## 3 Discussion
Currently, many strains resistant to multiple antibiotics remain susceptible to fosfomycin (FOS), even in geographic regions where its use is widespread. This antibiotic has shown efficacy, both in vitro and in vivo, in treating UTIs caused by ESBL-producing _Enterobacteriaceae_ and vancomycin-resistant enterococci [69]. In this study we showed that none of the isolates, including the multidrug-resistant ones, were resistant to FOS (as well as to ceftibuten, meropenem, and cefoxitin). Similar results with low rates of resistance or complete sensitivity to FOS have been obtained in other studiesat the Charlotte Maxeke Johannesburg Academic Hospital, among others [70; 71; 72]. These findings suggest that fosfomycin is still an effective treatment for urinary tract infections, but should be used with caution, and studies are needed to monitor the resistance status of strains isolated from patients with urinary tract infections. It has been previously described that bacterial biofilm protects bacteria from antibiotics and is involved in many diseases [73]. Biofilms are communities of bacteria encased in a polymeric matrix that protects resident bacteria from both the antibiotics and the host immune effectors, among many other stressors and potential means of eradication. Our results confirm the correlation between biofilm-forming ability and antibiotic resistance phenotype; however this tested was conducted under non-biofilm-forming conditions. Although the percentage of resistant strains in the CRA+/ \(-\) groups occurred at a similar level, biofilm-forming isolates were more likely to show resistance to more than one antibiotic. Bacteria residing within a biofilm are canonically highly resistant to antibiotic-mediated killing and are overall antimicrobial resistant, with resistance often exhibited toward multiple classes of antibiotics. Such a result was obtained in a study by [PERSON] et al. [74]. The authors also found no correlation of biofilm production with any of the virulence factors studied. Our study coincides with these observations, with the exception of the _ibeA_ gene (this sequence was not studied in the [PERSON] et al. experiment). Furthermore, in another study conducted in Poland, no relationship between the rate of biofilm formation and the presence of adhesion factors or toxin genes was determined [47]. Only the aerobactin gene was significantly associated with strong biofilm production. However, it is worth mentioning that in this study, the assessment of biofilm formation capacity was investigated with a test other than CRA. Biofilm is formed by the aggregation of bacterial cells, surrounded by a matrix composed of eDNA, proteins, and exopolysaccharide. These polysaccharides facilitate adhesion to the cell surface and protection from environmental stress. This is accompanied by a high concentration of 3', 5'-cyclic diguanosine monophosphate [c-di-GMP] [75]. Degradation of c-di-GMP occurs due to cyclic phosphodiesterase (PDE) activity of the Yybt protein [76]. Interestingly, [PERSON] et al. found that the bacterial regulator ppGpp is a potent inhibitor of the DHH/DHHA1 domain found in YybT, suggesting that YybT is under tight control during the stringent response [77]. In 2017, [PERSON] et al. described that if c-di-GMP regulates the production of biofilm matrix polysaccharides, Congo red staining can be used as an indirect measurement of elevated c-di-GMP production in bacteria [78]. Hence, if elevated levels of (p)ppGpp contributes to increased levels of c-di-GMP, we concluded that strains with black colonies on CRA may produce increased levels of the alarmones. Our study confirmed the assumption that CRA+ strains have significantly higher basal levels of alarmones (Figure 4). Moreover, in addition to the effect of (p)ppGpp on biofilm formation, alarmone production has also been linked to the expression of virulence factors or antibiotic tolerance. There are reports suggesting that UPEC strains with high intrinsic levels of (p)ppGpp exhibit higher levels of pathogenicity against the host [79; 80].