MALDI-TOF MS Biomarker Detection Models to Distinguish RTX Toxin Phenotypes of Moraxella bovoculi Strains Are Enhanced Using Calcium Chloride Supplemented Agar

Moraxella bovoculi is the bacterium most often cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of M. bovoculi contain operons encoding for a repeats-in-toxin (RTX) toxin, which is a known virulence factor of multiple veterinary pathogens. We explored the utility of MALDI-TOF MS and biomarker detection models to classify the presence or absence of an RTX phenotype in M. bovoculi. Ninety strains that had undergone whole genome sequencing were classified by the presence or absence of complete RTX operons and confirmed with a visual assessment of hemolysis on blood agar. Strains were grown on Tryptic Soy Agar (TSA) with 5% sheep blood, TSA with 5% bovine blood that was supplemented with 10% fetal bovine serum, 10 mmol/LCaCl2, or both. The formulations were designed to determine the influence of growth media on toxin production or activity, as calcium ions are required for toxin secretion and activity. Mass spectra were obtained for strains grown on each agar formulation and biomarker models were developed using ClinProTools 3.0 software. The most accurate model was developed using spectra from strains grown on TSA with 5% bovine blood and supplemented with CaCl2, which had a sensitivity and specificity of 93.3% and 73.3%, respectively, regarding RTX phenotype classification. The same biomarker model algorithm developed from strains grown on TSA with 5% sheep blood had a substantially lower sensitivity and specificity of 68.0% and 52.0%, respectively. Our results indicate that MALDI-TOF MS biomarker models can accurately classify strains of M. bovoculi regarding the presence or absence of RTX toxin operons and that agar media modifications improve the accuracy of these models.

A Rodentibacter heylii strain lacking all known RTX toxin genes is highly virulent in C57BL/6 and BALB/c mice in contrast to Muribacter muris

Background Rodentibacter (R.) heylii and Muribacter (M.) muris are frequently detected in laboratory rodents. Repeats in toxin (RTX) toxins are considered to be important virulent factors in R. pneumotropicus and R. heylii. As many R. heylii isolates do not carry genes encoding known RTX toxins we hypothesized that these isolates are at the most moderately virulent or even avirulent as M. muris . To test this hypothesis, we evaluated the virulence of R. heylii and M. muris strains negative for all known RTX toxin genes in experimental infections of C57BL/6 and BALB/c mice. Results Experimental intranasal infection with 10 8 colony forming units (CFU) of a pnx I-, pnx II- and pnx III-negative R. heylii strain resulted in 75% and 100% mortality of C57BL/6 and BALB/c mice, respectively. Infections of multiple internal organs such as lung and genito-urinary tract were recorded. Purulent bronchopneumonia was a common finding in lungs of early losses. Application of 10 4 CFU of the same R. heylii strain was neither associated with clinical signs nor dissemination, but with efficient colonization of the upper respiratory tract. Intranasal application of M. muris in different doses ranging from 10 4 to 10 8 CFU did not result in mortality or severe weight loss but efficient colonization and induction of systemic M. muris specific IgG in most animals. Conclusion The current study reveals high virulence of R. heylii strain SF27GVG carrying none of the known RTX toxin genes in wildtype mice. This result questions the validity of estimating virulence in the genus Rodentibacter by profiling of pnx toxin genes. Suitable colonization models for future investigations were established for R. heylii and M. muris . Application of M. muris was associated with a systemic IgG immune response and cultural detection in draining lymph nodes in most animals indicating infection and not sole colonization.