Evolution of mutations in the ftsI gene leading to amino acid substitutions in PBP3 in Haemophilus influenzae strains under the selective pressure of ampicillin and cefuroxime.

BACKGROUND: Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. One of the mechanisms of resistance to ß-lactams is the alteration of the transpeptidase region of penicillin binding protein 3 (PBP3) which is caused by mutations in the ftsI gene. It was shown that exposure to beta-lactams has a stimulating effect on increase of prevalence of H. influenzae strains with the non-enzymatic mechanism of resistance. OBJECTIVES: The aim of our study was to compare the mutational potential of ampicillin and cefuroxime in H. influenzae strains, determination of minimum inhibitory concentration and the evolution of mutations over time, focusing on amino acid substitutions in PBP3. METHODS: 30 days of serial passaging of strains in liquid broth containing increasing concentrations of ampicillin or cefuroxime was followed by whole-genome sequencing. RESULTS: On average, cefuroxime increased the minimum inhibitory concentration more than ampicillin. The minimum inhibitory concentration was increased by a maximum of 32 fold. Substitutions in the PBP3 started to appear after 15 days of passaging. In PBP3, cefuroxime caused different substitutions than ampicillin. CONCLUSIONS: Our experiment observed differences in mutation selection by ampicillin and cefuroxime. Selection pressure of antibiotics in vitro generated substitutions that do not occur in clinical strains in the Czech Republic.

The influence of biological relatedness on sexual dimorphism and sex classification based on external morphology of the frontal bone.

The most significant sexual differences in the human skull are located in the upper third of the face (the frontal bone), which is a useful research object, mainly in combination with virtual anthropology methods. However, the influence of biological relatedness on sexual dimorphism and frontal bone variability remains unknown. This study was directed at sexual difference description and sex classification using the form and shape of the external surface of the frontal bones from a genealogically documented Central European osteological sample (nineteenth to twentieth centuries). The study sample consisted of 47 cranial CT images of the adult members of several branches of one family group over 4 generations. Three-dimensional virtual models of the frontal bones were analyzed using geometric morphometrics and multidimensional statistics. Almost the entire external frontal surface was significantly different between males and females, especially in form. Significant differences were also found between this related sample and an unrelated one. Sex estimation of the biologically related individuals was performed using the classification models developed on a sample of unrelated individuals from the recent Czech population (Cechová et al. in Int J Legal Med 133: 1285 1294, 2019), with a result of 74.46% and 63.83% in form and shape, respectively. Failure of this classifier was caused by the existence of typical traits found in the biologically related sample different from the usual manifestation of sexual dimorphism. This can be explained as due to the increased degree of similarity and the reduction of variability in biologically related individuals. The results show the importance of testing previously published methods on genealogical data.

Biological relationships and frontal sinus similarity in skeletal remains with known genealogical data.

Frontal sinus analysis has potential utility for detecting biologically related individuals. However, the methodological approach to its evaluation, as well as its informative value, have been questioned. The aim of this work is to introduce a new approach to evaluating the frontal sinus using the 'external supraorbital line' (ESOL) and to determine whether there are sex differences within families in frontal sinus measurements and whether frontal sinus similarity reflects known genetic relationships in both measurements and morphology. We examined the skeletal remains of 41 adult individuals (25 males, 16 females), all members of one family over four generations (19th to 20th centuries), including individuals with very close consanguinity. CT images of skulls were acquired, and both the dimensions and morphology of the frontal sinuses were analyzed using their portions above the ESOL. No significant sex differences were found within families based on frontal sinus dimensions. Significant relationships were found between biological distance and the maximum height and morphology of the frontal sinuses. The greatest degree of similarity was found among closely related individuals. Additionally, in several cases, there was a greater degree of similarity between first cousins or grandparents and their grandchildren than among siblings or parents and their children. Total surface, volume and width are not significant indicators of relatedness. Known genetic relationships are also supported by individual morphological features. Variability within families with very close consanguineous relationships was lower than within families with common degrees of consanguinity, although differences are significant only for some variables.

Sex estimation using external morphology of the frontal bone and frontal sinuses in a contemporary Czech population.

Sex estimation is a task of utmost importance in forensic anthropology and bioarcheology. Along with the pelvic bone, the skull is the most important source of sexual dimorphism. On the human skull, the upper third of the face (i.e., the frontal bone) is one of the most significant sexually dimorphic structures useful in anthropological research, especially when studied by methods of virtual anthropology. This study was focused on sex estimation using the form and shape of the external surface of the frontal bone with or without the inclusion of its sinuses. The study sample consisted of 103 cranial CT images from a contemporary Czech population. Three-dimensional virtual models of the frontal bones and sinuses were analyzed using geometric morphometrics and multidimensional statistics: coherent point drift-dense correspondence analysis (CPD-DCA), principal component analysis (PCA), and support vector machine (SVM). The whole external frontal surface was significantly different between males and females both in form and shape. The greatest total success rate of sex estimation based on form was 93.2%, which decreased to 86.41% after crossvalidation, and this model identified females and males with the same accuracy. The best estimation based on shape reached a success rate of 91.26%, with slightly greater accuracy for females. After crossvalidation, however, the success rate decreased to 83.49%. The differences between sexes were significant also in the volume and surface of the frontal sinuses, but the sex estimation had only 64.07% accuracy after crossvalidation. Simultaneous use of the shape of the frontal surface and the frontal sinuses improved the total success rate to 98.05%, which decreased to 84.46% after crossvalidation.

A test of the Bulut et al. (2016) landmark-free method of quantifying sex differences in frontal bone roundness in a contemporary Czech sample.

The skull, along with the pelvic bone, serves an important source of clues as to the sex of human skeletal remains. The frontal bone is one of the most significant sexually dimorphic structures employed in anthropological research, especially when studied by methods of virtual anthropology. For this reason, many new methods have been developed, but their utility for other populations remains to be verified. In the present study, we tested one such approach-the landmark-free method of Bulut et al. (2016) for quantifying sexually dimorphic differences in the shape of the frontal bone, developed using a sample of the Turkish population. Our study builds upon this methodology and tests its utility for the Czech population. We evaluated the shape of the male and female frontal bone using 3D morphometrics, comparing virtual models of frontal bones and corresponding software-generated spheres. To do so, we calculated the relative size of the frontal bone area deviating from the fitted sphere by less than 1 mm and used these data to estimate the sex of individuals. Using our sample of the Czech population, the method estimated the sex correctly in 72.8% of individuals. This success rate is about 5% lower than that achieved with the Turkish sample. This method is therefore not very suitable for estimating the sex of Czech individuals, especially considering the significantly greater success rates of other approaches.