Antimicrobial and Anti-Biofilm Activity of Polymyxin E Alone and in Combination with Probiotic Strains of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 against Clinical Isolates of Selected Acinetobacter spp.: A Preliminary Study

Acinetobacter spp., the nosocomial pathogen, forms strong biofilms and is resistant to numerous antibiotics, causing persistent infections. This study investigates the antibacterial and anti-biofilm activity of polymyxin E alone and in combination with the cell-free supernatants (CFS) of the tested probiotic bacilli, Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 against the selected Acinetobacter spp. starins. Three isolates of Acinetobacter spp., designated as Acinetobacter spp. isolate 1; Acinetobacter spp. isolate 2, and Acinetobacter spp. isolate 3, were collected from patients with burns, wounds, and blood infections, respectively. Bacterial identification and antibiotic susceptibility testing were conducted using the VITEK2 system. Auto-aggregation and coaggregation of the tested bacilli strains with the selected Acinetobacter spp. isolates were evaluated. A disk diffusion assay was used to identify the microorganism’s susceptibility to the selected antibiotics, alone and in combination with the CFS of the bacilli. The MIC and MBIC (minimum inhibitory and minimum biofilm inhibitory concentrations) of polymyxin E combined with bacilli CFS were determined. Acinetobacter spp. isolates were (i) sensitive to polymyxin E, (ii) able to form a strong biofilm, and (iii) resistant to the tested antibiotics and the CFS of tested bacilli. Significant inhibition of biofilm formation was noticed when CFS of the tested bacilli were combined with polymyxin E. The bacilli CFS showed synergy with polymyxin E against planktonic cells and biofilms of the isolated pathogens.

A Klebsiella pneumoniae DedA family membrane protein is required for colistin resistance and for virulence in wax moth larvae

Ineffectiveness of carbapenems against multidrug resistant pathogens led to the increased use of colistin (polymyxin E) as a last resort antibiotic. A gene belonging to the DedA family encoding conserved membrane proteins was previously identified by screening a transposon library of K. pneumoniae ST258 for sensitivity to colistin. We have renamed this gene dkcA (dedA of Klebsiella required for colistin resistance). DedA family proteins are likely membrane transporters required for viability of Escherichia coli and Burkholderia spp. at alkaline pH and for resistance to colistin in a number of bacterial species. Colistin resistance is often conferred via modification of the lipid A component of bacterial lipopolysaccharide with aminoarabinose (Ara4N) and/or phosphoethanolamine. Mass spectrometry analysis of lipid A of the ∆dkcA mutant shows a near absence of Ara4N in the lipid A, suggesting a requirement for DkcA for lipid A modification with Ara4N. Mutation of K. pneumoniae dkcA resulted in a reduction of the colistin minimal inhibitory concentration to approximately what is found with a ΔarnT strain. We also identify a requirement of DkcA for colistin resistance that is independent of lipid A modification, instead requiring maintenance of optimal membrane potential. K. pneumoniae ΔdkcA displays reduced virulence in Galleria mellonella suggesting colistin sensitivity can cause loss of virulence.

Transcriptomics reveals how minocycline-colistin synergy overcomes antibiotic resistance in multidrug-resistant Klebsiella pneumoniae

Multidrug resistant gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using RNA-Seq, we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline.

Molecular Detection of Drug-Resistance Genes of blaOXA-23-blaOXA-51 and mcr-1 in Clinical Isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa has caused high rates of mortality due to the appearance of strains with multidrug resistance (MDR) profiles. This study aimed to characterize the molecular profile of virulence and resistance genes in 99 isolates of P. aeruginosa recovered from different clinical specimens. The isolates were identified by the automated method Vitek2, and the antibiotic susceptibility profile was determined using different classes of antimicrobials. The genomic DNA was extracted and amplified by multiplex polymerase chain reaction (mPCR) to detect different virulence and antimicrobial resistance genes. Molecular typing was performed using the enterobacterial repetitive intergenic consensus (ERIC-PCR) technique to determine the clonal relationship among P. aeruginosa isolates. The drug susceptibility profiles of P. aeruginosa for all strains showed high levels of drug resistance, particularly, 27 (27.3%) isolates that exhibited extensively drug-resistant (XDR) profiles, and the other isolates showed MDR profiles. We detected the polymyxin E (mcr-1) gene in one strain that showed resistance against colistin. The genes that confer resistance to oxacillin (blaOXA-23 and blaOXA-51) were present in three isolates. One of these isolates carried both genes. As far as we know from the literature, this is the first report of the presence of blaOXA-23 and blaOXA-51 genes in P. aeruginosa.

Antibiotic resistance profile of Klebsiella pneumoniae strains isolated in an infectious diseases clinic

Objective. To establish the resistance profile of Klebsiella pneumoniae (KP) strains isolated in the Craiova Infectious Diseases Clinic. Material and method. Retrospective study (January 2017-December 2018); KPs were identified using the automated Vitek 2 system, which subsequently established their susceptibility to antimicrobials (usual testing for 17 antibiotics, extended testing for another 9); for each strain the multiple antibiotic resistance index (MAR) was calculated (limits: 0-1); the information was entered into an Excel database. 45 strains were tested in 2017 and 290 in 2018. The percentage of MDR strains was calculated based on internationally accepted definitions. Results. 335 strains were identified, the vast majority isolated by sputum culture (192 strains - 57.31%). Demographic data: 330 strains (99%) were isolated in adult subjects, 190 (57%) in male patients, 185 (55%) in patients living in urban areas. The overall value of MAR was 0.37 (compared to 0.32 for all strains of isolated Gram-negative germs). Over 80% of KP strains were susceptible to Amikacin or Polymyxin E; susceptibilities between 60 and 79% were observed for Meropenem, Gentamicin, Tobramycin, Ciprofloxacin, Ertapenem, Trimethoprim-Sulfamethoxazole, Levofloxacin, Cefepima, and between 40 and 59% for Ceftriaxone, Cefoxitin, Ceftazidime, Minocycline and Im. Less than 39% of strains are susceptible to Ampicillin (± Sulbactam), Piperacillin (± Tazobactam), Ticarcillin (± Clavulanic acid), Aztreonam, Pefloxacin, Cefazolin, Nitrofurantoin. Resistance to the main classes of antibiotics shows lower values than national and european data for 3rd generation cephalosporins, fluoroquinolones and aminoglycosides, but a much higher percentage of resistance to carbapenems. Over 68% of isolates are multidrug-resistant (MDR); over 60% of the strains come from sources where there is a significant contact with antimicrobials. Conclusions. Over 80% of the isolated strains are sensitive to Amikacin or Polymyxin E; a percentage of over 29% of the strains demonstrates resistance to the carbapenem class in 2018; over 68% of isolates are multidrug-resistant; over 60% of the strains come from sources where there is significant contact with antimicrobials.

Clinical Experience of Personalized Phage Therapy Against Carbapenem-Resistant Acinetobacter baumannii Lung Infection in a Patient With Chronic Obstructive Pulmonary Disease

Overuse of antibiotics in clinical medicine has contributed to the global spread of multidrug-resistant bacterial pathogens, including Acinetobacter baumannii. We present a case of an 88-year-old Chinese man who developed hospital-acquired pneumonia caused by carbapenem-resistant A. baumannii (CRAB). A personalized lytic pathogen-specific single-phage preparation was nebulized to the patient continuously for 16 days in combination with tigecycline and polymyxin E. The treatment was well tolerated and resulted in clearance of the pathogen and clinical improvement of the patient’s lung function.

A rare case of Colistin induced maculopapular rash

Approximately 2–3% of hospitalized patients are known to experience an adverse drug reaction (ADR). Dermatologic ADRs account for 10–30% of ADRs, and are commonly reported to be associated with antibiotic use. The classes of antibiotics most commonly reported to cause cutaneous reactions are the penicillins, cephalosporins, and fluoroquinolones. Polymyxin E is known to cause such reactions, but rarely. Here, we report a case of a colistin- induced maculopapular rash in an 84-year-old male. To the best of our knowledge, this is the first case of colistin-induced maculopapular rash to be reported in India.