Uthaibhorn Singkham-In

Due to the possible coexistence of Klebsiella pneumoniae (KP) and Candida albicans (CA), strains of KP and CA with biofilm production properties clinically isolated from patients were tested. The production of biofilms from the combined organisms (KP+CA) was higher than the biofilms from each organism alone, as indicated by crystal violet and z-stack immunofluorescence. In parallel, the bacterial abundance in KP + CA was similar to KP, but the fungal abundance was higher than CA (culture method), implying that CA grows better in the presence of KP. Proteomic analysis was performed to compare KP + CA biofilm to KP biofilm alone. With isolated mouse neutrophils (thioglycolate induction), KP + CA biofilms induced less prominent responses than KP biofilms, as determined by (i) neutrophilic supernatant cytokines (ELISA) and (ii) neutrophil extracellular traps (NETs), using immunofluorescent images (neutrophil elastase, myeloperoxidase, and citrullinated histone 3), peptidyl arginine deiminase 4 (PAD4) expression, and cell-free DNA. Likewise, intratracheal KP + CA in C57BL/6 mice induces less severe pneumonia than KP alone, as indicated by organ injury (serum creatinine and alanine transaminase) (colorimetric assays), cytokines (ELISA), bronchoalveolar lavage fluid parameters (bacterial culture and neutrophil abundances using a hemocytometer), histology score (H&E stains), and NETs (immunofluorescence on the lung tissue). In conclusion, the biofilm biomass of KP + CA was mostly produced from CA with less potent neutrophil activation and less severe pneumonia than KP alone. Hence, fungi in the respiratory tract might benefit the host in some situations, despite the well-known adverse effects of fungi.

Because Pseudomonas aeruginosa is frequently in contact with Chlorhexidine (a regular antiseptic), bacterial adaptations are possible. In comparison with the parent strain, the Chlorhexidine-adapted strain formed smaller colonies with metabolic downregulation (proteomic analysis) with the cross-resistance against colistin (an antibiotic for several antibiotic-resistant bacteria), partly through the modification of L-Ara4N in the lipopolysaccharide at the outer membrane. Chlorhexidine-adapted strain formed dense liquid–solid interface biofilms with enhanced cell aggregation partly due to the Chlorhexidine-induced overexpression of psl (exopolysaccharide-encoded gene) through the LadS/GacSA pathway (c-di-GMP-independence) in 12 h biofilms and maintained the aggregation with SiaD-mediated c-di-GMP dependence in 24 h biofilms as evaluated by polymerase chain reaction (PCR). The addition of Ca²⁺ in the Chlorhexidine-adapted strain facilitated several Psl-associated genes, indicating an impact of Ca²⁺ in Psl production. The activation by Chlorhexidine-treated sessile bacteria demonstrated a lower expression of IL-6 and IL-8 on fibroblasts and macrophages than the activation by the parent strain, indicating the less inflammatory reactions from Chlorhexidine-exposed bacteria. However, the 14-day severity of the wounds in mouse caused by Chlorhexidine-treated bacteria versus the parent strain was similar, as indicated by wound diameters and bacterial burdens. In conclusion, Chlorhexidine induced psl over-expression and colistin cross-resistance that might be clinically important.

Carbapenem-resistant Klebsiella pneumoniae has globally emerged as an urgent threat leading to the limitation for treatment. K. pneumoniae carrying bla_OXA-48, which plays a broad magnitude of carbapenem susceptibility, is widely concerned. This study aimed to characterize related carbapenem resistance mechanisms and forage for new antibiotic combinations to combat bla_OXA-48-carrying K. pneumoniae. Among nine isolates, there were two major clones and a singleton identified by ERIC-PCR. Most isolates were resistant to ertapenem (MIC range: 2–>256 mg/L), but two isolates were susceptible to imipenem and meropenem (MIC range: 0.5–1 mg/L). All bla_OXA-48-carrying plasmids conferred carbapenem resistance in Escherichia coli transformants. Two ertapenem-susceptible isolates carried both outer membrane proteins (OMPs), OmpK35 and OmpK36. Lack of at least an OMP was present in imipenem-resistant isolates. We evaluated the in vitro activity of an overlooked antibiotic, azithromycin, in combination with other antibiotics. Remarkably, azithromycin exhibited synergism with colistin and fosfomycin by 88.89% and 77.78%, respectively. Bacterial regrowth occurred after exposure to colistin or azithromycin alone. Interestingly, most isolates were killed, reaching synergism by this combination. In conclusion, the combination of azithromycin and colistin may be an alternative strategy in dealing with bla_OXA-48-carrying K. pneumoniae infection during a recent shortage of newly effective antibiotic development.