Introduction

Aeromonads are ubiquitous in aquatic environments. Aeromonas dhakensis is recognised as a virulent species causing gastroenteritis, severe skin and soft tissue infection and bacteremia in humans [1]. Their pathogenic nature poses public health concerns especially with the rising of antimicrobial resistance (AMR). This study aimed to carry out antimicrobial susceptibility testing (AST) for A. dhakensis isolated from clinical and environmental sources [2,3,4,5,6].

Methods

A total of 118 A. dhakensis (clinical=95, water environments=19, food fish=4) were interpreted of AST for 17 antimicrobial agents using Microscan NM44 plates (Beckman Coulter, CA, USA), according to CLSI guideline M45-A3 2015 [7]. We further compared the susceptibility categorisation agreement of 6 antimicrobial agents available in EUCAST 2020 [8] and CLSI 2015 [7] to determine whether adoption of EUCAST would affect the susceptibility categorisation.

Results and Discussion

Based on the CLSI 2015, AMR patterns observed were imipenem (77.1%), doripenem (62.7%), meropenem (41.5%), trimethoprim/sulfamethoxazole (11%), cefotaxime (8.5%) and aztreonam (0.8%). Resistance rate to carbapenems in clinical strains was at least 2.5-fold higher than that of non-clinical (environmental and food fish) strains (doripenem: 71.6% vs 26.1%; imipenem: 87.4% vs 34.8%; meropenem: 48.4% vs 13%), suggesting that clinical empirical carbapenem therapy, should be used with caution before supporting antimicrobial susceptibility testing data are available. Application of the EUCAST 2020 increased the number of resistant isolates when compared to CLSI 2015 for ceftazidime (8.5% vs 5.9%), aztreonam (1.7% vs 0.8%) and ciprofloxacin (11% vs 0%).

Conclusion

This study revealed a high prevalence of carbapenem-resistance in A. dhakensis clinical isolates. Albeit the current relatively lower prevalence in environmental and aquaculture samples, constant monitoring of AMR Aeromonas is necessary for environment and food safety management. Harmonisation of MIC breakpoints for Aeromonas is needed for guiding therapy and surveillance for laboratories considering switching between CLSI and EUCAST.

ABSTRACT

Today the entire world is suffering from coronavirus (COVID-19) disease, with regards to the treatment of this many therapies used, amongst them antiviral therapy is on the top of the list. Many healthcare professionals and pharmaceutical companies focusing their path on the effectiveness of antiviral treatment conducted so many clinical trials of antiviral therapy. The effectiveness of antiviral drugs against coronavirus is still controversial. In the treatment of coronavirus, various antiviral drugs like remdesivir, HCQ, CQ, LPV/r, IFN, camostat mesylate, favipiravir, nitazoxanide, ribavirin, etc used and results of those drugs are analyzed based on their clinical reports. In present review mention various findings and the latest progress in antiviral therapy, to support ongoing numbers of clinical studies for better management against virus. Also, include detailed analysis of antiviral therapy results of various clinical studies with core discussion on the efficacy of antiviral treatments. In the treatment of coronavirus, it is necessary to summarize various clinical studies cover antiviral therapy to ensure the efficacy and safety of drugs or treatment for the betterment of making healthy mankind.

Bacteriocins are a large group of bioactive peptides ribosomally syntetized with antimicrobial activity against other bacteria. For these reasons, they could be used to inhibit or limit foodborne pathogen growth that increase also the shelf life of food.

We patented the composition of an edible and antimicrobial coating (Patent n° 102018000006424 at UIBM in Italy) in which it is possible to insert Nisin or its producer bacterium Lactococcus lactis to obtain the antimicrobial characteristic.

The composition of coating is very simple and affordable but above all an edible coating could contribute to reduce garbage amount and if coating is with producer L. lactis it could also be ingested like probiotic integrator by lactose intolerant people.

Our study was a 30 days long challenge test using a fruit and a meat product artificially contaminated with L. monocytogenes NCTC 10888 and wrapped with our two different edible coatings, one made antimicrobial using Nisin and the other using the producer L. lactis.

Results showed for both coatings high decrease of Listeria concentration indicating as bacteriocins like Nisin or its producer could be used in food packaging to control foodborne pathogens and related diseases.

Enterococcus faecalis is one of the species most strongly associated with cases of nosocomial infections. This pathogen is resistant to several antimicrobial classes, having an enormous capacity to acquire and transfer resistance genes. The objective of this work was to evaluate the level of antibiotic resistance Enterococcus faecalis isolates recovered from samples of food supplied to ornamental animals. A total of 57 samples of ornamental animal feed (birds, fish, mammals and reptiles) were collected between February and December 2020. Hundred and three Enterococcus faecalis putative isolates, obtained from Slanetz-Bartley and Kanamycin azide aesculin agar selective plates, were recovered from 15 birds, 9 from fish and 4 from reptile feed samples. The identification of isolates was confirmed by standard biochemical tests. Antimicrobial susceptibility was performed using 14 antimicrobial agents by the Kirby-Bauer disk diffusion method, according to the Clinical and Laboratory Standards Institute standards. Enterococcus faecalis isolates showed a higher prevalence of rifampicin resistance (77.7%). Additionally, these isolates also demonstrated resistance to erythromycin (48.5%) and ciprofloxacin (37.9%). None of the isolates showed resistance to gentamicin and streptomycin. Almost half of the isolates (47.6%) showed multidrug-resistance profile; 23.3% showed resistance to 3 different antimicrobial classes, 6.8% to 4 and 17.5% to 5 or more classes. In conclusion, these results indicated a significant presence of E. faecalis in the feeding of ornamental animals, as well as, multidrug-resistant isolates, becoming a public health problem given the proximity and interaction of humans with these animals.

Antimicrobial peptides (AMPs) appeared to be a new approach both against microorganisms and for regulation of inflammatory and repair process. To evaluate their potential usefulness in regenerative medicine we prepared different extracts of neutrophil-derived AMPs, from rabbit, ovine or porcine blood, which contained AMPs of different composition, mainly defensins, cathelicidins and the fragments thereof. Then, we assessed the influence of different AMPs extracts on activity of neutrophils and monocyte-derived macrophages (MDM) in vitro. For this purpose these cells were obtained from experimental animals, rabbits or sheep submitted to different orthopedic procedures such as insertion of titanium implant into the tibial defect, implantation of biomaterials into osteochondral or bone defect or osteochondral transplantation. Stimulation of cultured cells was autologous or heterologous dependently on origin of AMPs extract and the species of experimental animal. The neutrophil activity was assessed on the basis of release of enzymes from azurophilic and secondary granules and free radicals generation. MDM functional assessment was done on the basis of NO and superoxide generation and arginase activity, additionally morphological changes were evaluated in these cell cultures. Our results indicated that the origin of AMPs extract is crucial for its activity, autologous extracts stimulated anti-inflammatory responses, whereas heterologous extracts acted pro-inflammatory on neutrophils and macrophages in vitro. These results could be considered during introduction of new preparations in regenerative medicine.

The gene apt encodes the purine salvage enzyme adenine phosphoribosyltransferase that catalyzes the conversion of adenine and phosphoribosyl pyrophosphate into AMP and also plays a role in the uptake of adenine. We previously reported on the laboratory selection of stable vancomycin-intermediate Staphylococcus aureus (VISA) mutants (MM66-3 and MM66-4) from a hetero-VISA strain (MM66). We now report that one mutation observed in the VISA mutants characterized was in apt. Compared to MM66, growth of MM66-3 and MM66-4 in the presence of adenine and 2-fluoroadenine was less impaired than the MM66 VISA mutants, which indicated a greater reduction in the accumulation of these toxic compounds in the VISA mutants compared to MM66. In addition to increased adenine and 2-flouroadenine resistance, we also noted that several genes required for purine (purLFMNHD) and pyrimidine (pyrABFE) biosynthesis were upregulated in both MM66 VISA mutants compared to MM66. It has been reported that selection with 2-flouroadenine resulted in 2-flouroadenine reduced susceptibility (FARS) mutants harboring mutations in apt. To ascertain a role for apt mutations on vancomycin susceptibility, FARS mutants of MM66 were selected and their apt regions were sequenced. Suspected MM66 FARS mutants arose on media containing 5 mM 2-flouroadenine at a mutation frequency of 4.7 X 10^-7 and all randomly selected MM66 colonies demonstrated higher 2-flouroadenine MICs than MM66. In addition, all FARS MM66 mutants harbored one of a variety of mutations (e.g. deletion, insertion and nonsense mutations) within the apt gene. FARS mutants MM66-FARS-1 and MM66-FARS-6 demonstrated identical growth curves and slightly decreased tolerance to 5 mM adenine growth inhibition compared to the parent strain MM66. Furthermore, compared to MM66, all MM66-FARS mutants demonstrated increased resistance to Congo red, similar to apt mutants previously reported on. In contradiction to these findings, MM66-3 and MM66-4 actually demonstrated reduced resistance to Congo red compared to parent strain MM66. The FARS MM66 mutants also demonstrated increased distances grown on the vancomycin gradients investigated. These findings suggest that apt mutations can alter vancomycin susceptibility and suggests a role for altered purine metabolism in a VISA mechanism.

Aquaculture faces demanding challenges in the foreseeable future, especially the necessity to prevent and control infectious diseases outbreaks and the overuse of antibiotics, with the consequent emergence of antibiotic-resistant bacteria. Therefore, there is a serious urgence to develop alternative antimicrobial strategies to antibiotherapy. In this scenario, ribosomally synthesized antimicrobial peptides, i. e. bacteriocins, exert a high antimicrobial potency and low toxicity, not affecting gut commensal microbiota. Nisin Z (NisZ), a natural variant of nisin A (NisA), is a 34-residue-long lantibiotic bacteriocin, produced by several Lactococcus lactis strains, showing a remarkable inhibitory spectrum against several bacterial ichthyopathogens. In this work, two different multi-chromatographic procedures were used to purify NisZ from cell-free culture supernatants from Lactococcus lactis RBT18, isolated from cultured rainbow trout. In both cases, MALDI-TOF MS analyses of the peptides confirmed the presence of NisZ (3,330 Da), and the yields and antimicrobial activity results were compared. The first purification procedure, a modification of the protocol described by Cintas et al. (1995), resulted in an absorbance peak (eluted at 58% elution buffer, v/v) showing antimicrobial activity, with a yield of 77% and a 348,000-fold increase in the specific antimicrobial activity compared to that of the cell-free supernatant. The second purification procedure, based on the protocol described by Field et al. (2012), resulted in two antimicrobial active absorbance peaks (55 and 58% elution buffer, v/v, respectively), with a combined yield of 4% and a 16,000-fold-increase in the initial specific antimicrobial activity. In summary, our results show that while both procedures are suitable for NisZ purification the former is more appropriate and effective. Nevertheless, further research is necessary to increase production and optimize purification, to obtain economically viable yields that allow its use as an alternative to antibiotics in aquaculture.

The prevalence of chronic wounds (CW) is growing at an accelerated rate, as such wound dressings capable of assisting towards an effective healing process are desirable. To that effect, nanofibrous dressings with a structure resembling the extracellular matrix have been engineered via eletrospinning and functionalized with natural extracts, curcumin and piperine, endowed with antimicrobial, anti-inflammatory and antioxidant properties, highly desirable to an effective treatment of CW. To this day very little research has been presented on the use of piperine in healing processes.

In the present study, combinations of poly(vinyl alcohol) (PVA) and cellulosic compounds such as, cellulose acetate (CA) and cellulose nanocrystalline (CNC) were processed via electrospinning to guarantee porous, highlight intricated and flexible, biocompatible, biodegradable, and mechanically stable mats. PVA/CA and PVA/CNC mats were prepared at different ratios, 100/0, 90/10 and 80/20 v/v%, at 10 w/v% concentration in 75/25 v/v% acetic acid/water for PVA/CA and only in water for PVA/CNC. Processing electrospinning conditions were optimized to obtain uniform, continuous, bead free mats, with a flexible structure. The instant solubilization of the PVA portion of the mat in aqueous media was surpassed via crosslinking with glutaraldehyde (GA) vapor (less cytotoxic approach) during 7 h at 60ºC. Antimicrobial efficacy of selected biomolecules was evaluated against Staphylococcus aureus. The biomolecules were initially screened for their antibacterial efficacy by the determination of minimal inhibitory concentrations (MICs). Curcumin was the most effective biomolecule, requiring only 7.8-31.2 µg/mL to inhibit the grown of S. aureus, while piperine needed 31.2-125 µg/mL to induce the same effect. Mats were then functionalized with the antimicrobial agents at 2xMIC value and their antimicrobial action access. Data was very promising in revealing these biomolecules as potential substitutes of antibiotics in the fight against S. aureus bacteria.

The high resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious global public-health concerns. This has stimulated interest in the research of bio-based therapeutics with limited resistance, namely, antimicrobial peptides (AMPs) or essential oils (EOs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control for comparison purposes. The biomolecules were initially screened for their antibacterial activity using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation through scanning electron microscopy (SEM) observations of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000–1250 µg/mL) to induce the same effects as the AMPs (500–7.8 µg/mL). Pexiganan was the most effective biomolecule, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5-7.8 µg/mL), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were seen to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these biomolecules and allowed to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.

Antimicrobial photodynamic therapy (aPDT) is gaining a special importance as an effective approach against multidrug-resistant strains responsible of fatal infections. The addition of potassium iodide (KI), a non-toxic salt, is recognized to increase the aPDT efficiency of some photosensitizers (PSs) on a broad-spectrum of microorganisms. Until now, the literature survey only reported combinations of PSs and KI with a positive aPDT potentiation. Moreover, the possibility of extending this approach to cationic porphyrins was not evaluated. In order to gain a more comprehensive knowledge about this type of potentiation, in this work we decided to assess the effect of KI in the presence of a broad range of porphyrinic and non-porphyrinic PSs. For this evaluation were selected a series of cationic meso-tetraarylporphyrins charged at meso or β-pyrrolic positions and a set of non-porphyrinic dyes. The aPDT assays mediated by KI were performed using a bioluminescent Escherichia coli strain as a bacterial model. The results indicate that KI has the ability to potentiate the aPDT process mediated by some of the cationic PSs allowing a drastic reduction of the treatment time as well as of the PS concentration. However, the efficacy of some porphyrinic and non-porphyrinic PSs was not improved in the presence of the coadjuvant. This work helped to elucidate that for the series of compounds studied, the PSs capable to decompose the peroxyiodide into iodine (detectable through spectroscopy or by the iodine–starch test) are the most promising to be used in combination with KI, since their efficacy is complemented with the action of iodine. Although these studies confirm that the generation of ¹O₂ is an important factor in this process, the PS structure, aggregation behavior and affinity for the cell membrane are also important features to consider.