Medline, PubMed and the Cochrane databases were searched on epidemiology and diagnosis of Helicobacter pylori for the period of April 2011–March 2012. Several studies have shown that the prevalence ...of H. pylori infection is decreasing in adults and children in many countries. Various diagnostic tests are available, and most of them have high sensitivity and specificity. The Maastricht IV/Florence consensus report states that the urea breath test using 13C urea remains the best test to diagnose H. pylori infection. Among the stool antigen tests, the ELISA monoclonal antibody test is recommended. All these tests were used, either as a single diagnostic test or in combination, to investigate H. pylori infection among different populations throughout the world. Of particular interest, current improvements in high‐resolution endoscopic technologies enable increased diagnostic accuracy for the detection of H. pylori infection, but none of these techniques, at present, are specific enough for obtaining a real‐time diagnosis of H. pylori infection.
Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy ...of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic–protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.
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.
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.
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.
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.
•This is the first Croatian national study describing the molecular epidemiology of rotavirus strains.•The prevalence of the world’s ‘common’ G/P combinations was 88.8% overall.•A high prevalence of ...human–human reassortants (7.7%) was also detected.•The appearance of the G6 type was reported for the first time in Croatia.•There is a need for continuous surveillance of circulating rotavirus strains.
Rotavirus is the major cause of severe diarrhea in young children worldwide. In countries like Croatia, where rotavirus vaccine has not been introduced in the national immunization program, prospective surveillance is necessary to establish the diversity of rotavirus strains. The aim of this study was to describe the prevalence and geographical distribution of rotavirus strains in Croatia and to detect the possible emergence of novel strains.
The study was conducted among children ≤5 years of age with acute gastroenteritis at three hospitals located in different geographical regions of Croatia, during the years 2012 to 2014. Rotavirus was detected in stools using an immunochromatographic assay and then sent for further molecular analysis.
Genotyping of 822 rotaviruses showed that the predominant circulating strain was G1P8 (61.9%), followed by G2P4 (19.5%), G1P4 (3.9%), and G3P8 (2.9%). A high prevalence of reassortants among common human rotavirus genotypes was detected (7.7%). Possible zoonotic reassortants were found, including G8 and G6 strains. The latter is described for the first time in Croatia.
This study represents pre-vaccination data that are important for decisions regarding immunization strategies in Croatia. The high prevalence of ‘common’ rotavirus strains circulating in Croatia may advocate for rotavirus vaccine introduction, but further surveillance is necessary to monitor the possible emergence of novel genotypes.
•Hospitals from Turkey, Croatia, and Bosnia and Herzegovina were included.•Acinetobacter baumannii is highly resistant to colistin received by patients.•Colistin resistance is caused by point ...mutations in pmrCAB operon genes.•No plasmid-borne mcr genes were detected.•Findings point to within-hospital spread of colistin-resistant isolates.
To characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings.
A. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods.
Isolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country.
Colistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital.
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.
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