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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.

ABSTRACTAcinetobacter baumannii is a common cause of healthcare-associated infections and hospital outbreaks, particularly in intensive care units. Much of the success of A. baumannii relies on its genomic plasticity, which allows rapid adaptation to adversity and stress. The capacity to acquire novel antibiotic resistance determinants and the tolerance to stresses encountered in the hospital environment promote A. baumannii spread among patients and long-term contamination of the healthcare setting. This review explores virulence factors and physiological traits contributing to A. baumannii infection and adaptation to the hospital environment. Several cell-associated and secreted virulence factors involved in A. baumannii biofilm formation, cell adhesion, invasion, and persistence in the host, as well as resistance to xeric stress imposed by the healthcare settings, are illustrated to give reasons for the success of A. baumannii as a hospital pathogen.

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.

Abstract KLHL14 is a substrate-binding subunit of Cullin-RING ligase 3 ubiquitin ligase complex, highly enriched in thyroid since early embryonic development, together with its antisense RNA KLHL14-AS. We have previously demonstrated that Klhl14-AS is a competing endogenous RNA regulating several differentiation and survival factors in thyroid cancer, acting as tumor suppressor. Recently, also KLHL14 has been shown to function as tumor suppressor in diffuse large B-cell lymphoma and in malignant mesothelioma. Here we show that KLHL14 expression is strongly reduced in anaplastic thyroid cancer, the less differentiated and most aggressive type of thyroid neoplasia. Such reduction is reproduced in different in vivo and in vitro models of thyroid cancer, being invariably associated with loss of differentiation. When Klhl14 expression is rescued in thyroid transformed cells, it reduces the cell proliferation rate and increase the number of apoptotic cells. On the other side, Klhl14 loss of function in normal thyroid cells affects the expression of several regulatory as well as functional thyroid markers. All these findings suggest that KLHL14 could be considered as a novel tumor suppressor in thyroid cancer, by also revealing its physiological role in the maintenance of a fully differentiated and functional thyroid phenotype.

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.

Infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) remain a clinical challenge due to limited treatment options. Recently, cefiderocol, a novel siderophore cephalosporin, and sulbactam-durlobactam, a bactericidal β-lactam–β-lactamase inhibitor combination, have been approved by the Food and Drug Administration for the treatment of A. baumannii infections. In this review, we discuss the mechanisms of action of and resistance to cefiderocol and sulbactam-durlobactam, the antimicrobial susceptibility of A. baumannii isolates to these drugs, as well as the clinical effectiveness of cefiderocol and sulbactam/durlobactam-based regimens against CRAB. Overall, cefiderocol and sulbactam-durlobactam show an excellent antimicrobial activity against CRAB. The review of clinical studies evaluating the efficacy of cefiderocol therapy against CRAB indicates it is non-inferior to colistin/other treatments for CRAB infections, with a better safety profile. Combination treatment is not associated with improved outcomes compared to monotherapy. Higher mortality rates are often associated with prior patient comorbidities and the severity of the underlying infection. Regarding sulbactam-durlobactam, current data from the pivotal clinical trial and case reports suggest this antibiotic combination could be a valuable option in critically ill patients affected by CRAB infections, in particular where no other antibiotic appears to be effective.

The emergence of multidrug-resistance (MDR)—New Delhi metallo-beta-lactamase (NDM)-producing microorganisms—has become a serious concern for treating such infections. Therefore, we investigated the effective antimicrobial combinations against multidrug-resistant New Delhi metallo-beta-lactamase-producing strains of Enterobacterales. The tests were carried out using the 2D(two-dimensional) checkerboard method. Of 7 antimicrobials, i.e., doripenem (DRP), streptomycin (STR), cefoxitin (FOX), imipenem (IPM), cefotaxime (CTX), meropenem (MER), and gentamicin (GEN), 19 different combinations were used, and out of them, three combinations showed synergistic effects against 31 highly drug-resistant strains carrying blaNDM and other associated resistance markers. Changes in the minimum inhibitory concentration (MIC) values were interpreted using the test fractional inhibitory concentration index (FIC Index). The FIC Index values of these combinations were found in the range of 0.1562 to 0.5, which shows synergy, whereas no synergism was observed in the remaining antimicrobial combinations. We conclude that these antibiotic combinations can be analyzed in in vivo and pharmacological studies to establish an effective therapeutic approach.

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.

The spread of microorganisms causing health-care associated infection (HAI) is contributed to by their intrinsic tolerance to a variety of biocides, used as antiseptics or disinfectants. The natural monomeric stilbenoid resveratrol (RV) is able to modulate the susceptibility to the chlorhexidine digluconate (CHX) biocide in Acinetobacter baumannii. In this study, a panel of reference strains and clinical isolates of Gram-negative bacteria, Gram-positive bacteria and yeasts were analyzed for susceptibility to CHX and benzalkonium chloride (BZK) and found to be tolerant to one or both biocides. The carbonyl cyanide m-chlorophenylhydrazine protonophore (CCCP) efflux pump inhibitor reduced the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CHX and BZK in the majority of strains. RV reduced dose-dependently MIC and MBC of CHX and BZK biocides when used as single agents or in combination in all analyzed strains, but not CHX MIC and MBC in Pseudomonas aeruginosa, Candida albicans, Klebsiella pneumoniae, Stenotrophomonas maltophilia and Burkholderia spp. strains. In conclusion, RV reverts tolerance and restores susceptibility to CHX and BZK in the majority of microorganisms responsible for HAI. These results indicates that the combination of RV, CHX and BZK may represent a useful strategy to maintain susceptibility to biocides in several nosocomial pathogens.

Invasive Candida infections have become a global public health problem due to the increase of Candida species resistant against antifungal therapeutics. The glucocorticoid PYED-1 (pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1) has antimicrobial activity against various bacterial taxa. Consequently, it might be considered for the treatment of Candida infections. The antifungal activity of PYED-1 was evaluated against several fungal strains that were representative of the five species that causes the majority of Candida infections—namely, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei. PYED-1 exhibited a weak antifungal activity and a fungistatic effect on all five Candida species. On the other hand, PYED-1 exhibited a good anti-biofilm activity, and was able to eradicate the preformed biofilms of all Candida species analyzed. Moreover, PYED-1 inhibited germ tube and hyphae formation of C. albicans and reduced adhesion of C. albicans to abiotic surfaces by up to 30%.

Staphylococcus aureus is one of the major causes of hospital- and community-associated bacterial infections throughout the world, which are difficult to treat due to the rising number of drug-resistant strains. New molecules displaying potent activity against this bacterium are urgently needed. In this study, d- and l-deoxynojirimycin (DNJ) and a small library of their N-alkyl derivatives were screened against S. aureus ATCC 29213, with the aim to identify novel candidates with inhibitory potential. Among them, N-nonyloxypentyl-l-DNJ (l-NPDNJ) proved to be the most active compound against S. aureus ATCC 29213 and its clinical isolates, with the minimum inhibitory concentration (MIC) value of 128 μg/mL. l-NPDNJ also displayed an additive effect with gentamicin and oxacillin against the gentamicin- and methicillin-resistant S. aureus isolate 00717. Sub-MIC values of l-NPDNJ affected S. aureus biofilm development in a dose-dependent manner, inducing a strong reduction in biofilm biomass. Moreover, real-time reverse transcriptase PCR analysis revealed that l-NPDNJ effectively inhibited at sub-MIC values the transcription of the spa, hla, hlb and sea virulence genes, as well as the agrA and saeR response regulator genes.