The Organization for Economic Cooperation and Development predicted that 2.4 million people in Europe, North America and Australia may die from infections caused by resistant microorganisms in the next 30 years, with an associated economic cost of approximately US$3.5 billion per year. S. aureus – a common opportunistic pathogen resistant to multiple antibiotics – induced infections are among the most prevalent, being on the front of the line of the WHO concerns. Here, we report the modification of sodium alginate (SA) and gelatin (GN) microfibers, produced via wet-spinning technique, with Nisin Z, an antimicrobial peptide with a significant antibacterial activity against Gram-positive bacteria and low toxicity in humans. Wet-spun SA/GN microfibers were successfully produced at a 70/30% v/v polymer ratio, by extrusion within a calcium chloride (CaCl₂) 2wt% coagulation bath. SA-free fibers were obtained through chemical modification within PBS concentrated solutions and subsequently, SAGN and SA-free fibers were crosslinked with glutaraldehyde (labeled as SAGNCL and GNCL, respectively). Finally, Nisin Z was functionalized onto all the fibers at an average concentration of 178 µg/mL, via adsorption technique. Fibers were characterized via Fourier transform infrared spectroscopy, thermal analysis and brightfield microscopy, their degradation profile at physiological conditions (SBF, 28 days) and the Nisin Z release profile from the fibers were analyzed, and their effect against S. aureus was detected via time-kill kinetics assessments. SAGNCL and GNCL loaded with Nisin Z microfibers were capable of progressively eliminating the bacteria, reaching an inhibition superior to 99% after 48 h of culture. The Nisin Z-modified SA and SAGN were not as effective, losing their antimicrobial action after 6 h of incubation. Bacteria elimination was consistent with the release kinetics of Nisin Z from the fibers. Overall, data revealed the potential of Nisin Z in fighting S. aureus-induced infections, while loaded onto biodegradable crosslinked polymeric scaffolds.

With innate resistance to fluconazole (FLC) and rapid acquired resistance to other antifungal drugs, Candida krusei represents a potential multidrug-resistant pathogen. The mechanism of intrinsic FLC resistance in this yeast has been chiefly attributed to decreased susceptibility of FLC target, lanosterol 14α-demethylase (Erg11p), however, the role of efflux pump transporters remains controversial, and requires further investigation. Furthermore, although the overexpression of these transporters, including ATP-binding cassette 1 protein (Abc1p), results in multidrug-resistance phenotype, their inhibitors are limited. More so, there is currently no class of antifungal that specifically targets these transporters. Polyunsaturated fatty acids (PUFAs), including arachidonic acid (AA), which are known disruptors of the cellular membranes might function well as effective efflux pump inhibitors; however, this needs to be investigated. Hence, this study attempted to examine the influence of AA (with or without FLC) on the expression and activity of a representative ABC transporter, Abc1p, in C. krusei. This was carried out by exposing C. krusei biofilms to varying concentrations of AA alone or in combination FLC, and determining Abc1p expression and function using western blot analysis and Rhodamine 6G efflux assay, respectively. Our results demonstrate that Abc1p is overexpressed following exposure to FLC alone, but not in any treatments with AA. Furthermore, Abc1p exhibited increased functionality in the presence of FLC; however, this was severely diminished upon exposure to 1 mM AA, either alone or in combination with FLC. These findings demonstrate AA as a potential inhibitor of Abc1p expression, and subsequent activity. However, further research is necessary to fully delineate how AA influences this transporter.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), has been a major source of human suffering since antiquity. Presently, over 2 billion people are infected by M.tb worldwide, leading to an estimated 10 million active TB cases and 1.4 million deaths in 2020. Drug-resistant TB is becoming a major threat in the global TB control. Multidrug-resistant/rifampicin-resistant TB (MDR/RR TB) was diagnosed in an estimated 4.1% of new cases and about 19% of previously treated cases. Among these, approximately 6.2% of cases were extensively drug-resistant TB (XDR-TB). An estimated treatment success rate for MDR/RR-TB and XDR-TB was 54% and 30%, respectively. Treatment for MDR/RR-TB and XDR-TB requires a longer therapeutic duration with less effective, more expensive and toxic drugs, leading to a higher rate of treatment failure and mortality. To stop the global spread of MDR/RR-TB and XDR-TB, new anti-TB drugs or combined regimens are urgently needed. Recently, a combined therapeutic strategy consisting of an adjunct immunotherapy and anti-mycobacterial drugs has been proposed and investigated. In our current study, we found that extracellular vesicles isolated from M.tb-infected macrophages synergistically increased M.tb clearance in macrophages in combination with moxifloxacin, a key antibiotic against MDR-TB, in in vitro cell culture infection model and in vivo mouse model. We further demonstrated that extracellular vesicles isolated from M.tb-infected macrophages acted by activating host cytosolic RIG-I/MAVS-dependent pathway and LC3-associated M.tb-containing phagolysosome maturation in host cells. Our results shed light on the development of extracellular vesicle-based host-directed therapy against tuberculosis in humans.

The Burkholderia cepacia complex (Bcc), is a group of closely related bacterial pathogens which commonly infects people with the genetic disease cystic fibrosis. Bcc infections cause a severe drop in lung function and cepacia syndrome, a life-threatening type of sepsis. A species of the Bcc, Burkholderia cenocepacia is inherently resistant to many of the antibiotics currently used in the clinics, leading to persistent infections. One way of developing novel antibiotics is to repurpose approved drugs and develop them as antimicrobials. One example is auranofin; however, auranofin is inactive in many Gram-negative bacteria, possibly due to the presence of the highly impermeable outer membrane. We hypothesized that derivatives of auranofin could inhibit the growth of the multidrug resistant pathogen B. cenocepacia and these compounds could be possible candidates for further drug development. We have found two auranofin derivatives that are potent against B. cenocepacia and are comparable in activity to clinical antibiotics. The auranofin derivatives are bactericidal to both exponential and stationary phase cells and have lower minimum inhibitory concentrations (MICs) than clinical antibiotics. Further, these compounds can also kill persister cells created by other antibiotics. A continuously growing culture exposed to the drugs did not develop spontaneous resistance over the course of 24 days, showing resistance will not be easy to generate. Thus, the two auranofin derivatives have ideal characteristics to be further developed into antimicrobials to clear B. cenocepacia lung infections in cystic fibrosis patients.

Staphylococcus aureus is important pathogenic bacteria in both humans and animals. In animals, S. aureus is the main causative agent of mastitis and 30% of bovine mastitis cases are associated with S. aureus. Bovine mastitis is the most devasting disease in the dairy industry. The treatment, which includes the administration of antibiotics, has been only partially successful, this is due to the development of antibiotic resistance, and the ability of the bacteria to invade and hide in the host cells. Therefore, the present study aims to investigate the antibacterial activities of the Ikarugamycin compound against intracellular S. aureus, and to determine the cytotoxicity of the compound toward bovine mammary epithelial cells ( Mac-T cells ). Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of Ikarugamycin. In-vitro infection models’ assay was used to infected Mac-T cells with S. aureus. Ikarugamycin intracellular antibacterial activity assays were used to determine the bactericidal activity of Ikarugamycin against intracellular S. aureus. Alamar blue assay was used to evaluate the cytotoxicity of Ikarugamycin against Mac-T cells. S. aureus is susceptible to Ikarugamyicin with MIC value 0.6 μg/mL. Ikarugamycin at 4 MIC and 8MIC have bactericidal activity by reducing 3 and 5 log 10 CFU/ml of S.aureus in the first six-hour respectively. Moreover, the Ikarugamycin possesses intracellular antibacterial activity, at 5 μg/mL killed 90% of intracellular S. aureus. The concentration of Ikarugamycin that inhibits 50% of Mac-T cells (IC50) was 9.2 μg/mL, therefore the IC50 is far from the concentration required to kill 90% of intracellular S. aureus in Mac-T cells. The study highlighted that Ikarugamycin antibiotic can be used to deal with infections cause by intracellular and multi-drug resistance S. aureus in case of mastitis

People with cystic fibrosis experience re-occurring polymicrobial pulmonary infections that are a leading cause of mortality. Among the infecting organisms is the Gram-negative Burkholderia cenocepacia, which causes a rapidly progressing form of necrotizing pneumonia and bacteremia known as “cepacia syndrome”, with few treatment options due to high intrinsic antibiotic resistance. The application of next-generation sequencing has powered recent genome-wide explorations into antibiotic resistance. Here, we lay the foundation for such an exploration into the epidemic clinical isolate B. cenocepacia K56-2 with an in-depth survey into its resistance arsenal. We have characterized growth dose-response curves for a panel of 87 antimicrobials from over 30 diverse classes, including clinically relevant antibiotics. Despite many not causing full growth inhibition, we observed important differences and similarities in and among structural classes. For example, tetracyclines and quinolones were up to two orders of magnitude more potent than aminoglycosides and cationic peptides. Within the cephalosporins, while ceftazidime by itself has poor activity, the related siderophore conjugate, cefiderocol, possessed a 512-fold lower MIC. Furthermore, cross-reference to the Comprehensive Antibiotic Resistance Database (CARD 2020) explains many of the observed trends in antimicrobial activity. K56-2 putatively encodes over 250 unique known resistance genes, including carbapenemases, broad-spectrum efflux pumps, and outer membrane modification systems. Furthermore, we have constructed and validated a high-density barcoded transposon mutagenesis scheme to quantitatively profile genomic contributions to antimicrobial resistance. At sub-inhibitory concentrations of antibiotics, hypersusceptible mutants will be selectively killed, which can be monitored by next-generation sequencing and enumeration of unique DNA barcodes in each mutant. Current efforts are focused on cefiderocol, ceftazidime-avibactam, meropenem, and aztreonam; however, our high-throughput platform, will allow us to profile all 87 antimicrobials in the panel. We expect these studies to yield valuable insight into novel therapeutic avenues for treating infections caused by B. cenocepacia and related bacteria.

Staphylococcus aureus is an important pathogen, and the etiologic agent of more than 70% of ocular and peri-orbital infections causing severe tissue damage including permanent blindness. Ocular infections may be confounded by antibiotic resistance, and the breadth and nature of resistance among ocular S. aureus isolates is an area of active investigation. Therefore, we harnessed whole genome sequencing of 163 ocular S. aureus isolates from around the world and the CARD antimicrobial resistance database to investigate the prevalence of 21 classes of resistance genes. In order to identify emerging circulating resistance determinants that may be of particular importance among ocular S. aureus we utilized a collection of 116 publicly available non-ocular S. aureus genomes as a comparator strain set.

Among ocular and non-ocular isolates, antimicrobial efflux pumps associated with fluoroquinolone (Oc: N = 151, 92%; NonOc: N = 116, 100%) and tetracycline (Oc: N = 151, 92%; NonOc: N = 115, 99%) resistance were the most prevalent. However, aminoglycoside and macrolide efflux systems (NonOc: N = 109 (94%) vs Oc: N = 79, 48%; P < 0.0001), and the tetracycline resistance gene tetM (NonOc: N = 26 (23%) vs Oc: N = 2 (1%), P < 0.0001) were found more commonly among non-ocular isolates. Moreover, resistance determinants for daptomycin (N = 3, 2%), rifampin (N = 12, 11%), and trimethoprim/sulfamethoxazole (N = 10, 8%) were only present among non-ocular isolates. In contrast, blaZ-like b-lactamases were significantly more prevalent among ocular isolates (Oc: N = 115, 71%; NonOc: N = 33, 28%; P < 0.0001).

Antimicrobial resistance prediction software was able to detect significant differences in the antimicrobial resistance profiles between ocular and non-ocular S. aureus isolates perhaps reflecting different therapeutic selection pressures in these two groups, and supporting the need for further exploration of S. aureus isolates causing ocular disease.

Environmental acquisition of antimicrobial resistance is global threat to public health where wastewater treatment plants (WWTP) serve as a hub of carrying diverse range of antimicrobial resistant organism and provide suitable conditions for nonvertical transmission of genetic determinants of antimicrobial resistance. Despite of the advancement in wastewater treatment techniques, certain bacteria and antimicrobial resistant genes (ARGs) persist in the final effluent and are disseminated into the environment. We aimed at characterizing the core resistome of two WWTP influent and final effluent in a four-year longitudinal metagenomic study. We adapted read based approach to calculate the absolute abundance of ARGs and contig based approach to identify the host of the core ARGs. Moreover, we find the human fecal pollution in the both influent and effluent samples and calculated the metagenomic risk score.

We find a diversity of ARGs from 151 to maximum of 654 ARGs per site and the abundance ranges from 1.2 X 10^-1 to 2.4 copies of ARGs per copy of 16S rRNA gene. Of the total 1100 ARGs found in all sites, 73 ARGs in SeoNam WWTP and 75 in JungRang WWTP were found to be persistent in all four years. CrAssphage abundance and metagenomic risk score was higher in influent samples. ARGs diversity was negatively correlated to the core resistome while it was positively correlated with the crAssphage abundance (p<0.05). Abundance of some of the core ARGs got increased in effluent (p<0.01). Pseudomonas, Acinetobacter, Aeromonas and Mycobacterium were the major genera carrying core ARGs both in influent and effluent. The results of our study indicate that core resistome is sharing more than half of the total ARGs abundance and the presence of these ARGs on plasmid make them an environmental hazard. As core ARGs are not correlated to the either plasmids, chromosome or crAssphage, but their constant release in the environment can cause critical public health problems and needs to be addressed carefully.

Background: Enterococcus spp. are among the most frequently detected Gram-positive bacteria in the intestinal flora. These bacteria have developed many defense mechanisms that allow to survive in unfavorable conditions. In many cases Enterococcus spp. are resistant to commonly used antibiotics – vancomycin and teicoplanin (VRE - Vancomycin-Resistant Enterococcus) or gentamicin (HLAR -High-Level Aminoglycoside Resistance)and produce enzymes like gelatinase (gelE gene). An observed significant increase in the number of VRE-positive isolates and the ability to form a biofilm influence the spread of multi-drug resistant strains.

Aim: This study aimed to analyze the presence of vanA and vanB genes, which are responsible for the resistance to vancomycin and teicoplanin. Identification of genes responsible for the formation of biofilm (esp, fsrA, fsrB) and the production of the gelatinase enzyme (gelE) were also assumed.

Materials and Methods: Enterococcus bacteria isolated on D-Cocogel medium were collected from 56 hospital patients’rectal swabs. DNA from colonies of each strain was isolated using a heat-shock method in the TE buffer. The PCR reactions were conducted using specific primers to each of the evaluated genes: vanA, vanB, gelE, gelEfsr, esp, fsrA, fsrB.

Results: Vancomycin resistance was confirmed among 93% of tested Enterococcus strains. The vanA and 18% vanB genes were identified in 75% of the strains. The 12% of Enterococcus strains possessed the ability to produce gelatinase, among which 5% and 2% contained gelE gene, and gene gelEfsr, respectively. The remaining 5% of the strains have both genes gelEfsr and gelE. The 34% of tested bacteria strains showed the presence of analyzed genes responsible for biofilm production (gene esp in 58 % Enterococcus biofilm productive strains, and fsrA and fsrB genes among 42% strains). In all (seven) gelatinase production strains there was a parallel presence of genes fsrA and fsrB (responsible for biofilm formation).

PUMS funding grant no. 502-05-33014020-10254

Kefir is a fermented dairy beverage produced by the joint action of many species of lactic acid bacteria and yeast in milk. While barely known in Western Europe, kefir has long been made available and is widely consumed in Eastern Europe and Asia. Recent trends in healthy, homemade products, however, added to the beverage’s artisanal character and acclaimed beneficial properties, managed to attract consumer attention. Still, production remains mostly artisanal and fermentation environments are, therefore, seldomly controlled. While the inhibitory activity of kefir made with kefir grains – the traditional kefir starter culture – has already been investigated, its industrial counterpart – lyophilized cultures - has hardly received any attention in this regard. Thus, in order to assess the inhibitory activity of lyophilized kefir starter cultures and draw attention to the potential dangers of a contamination at household level, common foodborne bacteria were assessed in a challenge test. Representative strains of Listeria monocytogenes CECT 935, Staphylococcus aureus ATCC 25923, Escherichia coli DSMZ 682 and Salmonella enteritidis CECT 4300T were inoculated in kefir during its preparation. Viable cell counts were monitored for 120h after kefir preparation. Results revealed that all of the tested strains exhibited a peak in their growth at 24h of fermentation, presenting a potential hazard for the consumer. Still, inhibition was observed with S. enteritidis exhibiting the most prompt inhibition, followed by E. coli, L. monocytogenes and S. aureus.