Insights into the antimicrobial properties of a cationic steroid and antibiofilm performance in PDMS-based coatings to potentially treat urinary infections.

Currently, multidrug-resistant (MDR) infections are one of the most important threats, driving the search for new antimicrobial compounds. Cationic peptide antibiotics (CPAs) and ceragenins (CSAs) contain in their structures cationic groups and adopt a facially amphiphilic conformation, conferring the ability to permeate the membranes of bacteria and fungi. Keeping these features in mind, an amine steroid, DOCA-NH2, was found to be active against reference strains and MDR isolates of Gram-positive Enterococcus faecalis and Staphylococcus aureus and Gram-negative Escherichia coli and Pseudomonas aeruginosa. The compound was active against all the tested microorganisms, having bactericidal and fungicidal activity, displaying minimal inhibitory concentrations (MICs) between 16 and 128 µg mL-1. No synergy with clinically relevant antibacterial drugs was found. However, the compound was able to completely inhibit the biofilm formation of bacteria exposed to the MIC of the compound. For E. coli and E. faecalis, inhibition of biofilm formation occurred at half the MIC. Besides, DOCA-NH2 inhibited the dimorphic transition of Candida albicans at concentrations 4 times lower than the MIC, and can reduce the microorganism virulence and biofilm formation was significantly reduced at both MIC and half the MIC. Polydimethylsiloxane-based coatings containing DOCA-NH2 (0.5, 1.0, and 1.5 wt%) were prepared and tested against the E. coli biofilm formation under hydrodynamic conditions similar to those prevailing in ureteral stents. A biofilm reduction of approximately 80% was achieved when compared to the control.

A One-Year Systematic Study to Assess the Microbiological Profile in Oysters from a Commercial Harvesting Area in Portugal.

As filter-feeding animals farmed in water bodies exposed to anthropogenic influences, oysters can be both useful bioremediators and high-risk foodstuffs, considering that they are typically consumed raw. Understanding the dynamic of bacterial and viral load in Pacific oyster (Crassostrea gigas) tissues, hemolymph, outer shell surface biofilm, and farming water is therefore of great importance for microbiological risk assessment. A one-year survey of oysters collected from a class B production area (Canal de Mira, on the Portuguese western coast) revealed that these bivalve mollusks have a good depurating capacity with regard to bacteria, as Salmonella spp. and viable enterococci were not detected in any oyster flesh (edible portion) samples, despite the fact that these bacteria have regularly been found in the farming waters. Furthermore, the level of Escherichia coli contamination was clearly below the legal limit in oysters reared in a class B area (>230-≤4600 MPN E. coli/100 g). On the contrary, norovirus was repeatedly detected in the digestive glands of oysters sampled in autumn, winter, and spring. However, their presence in farming waters was only detected during winter.

BDDE-Inspired Chalcone Derivatives to Fight Bacterial and Fungal Infections.

The growing number of infectious diseases around the world threatens the effective response of antibiotics, contributing to the increase in antibiotic resistance seen as a global health problem. Currently, one of the main challenges in antimicrobial drug discovery is the search for new compounds that not only exhibit antimicrobial activity, but can also potentiate the antimicrobial activity and revert antibiotics' resistance, through the interference with several mechanisms, including the inhibition of efflux pumps (EPs) and biofilm formation. Inspired by macroalgae brominated bromophenol BDDE with antimicrobial activity, a series of 18 chalcone derivatives, including seven chalcones (9-15), six dihydrochalcones (16-18, and 22-24) and five diarylpropanes (19-21, and 25 and 26), was prepared and evaluated for its antimicrobial activity and potential to fight antibiotic resistance. Among them, chalcones 13 and 14 showed promising antifungal activity against the dermatophyte clinical strain of Trichophyton rubrum, and all compounds reversed the resistance to vancomycin in Enterococcus faecalis B3/101, with 9, 14, and 24 able to cause a four-fold decrease in the MIC of vancomycin against this strain. Compounds 17-24 displayed inhibition of EPs and the formation of biofilm by S. aureus 272123, suggesting that these compounds are inhibiting the EPs responsible for the extrusion of molecules involved in biofilm-related mechanisms. Interestingly, compounds 17-24 did not show cytotoxicity in mouse embryonic fibroblast cell lines (NIH/3T3). Overall, the results obtained suggest the potential of dihydrochalcones 16-18 and 22-24, and diarylpropanes 19-21, 25 and 26, as hits for bacterial EPs inhibition, as they are effective in the inhibition of EPs, but present other features that are important in this matter, such as the lack of antibacterial activity and cytotoxicity.

New diarylpentanoids and chalcones as potential antimicrobial adjuvants.

Antimicrobial resistance arises due to several adaptation mechanisms, being the overexpression of efflux pumps (EPs) one of the most worrisome. In bacteria, EPs can also play important roles in virulence, quorum-sensing (QS) and biofilm formation. To identify new potential antimicrobial adjuvants, a library of diarylpentanoids and chalcones was synthesized and tested. These compounds presented encouraging results in potentiating the activity of antimicrobials, being diarylpentanoid 13 the most promising. Compounds 9, 13, 16, 19, 22, and 23 displayed EP inhibitory effect, mainly in Staphylococcus aureus 272123. Compounds 13, 19, 22, and 23 exhibited inhibitory effect on biofilm formation in S. aureus 272,123 while 13 and 22 inhibited QS in the pair Sphingomonas paucimobilis Ezf 10-17 and Chromobacterium violaceum CV026. The overall results, demonstrated that diarylpentanoid 13 and chalcone 22 were active against all the resistance mechanisms tested, suggesting their potential as antimicrobial adjuvants.

Antibacterial and Antibiofilm Properties of Self-Assembled Dipeptide Nanotubes.

Over recent decades, multidrug-resistant pathogens have become a global concern, with WHO even considering it one of the biggest threats to global health, food security, and development today, which led to the search for alternative antibacterial agents. A special class is formed by peptides composed by the diphenylalanine motif whose antibacterial properties result from their supramolecular arrangement into nanotubes. However, several other dipeptides that also form nanotubes have been largely overlooked. Here, we present the antibacterial activity of four dipeptide nanotubes. The results point to diverse mechanisms through which dipeptide nanotubes exert their effect against bacteria. Antibacterial activity was similar for dipeptide nanotubes sufficiently wide to allow water flux while dipeptides displaying smaller channels were inactive. This suggests that two of the tested dipeptides, L-Phe-L-Phe (FF, diphenylalanine) and L-Leu-L-Ser (LS), are pore forming structures able to induce membrane permeation and affect cellular hydration and integrity. Of these two dipeptides, only FF demonstrated potential to inhibit biofilm formation. The amyloid-like nature and hydrophobicity of diphenylalanine assemblies are probably responsible for their adhesion to cell surfaces preventing biofilm formation and bacteria attachment.

Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal.

Birds are potential carriers of pathogens affecting humans and agriculture. Aiming to evaluate the occurrence of the top three most important foodborne pathogens in free-living birds in Portugal, we investigated 108 individual fecal samples from free-living birds and one pooled sample of gull feces (n = 50) for the presence of Escherichia coli (pathogenic and non-pathogenic), Salmonella spp. and Campylobacter spp. Virulence- and antimicrobial resistance- (AMR) associated genes were detected by PCR and Whole-Genome Sequencing (WGS), and phenotypic (serotyping and AMR profiles) characterization was performed. Overall, 8.9% of samples tested positive for pathogenic E. coli, 2.8% for Salmonella spp., and 9.9% for Campylobacter spp. AMR was performed on all pathogenic isolates and in a fraction of non-pathogenic E. coli, being detected in 25.9% of them. Ten of the tested E. coli isolates were multidrug-resistant (MDR), and seven of them were Extended-spectrum ß-lactamase (ESBL) producers. Among Salmonella (n = 3) and Campylobacter (n = 9), only one strain of C. jejuni was identified as MDR. Most of the identified serotypes/sequence types had already been found to be associated with human disease. These results show that free-living birds in Portugal may act as carriers of foodborne pathogens linked to human disease, some of them resistant to critically important antimicrobials.

Antimicrobial Activity of a Library of Thioxanthones and Their Potential as Efflux Pump Inhibitors.

The overexpression of efflux pumps is one of the causes of multidrug resistance, which leads to the inefficacy of drugs. This plays a pivotal role in antimicrobial resistance, and the most notable pumps are the AcrAB-TolC system (AcrB belongs to the resistance-nodulation-division family) and the NorA, from the major facilitator superfamily. In bacteria, these structures can also favor virulence and adaptation mechanisms, such as quorum-sensing and the formation of biofilm. In this study, the design and synthesis of a library of thioxanthones as potential efflux pump inhibitors are described. The thioxanthone derivatives were investigated for their antibacterial activity and inhibition of efflux pumps, biofilm formation, and quorum-sensing. The compounds were also studied for their potential to interact with P-glycoprotein (P-gp, ABCB1), an efflux pump present in mammalian cells, and for their cytotoxicity in both mouse fibroblasts and human Caco-2 cells. The results concerning the real-time ethidium bromide accumulation may suggest a potential bacterial efflux pump inhibition, which has not yet been reported for thioxanthones. Moreover, in vitro studies in human cells demonstrated a lack of cytotoxicity for concentrations up to 20 µM in Caco-2 cells, with some derivatives also showing potential for P-gp modulation.

Synthesis of a Small Library of Nature-Inspired Xanthones and Study of Their Antimicrobial Activity.

A series of thirteen xanthones 3-15 was prepared based on substitutional (appendage) diversity reactions. The series was structurally characterized based on their spectral data and HRMS, and the structures of xanthone derivatives 1, 7, and 8 were determined by single-crystal X-ray diffraction. This series, along with an in-house series of aminated xanthones 16-33, was tested for in-vitro antimicrobial activity against seven bacterial (including two multidrug-resistant) strains and five fungal strains. 1-(Dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one (7) and 1-(dibromomethyl)-3,4,6-trimethoxy-9H-xanthen-9-one (8) exhibited antibacterial activity against all tested strains. In addition, 3,4-dihydroxy-1-methyl-9H-xanthen-9-one (3) revealed a potent inhibitory effect on the growth of dermatophyte clinical strains (T. rubrum FF5, M. canis FF1 and E. floccosum FF9), with a MIC of 16 µg/mL for all the tested strains. Compounds 3 and 26 showed a potent inhibitory effect on two C. albicans virulence factors: germ tube and biofilm formation.

Occurrence of mcr-1 in Escherichia coli from rabbits of intensive farming.

The emergence of mobile colistin resistance genes (mcr) is yet another challenge in the fight against antimicrobial resistance, with reports proving the dissemination of these genes in different countries and different environments being of great concern. In the present study, we describe the recovery of three E. coli strains with mcr-1 gene in IncHI2 plasmids from intestinal content of necropsied meat rabbits reared in two intensive production systems in Portugal. Our findings are worrisome, given the high level of dependence on the usage of antibiotics in rabbit rearing and call for the development and implementation of an active surveillance system in this species.

Fecal contamination of wastewater treatment plants in Portugal.

Reutilization of effluents from wastewater treatment plants (WWTP) for non-potable applications is increasing due to the reduction of sustainable water resources. These products mostly come from municipal WWTP and also from slaughterhouses effluents. The microbiological certification of these products is mandatory before their discharge into the environment. This study evaluates if the treatment applied in WWTP to municipal waters or to poultry slaughterhouse effluents distributed over the Portuguese continental territory is efficient in reducing the microbiological risk associated with the reutilization of those wastewaters and sludges. Fecal indicators Escherichia coli and enterococci were evaluated in 42 and 24 wastewater samples from 14 municipal WWTP and 8 poultry slaughterhouse treatment plants, respectively, by the conventional culture method and a rapid Fluorescent in situ hybridization (FISH) technique. Bacterial enumeration in inflow water from most WWTP was rather high (generally >10(5) cells/ml), for both E. coli and Enterococcus spp., and the bacterial quantification by FISH was generally higher than enumeration by the conventional culture method. In both types of treatment plants studied, bacterial load from effluents and sludges was not statistically different from the inflows, indicating that the treatment applied seems to be equally unable to reduce the microbiological load of the effluents. These findings may jeopardize the safe reuse of treated wastewaters in agriculture and the quality of the water environment. Therefore, products like water, sewage sludge, and biosolids originated from the municipal and slaughterhouse WWTP studied should not be reutilized, and effluents treatment should be urgently reviewed.

MULTIDRUG RESISTANCE IN WILD BIRD POPULATIONS: IMPORTANCE OF THE FOOD CHAIN.

The presence of multidrug-resistant (MDR) Escherichia coli has recently been reported in wild birds (gulls and birds of prey) that had no apparent exposure to antimicrobials. Little work has been done to assess the role of the food chain in the emergence and spread of MDR E. coli . In this study, we evaluated the presence of MDR E. coli in 29 fecal samples collected from wild birds living in a rehabilitation center (the center receives injured animals found in their natural habitat) and in eight feed samples. In total, 166 E. coli isolates were obtained: 129 from cloacal swabs and 37 from raw feed samples. The antimicrobial resistance profile of these isolates was determined, and we found that 75 isolates showed resistance to five or more drugs, resulting in a total of 38 different antimicrobial resistance patterns. Subsequently, the molecular characterization of 36 isolates, performed by pulsed-field gel electrophoresis, revealed a great similarity between isolates collected from various species of birds and also between these last ones and the ones found in their feed samples.

Coagulase-Positive Staphylococcus: Prevalence and Antimicrobial Resistance.

Staphylococcus pseudintermedius is the most prevalent coagulase-positive Staphylococcus inhabitant of the skin and mucosa of dogs and cats, causing skin and soft tissue infections in these animals. In this study, coagulase-positive Staphylococcus species were isolated from companion animals, veterinary professionals, and objects from a clinical veterinary environment by using two particular culture media, Baird-Parker RPF agar and CHROMagar Staph aureus. Different morphology features of colonies on the media allowed the identification of the species, which was confirmed by performing a multiplex polymerase chain reaction (PCR). Among 23 animals, 15 (65.2%) harbored coagulase-positive Staphylococcus, being 12 Staphylococcus pseudintermedius carriers. Four out of 12 were methicillin-resistant S. pseudintermedius (MRSP). All veterinary professionals had coagulase-positive Staphylococcus (CoPS) species on their hands and two out of nine objects sampled harbored MRSP. The antimicrobial-resistance pattern was achieved for all isolates, revealing the presence of many multidrug-resistant CoPS, particularly S. pseudintermedius . The combined analysis of the antimicrobial-resistance patterns shown by the isolates led to the hypothesis that there is a possible crosscontamination and dissemination of S. aureus and S. pseudintermedius species between the three types of carriers sampled in this study that could facilitate the spread of the methicillin-resistance phenotype.

Molecular evidence of the close relatedness of clinical, gull and wastewater isolates of quinolone-resistant Escherichia coli.

Escherichia coli with reduced susceptibility to quinolones isolated from different environmental sources (urban wastewater treatment plants, n=61; hospital effluent, n=10; urban streams, n=9; gulls, n=18; birds of prey, n=17) and from hospitalised patients (n=28) were compared based on multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The habitats with the most diversified genotypes of quinolone-resistant E. coli, corresponding to the highest genetic diversity (H'), were wastewater and gulls. In addition, genetically distinct populations were observed in clinical samples and birds of prey, suggesting the influence of the habitat or selective pressures on quinolone-resistant E. coli. The close genetic relatedness between isolates of clinical origin and from gulls and wastewater suggests the existence of potential routes of propagation between these sources. The most common sequence types were ST131 and ST10, with ST131 being highly specific to patients, although distributed in all of the other habitats except birds of prey. The prevalence of antimicrobial resistance was significantly higher in isolates from patients and gulls than from other sources (P<0.01), suggesting that the effect of selective pressures met by isolates subjected to strong human impacts. The evidence presented suggests the potential circulation of bacteria between the environmental and clinical compartments, with gulls being a relevant vector of bacteria and resistance genes.

Prevalence of antimicrobial resistance in enteric Escherichia coli from domestic pets and assessment of associated risk markers using a generalized linear mixed model.

Antimicrobial resistance (AMR) is a growing global public health problem, which is caused by the use of antimicrobials in both human and animal medical practice. The objectives of the present cross-sectional study were as follows: (1) to determine the prevalence of resistance in Escherichia coli isolated from the feces of pets from the Porto region of Portugal against 19 antimicrobial agents and (2) to assess the individual, clinical and environmental characteristics associated with each pet as risk markers for the AMR of the E. coli isolates. From September 2009 to May 2012, rectal swabs were collected from pets selected using a systematic random procedure from the ordinary population of animals attending the Veterinary Hospital of Porto University. A total of 78 dogs and 22 cats were sampled with the objective of isolating E. coli. The animals' owners, who allowed the collection of fecal samples from their pets, answered a questionnaire to collect information about the markers that could influence the AMR of the enteric E. coli. Chromocult tryptone bile X-glucuronide agar was used for E. coli isolation, and the disk diffusion method was used to determine the antimicrobial susceptibility. The data were analyzed using a multilevel, univariable and multivariable generalized linear mixed model (GLMM). Several (49.7%) of the 396 isolates obtained in this study were multidrug-resistant. The E. coli isolates exhibited resistance to the antimicrobial agent's ampicillin (51.3%), cephalothin (46.7%), tetracycline (45.2%) and streptomycin (43.4%). Previous quinolone treatment was the main risk marker for the presence of AMR for 12 (ampicillin, cephalothin, ceftazidime, cefotaxime, nalidixic acid, ciprofloxacin, gentamicin, tetracycline, streptomycin, chloramphenicol, trimethoprim-sulfamethoxazole and aztreonam) of the 15 antimicrobials assessed. Coprophagic habits were also positively associated with an increased risk of AMR for six drugs, ampicillin, amoxicillin-clavulanic acid, cephamycin, ciprofloxacin, streptomycin, and trimethoprim-sulfamethoxazole. In summary, pets with a record of one or more previous quinolone treatments and exhibiting coprophagic habits were at an increased risk of harboring multidrug-resistant E. coli strains in their feces compared to pets without these characteristics. AMR is a serious global problem, and assessing the risk markers for the presence of drug-resistant bacteria in pets, a very close source of resistance determinants to humans, is essential for the implementation of safe handling procedures for companion animals and for the prudent selection of antimicrobial compounds in veterinary practice.

Spread of multidrug-resistant Enterococcus faecalis within the household setting.

Advances in veterinary medicine have resulted in the survival of many animals with severe illness or infectious diseases. In addition, increased usage of antimicrobial agents for veterinary purposes has contributed to the worldwide problem of increasing antimicrobial resistance. The objective of this study was to contribute to better understand the potential and implications for the spread of antimicrobial-resistant enterococci between pets receiving antimicrobial treatments and their owners. Three household aggregates (HA A, B, and C) were selected for this study. Information was collected on individual and clinical parameters of both humans and animals that cohabit. For this study, samples of feces, oral secretions, skin and fur of pets, as well as owners' feces and hands and exposed household surfaces and objects were also collected. All enterococci isolates were analyzed for antimicrobial susceptibility. Based on the antimicrobial resistance patterns and origin of isolates, ERIC-PCR analysis was performed on selected isolates to evaluate phylogenetic relationships. In all three HA, Enterococcus faecalis clonal spread was detected between pets and the respective owners, confirming the in-home interanimal species dissemination. Additionally, fecal enterococci colonization of other body parts of the same animal and dissemination of those same enterococci to household surfaces and objects were also observed. Our results demonstrate that enterococcal clones were found in pets in multiple body sites, their human cohabitants, and shared domestic objects.

Quinolone-resistant Escherichia coli isolated from birds of prey in Portugal are genetically distinct from those isolated from water environments and gulls in Portugal, Spain and Sweden.

The influence of geographic distribution and type of habitat on the molecular epidemiology of ciprofloxacin resistant Escherichia coli was investigated. Ciprofloxacin resistant E. coli from wastewater, urban water with faecal contamination and faeces of gulls, pigeons and birds of prey, from Portugal, Spain and Sweden were compared based on multi-locus sequence typing (MLST) and quinolone resistance genetic determinants. Multi-locus sequence typing allowed the differentiation of E. coli lineages associated with birds of prey from those inhabiting gulls and waters. E. coli lineages of clinical relevance, such as the complex ST131, were detected in wastewater, streams and gulls in Portugal, Spain and Sweden. Quinolone resistance was due to gyrA and parC mutations, although distinct mutations were detected in birds of prey and in wastewater, streams and gulls isolates. These differences were correlated with specific MLST lineages, suggesting resistance inheritance. Among the plasmid-mediated quinolone resistance genes, only aac(6')-ib-cr and qnrS were detected in wastewater, streams and gulls isolates, but not in birds of prey. The horizontal transfer of the gene aac(6')-ib-cr could be inferred from its occurrence in different MLST lineages.

Common phenotypic and genotypic antimicrobial resistance patterns found in a case study of multiresistant E. coli from cohabitant pets, humans, and household surfaces.

The objective of the study described in this article was to characterize the antimicrobial resistance profiles among E. coli strains isolated from cohabitant pets and humans, evaluating the concurrent colonization of pets, owners, and home surfaces by bacteria carrying the same antimicrobial-resistant genes. The authors also intended to assess whether household surfaces and objects could contribute to the within-household antimicrobial-resistant gene diffusion between human and animal cohabitants. A total of 124 E. coli strains were isolated displaying 24 different phenotypic patterns with a remarkable percentage of multiresistant ones. The same resistance patterns were isolated from the dog's urine, mouth, the laundry floor, the refrigerator door, and the dog's food bowl. Some other multiresistant phenotypes, as long as resistant genes, were found repeatedly in different inhabitants and surfaces of the house. Direct, close contact between all the cohabitants and the touch of contaminated household surfaces and objects could be an explanation for these observations.

Transfer of multidrug-resistant bacteria between intermingled ecological niches: the interface between humans, animals and the environment.

The use of antimicrobial agents has been claimed to be the driving force for the emergence and spread of microbial resistance. However, several studies have reported the presence of multidrug-resistant bacteria in populations exposed to low levels of antimicrobial drugs or even never exposed. For many pathogens, especially those organisms for which asymptomatic colonization typically precedes infection (e.g., Enterococcus spp. and Escherichia coli), the selective effects of antimicrobial use can only be understood if we considerer all biological and environmental pathways which enable these bacteria, and the genes they carry, to spread between different biomes. This ecological framework provides an essential perspective for formulating antimicrobial use policies, precisely because it encompasses the root causes of these problems rather than merely their consequences.