Antimicrobial peptides often show broad-spectrum activity due to a mechanism based on bacterial membrane disruption, which also reduces development of permanent resistance, a desirable characteristic ...in view of the escalating multidrug resistance problem. Host cell toxicity however requires design of artificial variants of natural AMPs to increase selectivity and reduce side effects. Kiadins were designed using rules obtained from natural peptides active against E. coli and a validated computational algorithm based on a training set of such peptides, followed by rational conformational alterations. In vitro activity, tested against ESKAPE strains (ATCC and clinical isolates), revealed a varied activity spectrum and cytotoxicity that only in part correlated with conformational flexibility. Peptides with a higher proportion of Gly were generally less potent and caused less bacterial membrane alteration, as observed by flow cytometry and AFM, which correlate to structural characteristics as observed by circular dichroism spectroscopy and predicted by molecular dynamics calculations.
Bacterium Acinetobacter baumannii is a leading cause of hospital infections. Over the last decade, its occurrence in natural environments outside hospital settings has been reported. The aim was to ...examine the survival of A. baumannii in water media exposed to different ranges of oxygen supply in order to predict its behaviour in the environment. The abundance of five A. baumannii isolates was monitored in nutrient-depleted and nutrient-rich water media in aerated, intermediate and anaerobic conditions (oxygen saturation 96, 56 and 0%, respectively). A. baumannii survived in both media in all tested oxygen concentrations for 50 days. In nutrient-rich water survival of A. baumannii was lowest in anaerobic conditions, while in nutrient-depleted water there was no difference in survival regardless of oxygen availability. A. baumannii formed translucent small colony variants as the fast response (after 1 day) and dormant cells as the prolonged response (after 14 days) to anaerobic conditions. Transmission electron microscopy (TEM) images showed the outer membrane of coccobacillus dormant cells was up to four times thicker than in regular cells. Once in the environment, A. baumannii is able to survive regardless of the availability of dissolved oxygen, which represents a serious public health concern.
Acinetobacter baumannii is an emerging hospital pathogen. Whereas A. baumannii isolated from patients or hospitals has been reported, there are few data regarding propagation of viable A. baumannii ...in the natural environment. This study investigates the occurrence and antimicrobial susceptibility of viable A. baumannii in municipal wastewater and its persistence through the wastewater treatment process. A total of 21 A. baumannii isolates were recovered at a secondary type of municipal wastewater treatment plant in Zagreb, Croatia: 15 from raw influent wastewater and six from final effluent. All isolates were carbapenem- and multidrug-resistant. Among 14 isolates tested for blaOXA genes, all harboured the constitutive blaOXA-51-like gene, while the acquired blaOXA-23-like and blaOXA-40-like genes were found in 10 and three isolates respectively. Six A. baumannii isolates recovered from effluent wastewater multiplied and survived in sterilised effluent wastewater up to 50 days. These findings support the idea that multidrug-resistant A. baumannii can occur and have the ability to survive in the environment.
Vancomycin-resistant Enterococcus faecium (VREfm) is an opportunistic pathogen among the highest global priorities regarding public and environmental health. Following One Health approach, we ...determined for the first time the antibiotic resistance and virulence genes, and sequence types (STs) affiliation of VREfm recovered simultaneously from marine beach waters, submarine outfall of a wastewater treatment plant and an offshore discharge of untreated sewage, and compared them with the surveillance VREfm from regional university hospital in Croatia to assess the hazard of their transmission and routes of introduction into the natural environment. Importantly, VREfm recovered from wastewater, coastal bathing waters and hospital shared similar virulence, multidrug resistance, and ST profiles, posing a major public health threat. All isolates carried the vanA gene, while one clinical isolate also possessed the vanC2/C3 gene. The hospital strains largely carried the aminoglycoside-resistance genes aac(6′)-Ie-aph(2″)-Ia, and aph(2″)-Ib and aph(2″)-Id, which were also predominant in the environmental isolates. The hyl gene was the most prevalent virulence gene. The isolates belonged to 10 STs of the clonal complex CC17, a major epidemic lineage associated with hospital infections and outbreaks, with ST117 and ST889 common to waterborne and hospital isolates, pointing to their sewage-driven dissemination.
To gain better insight into the diversity of accompanying taxons in the surveyed water matrices, microbiome taxonomic profiling was carried out using Illumina-based 16S rDNA sequencing and their resistome features predicted using the PICRUSt2 bioinformatics tool. An additional 60 pathogenic bacterial genera were identified, among which Arcobacter, Acinetobacter, Escherichia-Shigella, Bacteroides and Pseudomonas were the most abundant and associated with a plethora of antibiotic resistance genes and modules, providing further evidence of the hazardous effects of wastewater discharges, including the treated ones, on the natural aquatic environment that should be adequately addressed from a sanitary and technological perspective.
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•Hospital and environmental vancomycin-resistant Enterococcus faecium were studied.•VREfm enter marine environment by outfalls of treated and untreated wastewater.•VanA genotype predominates among hospital and waterborne isolates in Croatia.•Hospital and environmental VREfm carry similar virulence and resistance genes.•Microbiome analysis revealed accompanying pathogenic taxa and resistance genes.
According to the World Health Organization, bacterium
Acinetobacter baumannii
is the first on the critical priority list of pathogens in urgent need for new antibiotics. The increasing resistance of
...A. baumannii
to the last-line treatment options, including carbapenems, is a global problem. We report the molecular epidemiology of 12 carbapenem-resistant clinical isolates of
A. baumannii
collected from hospitalised patients in three neighbouring countries in Southeast Europe: Croatia, Serbia, and Bosnia and Herzegovina, giving an insight into the molecular characterisation and evolutionary history of the acquisition of resistance genes. Besides the
bla
OXA-23 gene, the endemic presence of OXA-72 oxacillinase of the same origin for more than a decade as the leading mechanism of carbapenem resistance in Southeast Europe was confirmed. To the best of our knowledge, this is the first paper that investigates and analyses the phylogenetic association of the most common mechanisms of resistance to carbapenems in clinical isolates of
A. baumannii
originating from three neighbouring countries in Southeast Europe.
The rapid and ongoing spread of carbapenemase-producing
has led to a global health threat. However, a limited number of studies have addressed this problem in the marine environment. We investigated ...their emergence in the coastal waters of the central Adriatic Sea (Croatia), which are recipients of submarine effluents from two wastewater treatment plants. Fifteen KPC-producing
(nine
, four
and two
) were recovered, and susceptibility testing to 14 antimicrobials from 10 classes showed that four isolates were extensively drug resistant (XDR) and two were resistant to colistin. After ERIC and BOX-PCR typing, eight isolates were selected for whole genome sequencing. The
isolates belonged to serotype O21:H27 and sequence type (ST) 2795, while
isolates were assigned to STs 37 and 534. Large-scale genome analysis revealed an arsenal of 137 genes conferring resistance to 19 antimicrobial drug classes, 35 genes associated with virulence, and 20 plasmid replicons. The isolates simultaneously carried 43-90 genes encoding for antibiotic resistance, while four isolates co-harbored carbapenemase genes
and
. The
was associated with IncL-type plasmids in
and
. Importantly, the
in four
isolates was located on ~40 kb IncP6 broad-host-range plasmids which recently emerged as
vesicles, providing first report of these
-bearing resistance plasmids circulating in
in Europe. This study also represents the first evidence of XDR and potentially virulent strains of KPC-producing
in coastal waters and the co-occurrence of
and
carbapenemase genes in this species. The leakage of these strains through submarine effluents into coastal waters is of concern, indicating a reservoir of this infectious threat in the marine environment.
Antimicrobial peptides (AMPs) are promising candidates for new antibiotic classes but often display an unacceptably high toxicity towards human cells. A naturally produced C-terminal fragment of ...PGLa, named PGLa-H, has been reported to have a very low haemolytic activity while maintaining a moderate antibacterial activity. A sequential tandem repeat of this fragment, diPGLa-H, was designed, as well as an analogue with a Val to Gly substitution at a key position. These peptides showed markedly improved in vitro bacteriostatic and bactericidal activity against both reference strains and multidrug resistant clinical isolates of Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells as assessed by haemolysis, cell viability, and DNA damage assays. The glycine substitution analogue, kiadin, had a slightly better antibacterial activity and reduced haemolytic activity, which may correlate with an increased flexibility of its helical structure, as deduced using molecular dynamics simulations. These peptides may serve as useful lead compounds for developing anti-infective agents against resistant Gram-negative and Gram-positive species.
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•PGLa-H is one of the smallest natural antimicrobial peptides but is poorly active.•Doubling its size in a tandem repeat enhanced peptide activity.•A rationally guided point mutation (Val15→Gly) improved peptide selectivity.•Cytotoxicity and genotoxicity towards host blood cells were low for both peptides.•Peptides were bactericidal for multidrug resistant clinical isolates.
The long-standing goal in the field of peptide antibiotics has been to design lead compounds that have a wide spectrum of excellent antibacterial activity but are nontoxic to human cells. ...Gram-negative and Gram-positive bacteria have very different membranes, which are additionally modified in some drug-resistant species, presenting a challenge for the design of a single membrane-active peptide able to adapt its conformation to various physical properties of membrane microenvironments. In this paper, we describe how a peptide sequence can be constructed starting from an adaptable dynamic turn tandem motif in a central location. The peptide, named flexampin, has been examined firstly by molecular dynamics simulations. It uses a flexible central motif and designed helix-forming cationic amphipathic arms to form a boomerang-like, L-shape, V-shape, and hairpin, super-secondary structures, whichever is the best in matching amphipathic and hydrophobic microenvironments it encounters. Secondly, activity measurements showed that flexampin is bactericidal at low micromolar concentrations against Gram-positive and Gram-negative strains including some multidrug resistant clinical isolates, while it is nontoxic for human circulating blood cells, does not cause DNA damage, and has good selectivity for bacterial cells in comparison to human cells. It is the first membrane-active peptide designed with the ability to self-adjust the orientation of its two cationic helical arms, 3D-hydrophobic moment, and dipole moment for obtaining a better grasp of anionic polar head groups at bacterial membrane surfaces.
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•In silico designed peptide can adjust its structure to membrane surface charge.•Flexible central hinge enables increased activity against bacterial strains.•Orientation & magnitude of dipole moment favor entrance into anionic membrane.•Molecular dynamics simulations connect structural changes to selectivity.
The aim of this study was to determine the susceptibility of hospital and environmental Acinetobacter baumannii isolate biofilms on ceramics and glass to common disinfectants benzalkonium chloride ...and chlorhexidine. For this purpose we developed a new method for biofilm cultivation and quantification on ceramics. The biofilm bacteria were more resistant to disinfectants than the planktonic populations, as more than 50 % of the biofilm population and none of the planktonic population survived 5-minute exposure. Furthermore, biofilm populations on ceramic tiles were significantly more resistant than those on glass coverslips, even though the amount of biofilm was practically the same on ceramics and glass. The reason for reduced susceptibility of A. baumannii biofilms on ceramics may be related to surface/disinfection interactions. Our findings suggest that biofilms on ceramic surfaces can be an important source of A. baumannii infection in hospital environments.
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