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IntroductionThe persistence of Acinetobacter baumannii in the contaminated environment is sustained by tolerance to biocides and ability to growth as biofilm. The aim of the study was to analyze the susceptibility of A. baumannii biofilms to chlorhexidine (CHX) and benzalkonium (BZK) biocides and the ability of natural monomeric stilbenoid resveratrol (RV) to modulate the phenomenon.MethodsBiofilm formation and preformed biofilm were tested by Crystal violet and tetrazolium salt reduction assay, respectively. Analysis of efflux pump (EP) expression during biofilm growth was performed by Real-time RT-PCR assays.ResultsCHX and BZK at ¼ and ½ MICs alone or in combination inhibited biofilm growth of A. baumannii ATCC 19606, 4190, and 3909 strains. RV at 32 mg/L and CHX and BZK at ¼ or ½ MICs showed a synergistic effect and completely inhibited biofilm formation in all A. baumannii strains. Similarly, RV at 32 mg/L and CHX and BZK at ½ MIC significantly inhibited air-liquid biofilm formation of A. baumannii ATCC 19606, 4190 and 3909 strains. The inactivation of AdeB and AdeJ RND EPs in A. baumannii ATCC19606 increased the susceptibility to CHX and BZK alone or in the presence of 32 mg/L RV. Concordantly, carbonyl cyanide m-chlorophenylhydrazine (CCCP) increased the susceptibility to CHX, BZK and RV and dose-dependently inhibited biofilm formation in A. baumannii ATCC 19606, 4190 and 3909 strains. RV at 32 mg/L inhibited basal and CHX-induced EP genes expression, while increased EP gene expression in the presence of BZK during A. baumannii ATCC19606 biofilm growth. In addition, CHX and BZK alone or in combination dose-dependently reduced preformed biofilm of all A. baumannii strains. The combination of RV with CHX and BZK additively decreased minimal biofilm eradicating concentrations in A. baumannii strains.ConclusionThese results demonstrate that: (i) CHX and BZK alone or in the presence of RV inhibit biofilm growth and preformed biofilm in A. baumannii; (ii) tolerance to CHX and BZK during biofilm growth is dependent on the activation of AdeB and AdeJ EPs; and (iii) the inhibitory effect of RV on biofilm growth is mediated by the inhibition of EP genes expression in A. baumannii.

IntroductionAntimicrobial-resistant pathogens are an ongoing threat to human and animal health. According to the World Health Organization (WHO), colistin is considered the last resort antibiotic against human infections due to multidrug-resistant Gram-negative organisms—including Acinetobacter baumanni, a priority-1 pathogen. Despite colistin being considered a last resort antibiotic, transferable bacterial resistance to this drug has been reported in humans and animals. This makes addressing colistin resistance a critical priority in public health efforts. The large PetN transferase membrane protein PmrC is responsible for colistin resistance due to its catalysed modification of lipid A of the external membrane. Despite its importance, this potential drug target was never characterised at a molecular level.MethodsThe recombinant production of large membrane proteins in their native forms is a bottleneck in modern molecular biology. In this study, we recombinantly produced PmrC and biophysically characterised it in solution. We employed in silico approaches, including virtual screening and molecular modelling, to identify PmrC ligands. The binding of these ligands to PmrC was measured using Microscale Thermophoresis (MST). The best ligand was tested for its ability to hamper colistin resistance in Acinetobacter baumannii clinical isolates. Finally, we checked that the identified compound was not cytotoxic at the used concentrations by haemolysis assays.ResultsWe successfully produced PmrC PetN transferase membrane protein in high yields and showed that PmrC is a stable α-β protein, with melting temperature Tm = 60°C. Based on the PmrC structural model, we identified a promising druggable cavity. Therefore, we used a structure-based virtual screening to identify potential inhibitors. A small molecule, here denominated as s-Phen, was proved to bind PmrC with μM affinity. Microbiological assays confirmed that the s-Phen can drastically reduce colistin minimum inhibitory concentration (MIC) in two A. baumannii-resistant isolates and that it is not cytotoxic. Importantly, PmrC binding pocket to s-Phen is highly conserved in all homologues of PmrC, regardless of the location of genes encoding for them and of their operons.DiscussionOur study provides a molecular characterisation of PmrC and demonstrates the importance of PmrC as a drug target and the strong potential of PmrC binding molecules to act as colistin adjuvants, operating as synergistic tools to combat multiresistant nosocomial pathogens.

IntroductionNon-baumannii Acinetobacter species are increasingly isolated in the clinical setting and the environment. The aim of the present study was to analyze a genome database of 837 Acinetobacter spp. isolates, which included 798 non-baumannii Acinetobacter genomes, in order to define the concordance of classification and discriminatory power of 7-gene MLST, 53-gene MLST, and single-nucleotide polymorphism (SNPs) phylogenies.MethodsPhylogenies were performed on Pasteur Multilocus Sequence Typing (MLST) or ribosomal Multilocus Sequence Typing (rMLST) concatenated alleles, or SNPs extracted from core genome alignment.ResultsThe Pasteur MLST scheme was able to identify and genotype 72 species in the Acinetobacter genus, with classification results concordant with the ribosomal MLST scheme. The discriminatory power and genotyping reliability of the Pasteur MLST scheme were assessed in comparison to genome-wide SNP phylogeny on 535 non-baumannii Acinetobacter genomes assigned to Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter seifertii, and Acinetobacter lactucae (heterotypic synonym of Acinetobacter dijkshoorniae), which were the most clinically relevant non-baumannii species of the A. baumannii group. The Pasteur MLST and SNP phylogenies were congruent at Robinson-Fould and Matching cluster tests and grouped genomes into four and three clusters in A. pittii, respectively, and one each in A. seifertii. Furthermore, A. lactucae genomes were grouped into one cluster within A. pittii genomes. The SNP phylogeny of A. nosocomialis genomes showed a heterogeneous population and did not correspond to the Pasteur MLST phylogeny, which identified two recombinant clusters. The antimicrobial resistance genes belonging to at least three different antimicrobial classes were identified in 91 isolates assigned to 17 distinct species in the Acinetobacter genus. Moreover, the presence of a class D oxacillinase, which is a naturally occurring enzyme in several Acinetobacter species, was found in 503 isolates assigned to 35 Acinetobacter species.ConclusionIn conclusion, Pasteur MLST phylogeny of non-baumannii Acinetobacter isolates coupled with in silico detection of antimicrobial resistance makes it important to study the population structure and epidemiology of Acinetobacter spp. isolates.

The management of infections caused by Acinetobacter baumannii is hindered by its intrinsic tolerance to a wide variety of biocides. The aim of the study was to analyze the role of different A. baumannii efflux pumps (EPs) in tolerance to chlorhexidine (CHX) and benzalkonium (BZK) and identify non-toxic compounds, which can restore susceptibility to CHX and BZK in A. baumannii. A. baumannii ATCC 19606 strain was tolerant to both CHX and BZK with MIC and MBC value of 32 mg/L. CHX subMIC concentrations increased the expression of adeB and adeJ (RND superfamily), aceI (PACE family) and amvA (MFS superfamily) EP genes. The values of CHX MIC and MBC decreased by eightfold in ΔadeB and twofold in ΔamvA or ΔaceI mutants, respectively, while not affected in ΔadeJ mutant; EPs double and triple deletion mutants showed an additive effect on CHX MIC. CHX susceptibility was restored in double and triple deletion mutants with inactivation of adeB gene. BZK MIC was decreased by fourfold in ΔadeB mutant, and twofold in ΔamvA and ΔaceI mutants, respectively; EPs double and triple deletion mutants showed an additive effect on BZK MIC. BZK susceptibility was recovered in ΔadeB ΔaceI ΔadeJ and ΔamvA ΔadeB ΔadeJ triple mutants. The structural comparison of AdeB and AdeJ protomers showed a more negatively charged entrance binding site and F-loop in AdeB, which may favor the transport of CHX. The carbonyl cyanide m-chlorophenylhydrazine protonophore (CCCP) EP inhibitor reduced dose-dependently CHX MIC in A. baumannii ATCC 19606 and in ΔadeJ, ΔaceI, or ΔamvA mutants, but not in ΔadeB mutant. Either piperine (PIP) or resveratrol (RV) at non-toxic concentrations inhibited CHX MIC in A. baumannii ATCC 19606 parental strain and EPs gene deletion mutants, and CHX-induced EP gene expression. Also, RV inhibited BZK MIC and EP genes expression in A. baumannii ATCC 19606 parental strain and EPs mutants. These results demonstrate that tolerance to CHX and BZK in A. baumannii is mediated by the activation of AdeB, AceI and AmvA EPs, AdeB playing a major role. Importantly, inhibition of EP genes expression by RV restores CHX and BZK susceptibility in A. baumannii.