The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA–PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

Novel antibiotic combinations may act synergistically to inhibit the growth of multidrug-resistant bacterial pathogens but predicting which combination will be successful is difficult, and standard antimicrobial susceptibility testing may not identify important physiological differences between planktonic free-swimming and biofilm-protected surface-attached sessile cells. Using a nominally macrolide-resistant model Klebsiella pneumoniae strain (ATCC 10031) we demonstrate the effectiveness of several macrolides in inhibiting biofilm growth in multi-well plates, and the ability of azithromycin (AZM) to improve the effectiveness of the antibacterial last-agent-of-choice for K. pneumoniae infections, colistin methanesulfonate (CMS), against biofilms. This synergistic action was also seen in biofilm tests of several K. pneumoniae hospital isolates and could also be identified in polymyxin B disc-diffusion assays on azithromycin plates. Our work highlights the complexity of antimicrobial-resistance in bacterial pathogens and the need to test antibiotics with biofilm models where potential synergies might provide new therapeutic opportunities not seen in liquid culture or colony-based assays.

A therapeutic combination of azithromycin (AZM) and colistin methanesulfonate (CMS) was shown to be effective against both non-PDR and PDR Klebsiella pneumoniae biofilms in vitro. These anti-biofilm effects, however, may not correlate with effects observed in standard plate assays, nor will they representative of in vivo therapeutic action. After all, biofilm-associated infection processes are also impacted by the presence of wound bed components, such as host cells or wound fluids, which can all affect the antibiotic effectiveness. Therefore, an in vitro wound model of biofilm infection which partially mimics the complex microenvironment of infected wounds was developed to investigate the therapeutic potential of an AZM-CMS combination against XDR K. pneumoniae isolates. The model consists of a 3D collagen sponge-like scaffold seeded with HEK293 cells submerged in a fluid milieu mimicking the wound bed exudate. Media that were tested were all based on different strengths of Dulbecco’s modified Eagles/high glucose medium supplemented with fetal bovine serum, and/or Bacto Proteose peptone. Use of this model confirmed AZM to be a highly effective antibiofilm component, when applied alone or in combination with CMS, whereas CMS alone had little antibacterial effectiveness or even stimulated biofilm development. The wound model proposed here proves therefore, to be an effective aid in the study of drug combinations under realistic conditions.

The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here we investigated how different biocides affected the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides including alcohols, hydrogen peroxide, quaternary ammonium compounds, and polymeric guanidines were evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA-PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after four weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain which demonstrates the potential of PHMG-Cl as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

Summary. Objective: to improve the results of surgical treatment of destructive forms of acute cholecystitis, taking into account the sensitivity of the isolated microflora to antibiotics. Materials and methods. 347 patients with acute cholecystitis (AC) have been operated in the surgical department of Kiev Regional Clinical Hospital in 2016 – 2019. The control group included 275 AC patients operated on in 2013–2015, similar in sex, age, comorbidities, treatment and surgical tactics to each other, but with an empirical choice of antibiotic therapy. Bacteriological studies were performed to 237 patients with destructive forms of acute cholecystitis, where 531 microflora isolates were seeded. Results and discussion. The AC treatment group patients (n=347) were arranged according to Tokyo guidelines (2013) into 3 grades. Mild course (grade I, n=38, 11 %), microbiological studies were not conducted. Antibiotic prophylaxis with beta-lactam antibiotics was used. At moderately severe AC (grade II, n = 260, 75 %) escalation antibiotic therapy was carried out. At severe disease (grade III, n = 49, 14 %) de-escalation antibiotic therapy was performed. Most often intestinal group microflora was sown. The highest sensitivity of microflora was marked for linezolid - 100 %. Also high sensitivity of microflora was revealed to beta-lactam antibiotics - 83.3 % (penicillins, cephalosporins, monobactams, and carbapenems). The highest resistance was marked for unprotected penicillins: penicillin — 67.7 %, ampicillin — 83.3 %, amoxicillin — 100 %, oxacillin — 100 %; among cephalosporins: cefixime — 75.0 %, cefuroxime — 66.7 %; among fluoroquinolones: lomefloxacin and pefloxa-cin — 100 % resistance to seeded aerobic microflora. In the control group (n=275) purulent complications were noted in 9.8 % (27 patients), fatal cases — 6, the total mortality rate was 2.18 %, postoperative — 1.45 % (4 patients). In the study group (n = 347), the number of purulent complications was reduced to 4 % (p˂0.01), the total mortality rate was reduced to 0.58 % (p˂0.05). Conclusions: 1. Acute obstructive cholecystitis, acute emphysematous (gas) cholecystitis and AC complicated by choledocholithiasis or cholangitis are referred to the local abdominal infectious process. 2. Infection is not a leading factor on first stage for emphysematous, vascular and post-traumatic forms of AC, but it develops in a destructive gall bladder on second stage. 3. For the patients of the first grade by Tokio guideline 2013 we recommend the use of antibiotic prophylaxis. Under moderate grade — escalation antibiotic therapy and under severe grade of the AC — de-escalation antibiotic therapy, followed by the use of selective antibacterial agents should be administered. 4. The highest selective sensitivity of seeded microflora in patients with destructive forms of AC was marked for oxazolidinones (linezolid) — (100 %), as well as for beta-lactam antibiotics (83.3 %). 5. The control group patients had decreased number of suppurations (from 9.8 to 4 %; p<0.01), and decreased mortality rate (from 2.18 to 0.58 %; p<0.05).