, OR = 10.562, CI95% = 1.057–105.521), and correlated with old age (P = 0.030) and tumour size (P = 0.053). Bacterial abundance was significantly different between groups (P = 0.001). Conclusion Our findings could improve the control and promote our understanding of opportunistic bacteria and their relevance to malignant phenotypes.

Development of a molecular biological assay for the detection of markers related to decreased susceptibility to macrolides and lincomycin in Mycoplasma hyorhinis

The control of Mycoplasma hyorhinis infection relies mainly on antimicrobial therapy. However, the antibiotic susceptibility testing of the bacteria is usually not performed before applying the treatment, and thus therapeutic failures are not uncommon. In the case of M. hyorhinis, several antibiotic-resistance-related single nucleotide polymorphisms (SNPs) are known but assays for their detection have not been described yet. The aims of the present study were to investigate macrolide- and lincomycin-resistance-related SNPs in Hungarian M. hyorhinis isolates and to develop mismatch amplification mutation assays (MAMA) to detect the identified resistance markers. Minimal inhibitory concentrations (MIC) of different drugs and whole genome sequences of 37 M. hyorhinis isolates were used to find the resistance-related mutations. One MAMA assay was designed to detect the mutation of the 23S rRNA gene at nucleotide position 2058 (Escherichia coli numbering). For further evaluation, the assay was challenged with 17 additional isolates with available MIC data and 15 DNA samples from clinical specimens. The genotypes of the samples were in line with the MIC test results. The developed assay supports the practice of targeted antibiotic usage; hence it may indirectly reduce some bacterial resistance-related public health concerns.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights as an adhesin in Mycoplasma hyorhinis adhesion to epithelial cells as well as a plasminogen receptor mediating extracellular matrix degradation

Mycoplasma hyorhinis infects pigs causing polyserositis and polyarthritis, and has also been reported in a variety of human tumor tissues. The occurrence of disease is often linked with the systemic invasion of the pathogen. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), one of the key enzymes of glycolysis, was reported as a surface multifunctional molecule in several bacteria. Here, we investigated whether GAPDH could manifest binary functions; as an adhesin to promote colonization as well as a plasminogen receptor functioning in extracellular matrix (ECM) degradation to promote systemic invasion. The surface localization of GAPDH was observed in M. hyorhinis with flow cytometry and colony blot analysis. Recombinant GAPDH (rGAPDH) was found to be able to bind porcine-derived PK-15 and human-derived NCI-H292 cells. The incubation with anti-GAPDH antibody significantly decreased the adherence of M. hyorhinis to both cell lines. To investigate its function in recruiting plasminogen, firstly, the interaction between rGAPDH and plasminogen was demonstrated by ELISA and Far-Western blot assay. The activation of the rGAPDH-bound plasminogen into plasmin was proved by using a chromogenic substrate, and furtherly confirmed to degrade extracellular matrix by using a reconstituted ECM. Finally, the ability of rGAPDH to bind different ECM components was demonstrated, including fibronectin, laminin, collagen type IV and vitronectin. Collectively, our data imply GAPDH as an important adhesion factor of M. hyrohinis and a receptor for hijacking host plasminogen to degrade ECM. The multifunction of GAPDH to bind both plasminogen and ECM components is believed to increase the targeting of proteolysis and facilitate the dissemination of M. hyorhinis.

Altered Nasal Microbiota Composition Associated with Development of Polyserositis by Mycoplasma hyorhinis

Fibrinous polyserositis in swine farming is a common pathological finding in nursery animals. The differential diagnosis of this finding should include Glaesserella parasuis (aetiological agent of Glässer’s disease) and Mycoplasma hyorhinis, among others. These microorganisms are early colonizers of the upper respiratory tract of piglets. The composition of the nasal microbiota at weaning was shown to constitute a predisposing factor for the development of Glässer’s disease. Here, we unravel the role of the nasal microbiota in the subsequent systemic infection by M. hyorhinis, and the similarities and differences with Glässer’s disease. Nasal samples from farms with recurrent problems with polyserositis associated with M. hyorhinis (MH) or Glässer’s disease (GD) were included in this study, together with healthy control farms (HC). Nasal swabs were taken from piglets in MH farms at weaning, before the onset of the clinical outbreaks, and were submitted to 16S rRNA gene amplicon sequencing (V3–V4 region). These sequences were analyzed together with sequences from similar samples previously obtained in GD and HC farms. Animals from farms with disease (MH and GD) had a nasal microbiota with lower diversity than those from the HC farms. However, the composition of the nasal microbiota of the piglets from these disease farms was different, suggesting that divergent microbiota imbalances may predispose the animals to the two systemic infections. We also found variants of the pathogens that were associated with the farms with the corresponding disease, highlighting the importance of studying the microbiome at strain-level resolution.