Phenotypic and Molecular Characterization of a Hospital Outbreak Clonal Lineage of Salmonella enterica Subspecies enterica serovar Mikawasima Containing blaTEM-1B and blaSHV-2 That Emerged on a Neonatal Ward, During the COVID-19 Pandemic.

Nontyphoid salmonella can cause severe infections in newborns and is therefore declared a pathogen of major health significance at this age. The aim of the study was molecular and antimicrobial characterization of ß-lactamase-producing Salmonella Mikawasima outbreak clone on a Neonatal ward, University Hospital of Split (UHS), Croatia during the COVID-19 pandemic. From April 2020, until April 2023, 75 nonrepetitive strains of Salmonella Mikawasima were isolated from stool specimens and tested for antimicrobial resistance. All 75 isolates were resistant to ampicillin and gentamicin, while 98% of isolates were resistant to amoxicillin/clavulanic acid. A high level of resistance was observed to third-generation cephalosporins (36% to ceftriaxone and 47% to ceftazidime). Extended-spectrum ß-lactamase production was phenotypically detected by double-disk synergy test in 40% of isolates. Moderate resistance to quinolones was detected; 7% of isolates were resistant to pefloxacin and ciprofloxacin. All isolates were susceptible to carbapenems, chloramphenicol, and co-trimoxazole. Fourteen representative isolates, from 2020, 2021, 2022, and 2023, were analyzed with PFGE and all of them belong to the same clone. Whole-genome sequencing (WGS) analysis of three outbreak-related strains (SM1 and SM2 from 2020 and SM3 from 2023) confirmed that these strains share the same serotype (Mikawasima), multilocus sequence typing profile (ST2030), resistance genes [blaTEM-1B, aac(6')-Iaa, aac(6')-Im, and aph(2'')-Ib)] and carry incompatibility group C (IncC) plasmid. Furthermore, the gene blaSHV-2 was detected in SM1 and SM2. In summary, WGS analysis of three representative strains clearly demonstrates the persistence of ß-lactamase-producing Salmonella Mikawasima in UHS during the 4-year period.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia / Caracterización molecular de genes de resistencia a la colistina en un clon ST101/KPC-2 de alto riesgo de Klebsiella pneumoniae en un Hospital Universitario de Split, Croacia

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.(AU)

Molecular characterisation of colistin and carbapenem-resistant clinical isolates of Acinetobacter baumannii from Southeast Europe.

OBJECTIVES: To characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Microbiome profiling and characterization of virulent and vancomycin-resistant Enterococcus faecium from treated and untreated wastewater, beach water and clinical sources.

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.

A RARE PRESENTATION OF GENITOURINARY TUBERCULOSIS MIMICKING ABDOMINAL TUMOR.

Genital tuberculosis is a rare and unexpected disease in European countries including Croatia. Diagnosis of female genital tract tuberculosis is challenging and is rarely pin-pointed by clinical symptoms because of their low specificity. The authors decided to present a case of genitourinary tuberculosis in a young, immunocompetent fertile woman with high clinical suspicion of abdominal tumor mass. Although considered a desease of the past, rare clinical presentation of genital tuberculosis should be expected and taken into account.

Multidisciplinary Approach to Agricultural Biomass Ash Usage for Earthworks in Road Construction.

Agricultural biomass has great bioenergy potential due to its availability, and it is a carbon-free energy source. During biomass incineration, biomass ash is formed, which is still considered as a waste without proper disposal and management solutions. Various biomass ash utilization options were investigated, mainly concerning engineering issues (the mechanical characterization of newly produced building materials or products), and there is a lack of knowledge of environmental issues arising from this "waste" material utilization in civil engineering practice. The main aim of this research is discussion of a different agricultural biomass characteristics as a fuel, the impact of agricultural biomass ashes (ABA) on the mechanical properties of stabilized soil with a particular emphasis on the environmental impacts within this kind of waste management. The results of this study indicate improved geotechnical characteristics of low-plasticity clay stabilized by lime/ABA binder. In addition to mechanical characterization for materials embedded in road embankments and subgrades, appropriate environmental risk assessment needs to be performed, and the results of this study indicate that the amount of ABAs added to the soil for roadworks should not have adverse effects on the soil fauna in the surrounding environment.

Submarine Outfalls of Treated Wastewater Effluents are Sources of Extensively- and Multidrug-Resistant KPC- and OXA-48-Producing Enterobacteriaceae in Coastal Marine Environment.

The rapid and ongoing spread of carbapenemase-producing Enterobacteriaceae 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 Enterobacteriaceae (nine Escherichia coli, four Klebsiella pneumoniae and two Citrobacter freundii) 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 E. coli isolates belonged to serotype O21:H27 and sequence type (ST) 2795, while K. pneumoniae 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 bla KPC-2 and bla OXA-48. The bla OXA-48 was associated with IncL-type plasmids in E. coli and K. pneumoniae. Importantly, the bla KPC-2 in four E. coli isolates was located on ~40 kb IncP6 broad-host-range plasmids which recently emerged as bla KPC-2 vesicles, providing first report of these bla KPC-2-bearing resistance plasmids circulating in E. coli in Europe. This study also represents the first evidence of XDR and potentially virulent strains of KPC-producing E. coli in coastal waters and the co-occurrence of bla KPC-2 and bla OXA-48 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.

Identification of Recent Tuberculosis Exposure Using QuantiFERON-TB Gold Plus, a Multicenter Study.

We investigated whether the difference of antigen tube 2 (TB2) minus antigen tube 1 (TB1) (TB2-TB1) of the QuantiFERON-TB gold plus test, which has been postulated as a surrogate for the CD8+ T-cell response, could be useful in identifying recent tuberculosis (TB) exposure. We looked at the interferon gamma (IFN-γ) responses and differences in TB2 and TB1 tubes for 686 adults with QFT-plus positive test results. These results were compared among groups with high (368 TB contacts), low (229 patients with immune-mediated inflammatory diseases [IMID]), and indeterminate (89 asylum seekers or people from abroad [ASPFA]) risks of recent TB exposure. A TB2-TB1 value >0.6 IU·ml-1 was deemed to indicate a true difference between tubes. In the whole cohort, 13.6%, 10.9%, and 11.2% of cases had a TB2>TB1 result in the contact, IMID, and ASPFA groups, respectively (P = 0.591). The adjusted odds ratios (aORs) for an association between a TB2-TB1 result of >0.6 IU·ml-1 and risk of recent exposure versus contacts were 0.71 (95% confidence interval [CI], 0.31 to 1.61) for the IMID group and 0.86 (95% CI, 0.49 to 1.52) for the ASPFA group. In TB contact subgroups, 11.4%, 15.4%, and 17.7% with close, frequent, and sporadic contact had a TB2>TB1 result (P = 0.362). The aORs versus the close subgroup were 1.29 (95% CI, 0.63 to 2.62) for the frequent subgroup and 1.55 (95% CI, 0.67 to 3.60) for the sporadic subgroup. A TB2-TB1 difference of >0.6 IU·ml-1 was not associated with increased risk of recent TB exposure, which puts into question the clinical potential as a proxy marker for recently acquired TB infection. IMPORTANCE Contact tuberculosis tracing is essential to identify recently infected people, who therefore merit preventive treatment. However, there are no diagnostic tests that can determine whether the infection is a result of a recent exposure or not. It has been suggested that by using the QuantiFERON-TB gold plus, an interferon gamma (IFN-γ) release assay, a difference in IFN-γ production between the two antigen tubes (TB2 minus TB1) of >0.6 IU·ml-1 could serve as a proxy marker for recent infection. In this large multinational study, infected individuals could not be classified according to the risk of recent exposure based on differences in IFN-γ in TB1 and TB2 tubes that were higher than 0.6 IU·ml-1. QuantiFERON-TB gold plus is not able to distinguish between recent and remotely acquired tuberculosis infection, and it should not be used for that purpose in contact tuberculosis tracing.

Endemicity of OXA-23 and OXA-72 in clinical isolates of Acinetobacter baumannii from three neighbouring countries in Southeast Europe.

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 blaOXA-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.

Molecular Characterization and Survival of Carbapenem-Resistant Acinetobacter baumannii Isolated from Hospitalized Patients in Mostar, Bosnia and Herzegovina.

Increasingly difficult treatment of multidrug-resistant (MDR) bacteria has become a global problem of the 21st century. Within a group of multiresistant bacteria, the Acinetobacter baumannii convincingly occupies the position at the top of the group designated as ESKAPE pathogens. In this study, 61 isolates of A. baumannii were recovered from different samples originating from various departments of the University Clinical Hospital Mostar during 2018. All of the isolates were identified using conventional phenotypic methods and the VITEK® 2 Compact System, and were confirmed by MALDI-TOF mass spectrometry. The minimum inhibitory concentrations (MICs) were determined by the microbroth dilution method using MICRONAUT-S MDR MRGN-Screening and VITEK 2 Compact System. All strains were resistant to carbapenems and classified in eight different resistotypes according to their antibiotic resistance and macrorestriction pulsed-field gel electrophoresis profiles, with all belonging to IC II. One isolate displayed resistance to colistin (MIC ≥16 mg/L). The presence of blaOXA genes encoding OXA-type carbapenemases was investigated by multiplex PCR and the Eazyplex® SuperBugAcineto system and showed 100% compatibility with the detection of acquired oxacillinases. Molecular characterization of the isolates tested in this study revealed the OXA-23- and OXA-40-like groups of acquired oxacillinases. Sequencing of two PCR products of the OXA-40-like group confirmed the presence of OXA-72. Survival assays with two selected isolates of A. baumannii encoding different mechanisms of carbapenem resistance revealed that one isolate was able to survive on a fragment of white laboratory coat during 90 days of monitoring. To the best of our knowledge, this is the first article to present the results of a comprehensive phenotypic, genotypic, and molecular analysis of A. baumannii isolates from the leading clinical hospital center in the southwestern part of Bosnia and Herzegovina, including data for the survival of this pathogen on the white laboratory coats used as compulsory medical clothing.

Molecular Characterization of ß-Lactam Resistance and Antimicrobial Susceptibility to Possible Therapeutic Options of AmpC-Producing Multidrug-Resistant Proteus mirabilis in a University Hospital of Split, Croatia.

This study was performed to elucidate genetic relatedness and molecular resistance mechanisms of AmpC-producing multidrug-resistant Proteus mirabilis isolates in University Hospital of Split (UHS), and define efficient antibiotics in vitro. A total of 100 nonrepeated, consecutive, amoxicillin/clavulanate- and cefoxitin-resistant P. mirabilis isolates were collected, mostly from urine (44%) and skin and soft-tissue samples (30%). They were all positive in cefoxitin Hodge test and negative for extended spectrum beta-lactamase production. Pulsed field gel electrophoresis identified four clusters and two singletons, with 79% of isolates in dominant cluster. Molecular characterization and I-CeuI analysis of representatives revealed blaCMY-16 gene located on chromosome, and insertion element ISEcp1 positioned 110 pb upstream of blaCMY-16 starting codon. They also harbored blaTEM-1, except one with blaTEM-2. They were all resistant to trimethoprim-sulfamethoxazole, all but one to quinolones, and 81% to all aminoglycosides, while 77% were susceptible (S) and 22% intermediate (I) to piperacillin/tazobactam, and 4% were S and 68% I to cefepime. Only 15% were S to ceftolozane/tazobactam. Meropenem, ertapenem, ceftazidime/avibactam, temocillin, and fosfomycin were 100% efficient in vitro. This is the first report of blaCMY-16 gene in P. mirabilis from hospital samples in Croatia. The findings are in accordance with Italian and Greek reports. The clonal nature of outbreak suggests the high potential of clonal spread. Alternative agents should be considered to spare carbapenem usage.

The Spectrum of Design Solutions for Improving the Activity-Selectivity Product of Peptide Antibiotics against Multidrug-Resistant Bacteria and Prostate Cancer PC-3 Cells.

The link between the antimicrobial and anticancer activity of peptides has long been studied, and the number of peptides identified with both activities has recently increased considerably. In this work, we hypothesized that designed peptides with a wide spectrum of selective antimicrobial activity will also have anticancer activity, and tested this hypothesis with newly designed peptides. The spectrum of peptides, used as partial or full design templates, ranged from cell-penetrating peptides and putative bacteriocin to those from the simplest animals (placozoans) and the Chordata phylum (anurans). We applied custom computational tools to predict amino acid substitutions, conferring the increased product of bacteriostatic activity and selectivity. Experiments confirmed that better overall performance was achieved with respect to that of initial templates. Nine of our synthesized helical peptides had excellent bactericidal activity against both standard and multidrug-resistant bacteria. These peptides were then compared to a known anticancer peptide polybia-MP1, for their ability to kill prostate cancer cells and dermal primary fibroblasts. The therapeutic index was higher for seven of our peptides, and anticancer activity stronger for all of them. In conclusion, the peptides that we designed for selective antimicrobial activity also have promising potential for anticancer applications.

Influence of Clogging and Unbound Base Layer Properties on Pervious Concrete Drainage Characteristics.

This paper aims to assess the influence of clogging on paving material (pervious concrete) drainage characteristics as well as the influence of the properties of an unbound base layer on drainage characteristics of the whole paving system. The clogging influence has been studied measuring the drainage characteristics on pervious concrete flags before and after their clogging, according to ASTM C1701-09. Additionally, the drainage characteristics of uncontaminated pervious concrete as a paving material was assessed using the falling head method. To assess the influence of properties of an unbound base course (UBC) on drainage characteristics of the whole paving system, the unbound base layer was compacted in two different levels of compaction and the drainage characteristics were measured (according to ASTM C1701-09). It is concluded that pervious concrete prepared with a smaller aggregate fraction is more prone to clogging. Regarding the influence of UBC, it is important to find a balance between pervious concrete infiltration and UBC exfiltration rate, particularly in a case of pervious concrete flags made of coarse aggregate.

Vancomycin-resistant Enterococcus faecium COLONIZATION and Clostridium difficile infection in a HEMATOLOGIC patient.

Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have emerged as significant nosocomial pathogens and patients with impaired host defenses are at a particular risk of VRE infection. The most common occurrence is asymptomatic colonization of the gastrointestinal tract that can persist for a long time and serve as a reservoir for transmission of VRE to other patients. We present a case of a patient who was diagnosed with acute myelogenous leukemia and suffered from bone marrow aplasia following induction therapy. The patient received prolonged broad-spectrum antimicrobial therapy. During hospital stay, the patient developed Clostridium difficile infection (CDI) and was found to be colonized with a strain of Enterococcus faecium resistant to vancomycin during therapy for CDI. This case also highlights the role of risk factors that could contribute to development of resistance, particularly CDI. Early detection of VRE colonization or infection is a crucial component in hospital program designed to prevent transmission of nosocomial infections. Surveillance cultures of such patients should be mandatory.

Feasibility of Agricultural Biomass Fly Ash Usage for Soil Stabilisation of Road Works.

Agricultural biomass ash is a waste material produced by incineration of residue from fields after harvesting crops. The use of agricultural biomass in industry produces large quantities of ash that represent an ecological problem. Another ecological problem is the dependency of road building on natural materials, which has been traditionally used for all pavement layers. Today, roads are built on less accessible and suitable terrains, increasing the need for improving the mechanical characteristics of locally available materials by various means of stabilisation. Within this research, three agricultural biomass fly ashes are used as lime substitutes for hydraulically stabilised soil. The purpose of this research is evaluation of potential use of agricultural biomass fly ash for the soil stabilisation of road works, i.e., for embankment and subgrade purposes. The results indicate that there is a potential of using barley, sunflower seed shells and wheat fly ash as lime substitutes in the soil stabilisation of road works. The strength characteristics of stabilised soil incorporating biomass fly ash are highly dependent on its chemical composition. Using a three-dimensional digital image correlation technique, it is concluded that the elastic properties of stabilised soil correlate to a fracture mechanism that can be efficiently defined by this modern research tool.

Survival of emerging pathogen Acinetobacter baumannii in water environment exposed to different oxygen conditions.

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.

Designed peptide with a flexible central motif from ranatuerins adapts its conformation to bacterial membranes.

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.

Antibacterial Activity Affected by the Conformational Flexibility in Glycine-Lysine Based α-Helical Antimicrobial Peptides.

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.