Studies show that reduced sphingosine levels in bronchiectasis patients' airways weaken bacterial clearance, making them more susceptible to infections by PA. Additionally, bactericidal/permeability-increasing protein (BPI) [9] can trigger autoimmune responses, further compromising the immune system. Low levels of the anti-inflammatory regulator PPARγ [10] and common mutations in the MucA gene [11], which promote a mucoid phenotype in the bacteria, are also linked to chronic PA infections. The patient's prolonged hospital stays and repeated use of antibiotics, inhaled steroids, and systemic intravenous steroids have led to PA infections. This mechanism may involve the aforementioned factors.
The composition of these stones includes calcium phosphate (85–90%) and calcium carbonate (10–15%) [6]. However, in this case, elemental analysis of the patient's broncholiths revealed the presence of C, N, O, P, S, and Zn, but no Ca. The complexity of broncholith formation mechanisms and individual variations may contribute to this anomaly. The patient had been on long-term intravenous steroids, used non-invasive ventilation, and had limited sun exposure, leading to severe osteoporosis, as confirmed by later bone density tests. These factors might contribute to the formation of broncholiths in a calcium-deficient environment. Bronchiectasis also damages the ciliary structure, which causes a lot of mucus and dead cells that are full of protein, lipids, and carbohydrate breakdown products to build up. This causes high levels of C, N, O, and S. This fits with what we saw when we looked at the elements in the broncholiths: chronic inflammatory stimulation could also cause the deposition of P and Zn.
It remains unclear whether microbial infections lead directly to broncholithiasis or whether the microorganisms colonize after stone formation. Patient infections with PA are not usually associated with broncholithiasis. However, this strain was associated with the ST270 genotype, which is a rare report worldwide. Further studies are needed to determine whether this genotype forms broncholithiasis. Mayuri A. Kamble [12] said that broncholithiasis occur before bronchiectasis because untreated bronchiectasis predisposes to calcium deposition. Conversely, it is also believed that bronchial cholelithiasis leads to complications such as bronchiectasis, bronchopleural fistula and lobar pneumonia [6]. Thus, the causal relationship between bronchial cholelithiasis and bronchiectasis remains unresolved. Because broncholithiasis are rarely reported, to verify these relationships, we need more cases and time.
This strain of PA was resistant to ceftazidime, meropenem, and levofloxacin and sensitive to polymyxin B. We found it resistant to cefotaxime, meropenem, and levofloxacin (Table S1). In our samples, regions of concentrated tRNA genes were in physical proximity to drug-resistant genes, suggesting the possibility of horizontal gene transfer (HGT). Integrative and transposable elements (e.g., plasmids, transposons, and integrons), which often carry drug resistance genes, target tRNA loci, making them hotspots for bacterial HGT. Furthermore, the presence of two drug-resistant pathways, namely secondary metabolite synthesis, transport, and degradation (Q) and intracellular transport, secretion, and vesicular transport (U), in the second and third circles mapped in this region of Fig. 5, further reinforces the region's significance in the transmission of drug-resistant genes.
We observed a thick biofilm (Fig. 2A) during the microbial cultivation process, classifying this strain as a high-mucoid PA. It produces a large amount of mucoid material, alginate, around the bacterial cell to form biofilms. This makes it difficult for antimicrobial agents to come into contact with the bacteria and also reduces the metabolic rate of bacteria within the biofilm, effectively resisting phagocytosis by phagocytic cells and the action of antimicrobial drugs, allowing the bacteria to continue to colonize. There is a biofilm formation pathway for PA on KEGG, which includes 90 genes [13]. Preliminary screening identified nonsense mutations in the PilI and ChpA genes, leading to the premature appearance of stop codons, which suppress protein expression. Type IV pili, which aggregate bacteria into microcolonies through twitching motility, heavily relies on PilI. The loss of this gene's encoded protein impacts colony formation, resulting in the formation of only small colonies on our plates. ChpA also influences the twitching motility of type IV pili. The deletion mutation of ChpA completely eliminates the wild-type twitching motility, resulting in the absence of motile cells at the edges of mutant colonies. This can also explain why this bacteria grows slowly, produces few colonies, and is difficult to culture.
Antibiotic resistance is associated with overexpression of AmpD and LysR, as well as mutations in the regulatory genes AmiD, DacC, and DacB, according to KEGG annotation. AmpD, as a type of β-lactamase, is overexpressed in response to signals produced from peptidoglycan degradation caused by antibiotic action. This increases the breakdown of β-lactam antibiotics, rendering them ineffective at killing or inhibiting bacterial growth, thereby leading to bacterial resistance to these drugs [14]. LysR binds to specific DNA sequences, increasing antibiotic resistance genes, regulating bacterial metabolic pathways and physiological states, helping PA adapt to adverse conditions [15]. DacC and DacB [16, 17] belong to the carboxypeptidase family, enzymes that act on the peptidoglycan layer within bacterial cell walls. The peptidoglycan layer is a crucial component for the integrity of bacteria and their cell walls. Mutations in the DacC and DacB gene impair their function, compromising the integrity of the bacterial cell wall, and preventing β-lactam antibiotics from effectively binding to their targets, thereby leading to resistance. The same principle applies to AmiD [18, 19].