**3. Discussion**

Until recently, it was generally believed that TB was caused by a pathogen with uniform characteristics, since its genome was considered to be highly conserved and without horizontal gene transfer [22]. However, studies have demonstrated that small mutations and changes at the regulatory level, often caused by selective environmental pressure and the interaction with the host organism, might induce significant changes in the mycobacterial phenotype. Experiments on animal and cell models and population studies have revealed the existence of hyper-, hypo- and mildly virulent MTBC strains and their different resistance to antibiotics [24,28–31]. Such differences in virulence and drug resistance profile of mycobacteria have a negative impact on the efficacy of treatment, making it difficult to achieve the goal of reducing TB incidence and mortality, especially from Beijing strains.

In Poland, patients with Beijing-TB are diagnosed each year [21,25,32]. By using spoligotyping for MTBC identification, it was possible to track changes in the molecular structure of mycobacteria isolated from patients in Poland. Spoligotyping enabled the identification of TB patients shedding lineage 2 mycobacterial strains of different phylogenetic subtypes. Until now, many Beijing-TB epidemics, caused by the ancient Beijing 1 genotype, have been reported around the world, and therefore, it was possible to better understand and characterize this pathogen. Its increased virulence, multidrug resistance, and the cause of treatment failure and TB relapse have been proven [15,18]. Today, the Beijing 1 genotype accounts for approx. 93% of all Beijing strains registered in the global database (source: http://www.pasteur-guadeloupe.fr:8081/SITVIT\_ONLINE/, accessed on 17 March 2022).

Still, little is known about tuberculosis caused by rare Beijing (Beijing-like) genotypes [33]. Findings reported to date sugges<sup>t</sup> that Beijing sublineages may evolve into highly pathogenic clones. However, there are no population studies or demographic, epidemiological or clinical data on patients with Beijing-like TB, and the only source providing selective information is SITVIT, an online *Mycobacterium tuberculosis* molecular markers database.

In most countries, modern Beijing strains are much more widespread than the ancient ones, which suggests their active spread. The exception is Japan, where the ancient W genotype is dominant [34]. Although the modern and ancient Beijing strains appear to be closely related at the genetic level, there are marked differences in their drug resistance profiles and their ability to spread and cause disease [23,35]. Moreover, the increased ability of Beijing strains to circumvent the immunity induced by the BCG vaccine has been proven, and studies by Kremer et al. have demonstrated that modern Beijing-like strains are isolated more often from BCG vaccinated patients than from unvaccinated individuals [17,18,36]. The observation that the interaction of modern Beijing strains with the host's immune system is different from that of the ancient genotypes was further confirmed by van Laarhoven et al., who described differences in the induction of pro-inflammatory cytokines for both Beijing sublineages [37].

In this paper, we presented two cases of patients infected with Beijing-like strains representing subtypes 541 and 265. At the time of this analysis, in the SITVIT2 online spoligotype database, there were 16 MTB strains with the Beijing 541 genotype and 81 isolates with the Beijing 265 genotype, which accounted for 0.1% and 0.7%, respectively, of all Beijing strains registered worldwide (n = 10,850). The occasional isolation of atypical Beijing mycobacteria from patients means that little is known about the effect of these pathogens on the course of treatment and prognosis. Only a few relevant reports have been published, including two from Colombia, describing cases of tuberculosis caused by the modern Beijing subtype 190 mycobacteria [38,39]. Another report described the case of a 15-year-old female patient infected with this subtype of multidrug-resistant MT who died after unsuccessful treatment [40]. The first patient described in our report was infected with Beijing type 541 mycobacteria, and despite the multidrug resistance of the strain, therapeutic success was achieved. The second patient, infected with the Beijing 265 strain, is still AFB-positive despite long-term antimycobacterial therapy, which is confirmed by

the results of microbiological tests carried out at the beginning of 2022 (MTBC culture, January 2022).

Our study has some limitations because although treatment failure is generally more common in patients with Beijing-type TB, there are a number of other factors that may additionally contribute to this, such as ethnicity, age, and sex [41,42]. It is not entirely possible to conclude whether treatment failure is associated with the genotype or with the drug resistance of mycobacteria, inappropriate adherence to the therapeutic regimen, or other factors that make the problem complex. Therefore, findings from our study must be interpreted with caution, as they do not clearly explain whether the high virulence and multidrug resistance of the Beijing 265 strain observed in our study is only episodic or is a feature closely related to this specific genotype.
