Prior to the discovery of penicilin in 1928, metal-based antimicrobials were the go-to method to treat bacterial infections. Despite their effectiveness, the lack of awareness surrounding their responsible usage could lead to undesired side effects by means of exposure to toxic metal species at high levels. Nowadays in the current antimicrobial resistance era, where the incidence of bacterial strains that have developed resistance to most antibiotics is common, metal-based antimicrobials (MBA) have re-emerged as an alternative to manage infections. A variety of MBAs are being investigated for their antimicrobial activities, with a focus on silver- and copper-based products as they are already on the market. However, despite their effectiveness, their underlying biomolecular mechanisms of action are not fully established. Shedding light onto these metal-bacteria interactions would provide certainty to the exact biological processes that confer effectiveness to each metal species and would help identify new possible cellular targets to design sustainable antimicrobial strategies. Here we explore the cell response profile of Escherichia coli K12 BW25113 when challenged to grow planktonically in the presence of sublethal concentrations of silver nitrate, copper sulfate, gallium nitrate and tetrachloroauric acid. Growth curves using a gradient of metal salt concentrations were run to determine an adequate concentration that would partially hinder growth while remaining within exponential growth phase. Three biological trials per treatment (silver, copper, gallium, gold, and each of their respective unchallenged controls) were processed for RNAseq. Differentially expressed genes analysis shows shared and unique up-regulated genes per treatment, featuring core biological processes being affected such as response & homeostasis of metal ions, transmembrane transport, sulfate homeostasis and amino acid biosynthesis. This brings us closer to a better understanding of how metals and bacteria interact. Such knowledge may help in the development of novel antimicrobial strategies that balance effectiveness against antibiotic-resistant bacterial infections and minimum cytotoxicity to the host organism.

Although antimicrobial resistance has traditionally been associated with exposure to high therapeutic concentrations of antibiotics, the contribution of subclinical concentrations has recently been demonstrated to select for multidrug-resistant mutants. Nonetheless, this is an unexplored topic under gastrointestinal pathogens. In this respect, the reports of multidrug resistance rates for Aliarcobacter butzleri, an emerging enteropathogen whose ubiquity represents a significant concern for public health, translate into a potential compromise in the treatment of infections caused by this microorganism. Fluoroquinolones are one of the recommended classes of antibiotics for infections by this bacterium. However, ciprofloxacin is classified by the WHO as having the highest priority among the critically important antimicrobials and is a member of the Watch category. Aiming to unveil the role of low antibiotic concentrations on the resistance potential of A. butzleri, three strains isolated from distinct origins were phenotypically characterized regarding resistance to ciprofloxacin and submitted to a 12-day adaptative laboratory evolution in the presence of subinhibitory concentrations of this fluoroquinolone. Following experimental evolution, the susceptibility of the evolved populations to ciprofloxacin was assessed, as was their cross-resistance profile to antibiotics belonging to classes, biocides, heavy metals, and ethidium bromide. In all cases, multidrug-resistant mutants emerged upon exposure to concentrations below the levels reported in ciprofloxacin-polluted ecosystems, with changes in the susceptibility to ethidium bromide observed, suggesting that the efflux pumps activity is involved in the resistance phenotypes. From a One-Health perspective, these findings support the relevance that low antibiotic concentrations may have in worsening the antibiotic resistance problem, especially for environmental pathogens such as A. butzleri.

Antimicrobial resistance (AMR) is a major threat to global health, posing a serious challenge for treating bacterial infections[1]. One of the mechanisms that may be behind AMR is the increased efflux of antibiotics from bacteria by specialized membrane transporters[2] . Thus, targeting efflux pumps is a promising approach to combat AMR and restore the effectiveness of antibiotics[3] . In turn, the hybridization of antibiotics with efflux pump inhibitors could lead to improved antimicrobial activity and increased efficacy against drug-resistant bacteria. Taking this into account, in this study, we hybridized two fluoroquinolones, ciprofloxacin or norfloxacin, with phenothiazines, a class of compounds with known efflux pump inhibitory activity, to develop novel molecules with dual action[4] . The hybrid molecules were synthesized using nucleophilic substitution reactions and were converted to maleate salts to improve their water-solubility. The antimicrobial activity of fluoroquinolones and their hybrids was evaluated, focusing on minimum inhibitory concentration, time-kill curves, post-antibiotic effects, mutation frequency, efflux pump inhibitory activity, and anti-biofilm activity. Six of the eight synthesized hybrids were more effective at killing bacteria and inhibiting biofilm formation than the reference fluoroquinolone. Moreover, these new compounds reduced mutation frequency compared to the reference fluoroquinolone and improved ethidium bromide accumulation, demonstrating that the hybrid compounds may inhibit efflux pumps. These results may contribute to ongoing efforts to develop innovative strategies to combat bacterial infections and provide potential alternatives in the fight against antimicrobial resistance

Funding: João L. Serrano acknowledges a doctoral fellowship grant from the FCT (SFRH/BD/148028/2019). This research was funded by the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES. The NMR spectrometers are part of the Portuguese NMR Network (PTNMR) and are partially supported by the Infrastructure Project No. 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).

Acknowledgments: Staphylococcus aureus SA1199 and SA1199A were kindly provided by Professor Dr. Lorena Tuchscherr de Hauschopp (Jena University Hospital-Germany).

Conflicts of Interest: The authors declare no conflict of interest.

References

1. EClinicalMedicine Antimicrobial resistance: a top ten global public health threat. eClinicalMedicine 2021, 41, 101221, doi:10.1016/j.eclinm.2021.101221.
2. Barnabas, V.; Kashyap, A.; Raja, R.; Newar, K.; Rai, D.; Dixit, N.M.; Mehra, S. The Extent of Antimicrobial Resistance Due to Efflux Pump Regulation. ACS Infect. Dis. 2022, 8, 2374–2388, doi:10.1021/acsinfecdis.2c00460.
3. Sharma, A.; Gupta, V.K.; Pathania, R. Efflux pump inhibitors for bacterial pathogens: From bench to bedside. Indian J. Med. Res. 2019, 149, 129–145, doi:10.4103/ijmr.IJMR_2079_17.
4. Grimsey, E.M.; Piddock, L.J. V Do Phenothiazines Possess Antimicrobial and Efflux Inhibitory Properties? FEMS Microbiol. Rev. 2019, 43, 577–590, doi:10.1093/femsre/fuz017.

Background: This study aimed to assess the antimicrobial resistance profiles of urinary tract infection (UTI) collected from individuals of various age groups, both male and female.

Methods: A total of 266 urine samples from a diverse range of individuals were included in this investigation. Midstream urine (MSU) specimens were collected and promptly transported to the laboratory. Within two hours of collection, these samples were processed on Cysteine Lactose Electrolyte Deficient (CLED) medium. Following bacterial growth on the culture medium, species identification was carried out based on colony morphology, Gram staining, and biochemical characteristics. Antimicrobial susceptibility testing, Extended Spectrum β - Lactamase (ESBL) detection, and Carbapenemase detection were performed using the VITEK 2 Compact system (Biomerieux, France).

Results: A total of 61 (22.93%) significant UTIs were identified in both male and female patients. These UTIs were caused by 48 (78.69%) gram-negative bacteria, 7 (11.48%) gram-positive bacteria, and 6 (9.84%) Candida species. Escherichia coli was the predominant bacterium among all the uropathogens isolated (37.70%), followed by Klebsiella pneumoniae (26.22%) and Pseudomonas aeruginosa (11.47%). The overall antibiotic susceptibility patterns revealed that tetracycline exhibited the highest resistance rate (88.89%) among all the gram-negative and gram-positive uropathogens, followed by ceftriaxone (77.14%). The most effective drugs were found to be amikacin (89.36%) and colistin (97.30%) against both gram-negative and gram-positive uropathogens. Notably, colistin susceptibility was observed exclusively in cases of multidrug resistance (MDR) and pandrug resistance (PDR).

Conclusions: This study indicates that gram-negative bacteria, particularly E. coli, are responsible for a higher number of UTIs compared to gram-positive uropathogens. Amikacin and colistin were identified as the most effective antibiotics against both gram-negative and gram-positive uropathogens.

Antimicrobial resistance (AMR) is actually one of the principal public health threats. Pathogens, like bacteria virus or fungi, are becoming resistant to conventional antibiotics and new tools are needed to fight their infections, especially at a nosocomial level. Antimicrobial peptides (AMP) are studied as a promising response because they are the first line of defence of the immune system in all organisms. They have usually a broad spectrum of activity and their common target is the cell membrane of the pathogen. Our work is focused on AMPs identified in Antarctic fishes, like chionodracine and trematocine, which are adapted to live in an extreme environment and, therefore, have evolved particular physiological arrangements. Our aim is to find new weapons to fight especially bacteria identified with the acronym ESKAPE (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) that are among the most common causes of life-threatening infections acquired in health facilities. Usually, the identified peptides are not highly active against human pathogens but, using them as a scaffold and with few amino acids mutations, we have been able to generate molecules with interesting bactericidal activity. Chionodracine mutants showed, as an example, MIC and MBC values between 0.6 (0,002 mg/mL) and 5 µM (0,016 mg/mL) against clinical isolates from ESKAPE pathogens, whereas trematocine values ranged between 3 (0,008 mg/mL) and 10 µM (0,032 mg/mL). Successively, we studied their selectivity towards cell membranes, and cytotoxicity versus human cell lines. Moreover, we determined in vitro their toxicity versus mammalian erythrocytes, and also in vivo using an invertebrate model (insect pupae). In conclusion, these AMPs could be considered as new potential drugs for pharmacological applications.

Chromobacterium violaceum is a gram-negative bacteria, intrinsically resistant to multiple antibiotics, considered as a pathogen of emerging importance. C. violaceum pre-exposed to a phytochemical, berberine (0.5-20 ppm), when allowed to attack the model host Caenorhabditis elegans, could kill lesser worms than its berberine-non-exposed control counterpart. While all the test concentrations between 5-20 ppm were able to attenuate this pathogen’s virulence, 10 ppm had the maximum (60±10%; p<0.001) virulence-attenuating effect. Interestingly, concentrations higher than 10 ppm had lesser anti-virulence effect. Berberine’s anti-virulence effect between 5-20 ppm can be said to follow an inverted-U shaped pattern of dose-response relation. Berberine at none of these concentrations had any effect on bacterial growth or quorum sensing-regulated violacein production. Whole transcriptome of berberine-treated C. violaceum revealed 9 genes to be differently expressed (FDR<0.01; log FC>2). Among the down-regulated genes were those (nirK: copper containing nitrite reductase; and norB: nitric oxide reductase large subunit) pertaining to nitrogen metabolism. Co-occurrence analysis shows both the down-regulated target genes to be absent from humans. Further investigation on targetability of these genes with respect to development of novel anti-virulence agents, and correlation of nitrogen metabolism with virulence of C. violaceum is warranted. Repeated exposure of C. violaceum to berberine (ten subculturings in 10-20 ppm berberine containing media) did not induce resistance against this phytochemical in the bacteria, as this berberine-habituated C. violaceum still could kill lesser worms than the berberine non-exposed control.

Self‑medication use of antibiotics has become a prevalent reason for the development of antibiotics resistance one of the growing global health risks. It is the process by which people choose and take medications to cure ailments or symptoms that they have recognised for themselves. The potency of antibiotics may decrease as antibiotics are used as primary care for the treatment of infectious ailments which may be due to increase in level of antimicrobial resistance (AMR) throughout the globe. Antibiotics resistance has become a major worry, particularly in low income nations. The prevalence and pattern of self-medication use is not well documented, hence a questionnaire survey was conducted in a random sample of the population of Ernakulum district. This study aims to evaluate the use of self-medication with antibiotics and related factors in the Ernakulum district, Kerala. The survey tool included questions regarding age, gender, education, Occupation, name of antibiotics, number of times antibiotics administered in the past 6 months, reason for stopping antibiotics, source of antibiotics, reason for antibiotics self-medications, disposal method of leftover antibiotics etc. Sore throat (34%), cough (26%) are one of the main symptoms for the self-usage of antibiotics. Various antibiotics, including amoxicillin, ciprofloxacin, and azithromycin were commonly used for self-medication. Reasons for antibiotic self-medication was cost saving (3.8%), previous successful experiences (7.7%), and convenience (11.5%).Information regarding self-medication were primarily obtained from past prescriptions from doctors (64.32%) and from pharmacies (23%).15% people disposed antibiotics to household bins. 26.5% of the participants were not aware about the functions of antibiotics. socioeconomic factors such as age and gender is not correlated with the self-usage of antibiotics. Self-medication use of antibiotics is dangerous, its illogical and improper use of antibiotics may lead to antibiotics resistance due to overuse of drugs, hence should be taken under proper supervision by expert. Health care providers must be more responsible and should provide a free training and guide to general public highlighting the adverse effects of self-medicated drugs.

Several studies have reported an increased frequency of colonization and/or infection with antibiotic-resistant bacteria (ARB) during the COVID-19 pandemic. Extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) are a group of bacteria with intrinsic resistance to multiple antibiotics, including penicillins, cephalosporins, and monobactams. These pathogens are easy to spread and can cause difficult-to-treat infections. Here, we summarize the available evidence on the impact of the COVID-19 pandemic on infections caused by ESBL-PE. Using specific criteria and keywords, we searched PubMed, MEDLINE, and EMBASE for articles published up to 30 March 2023 on potential changes in the epidemiology of ESBL-E since the beginning of the COVID-19 pandemic. We identified eight studies that documented the impact of COVID-19 on ESBL-E. Five studies were focused on assessing the frequency of ESBL-PE in patient-derived specimens, and three studies investigated the epidemiological aspects of ESBL-PE infections in the context of the COVID-19 pandemic. Some of the studies that were focused on patient specimens reported a decrease in ESBL-PE positivity during the pandemic, whereas the three studies that involved patient data (1829 patients in total) reported a higher incidence of ESBL-PE infections in patients hospitalized for COVID-19 compared with those with other conditions. There are limited data on the real impact of the COVID-19 pandemic on the epidemiology of ESBL-PE infections; however, patient-derived data suggest that the pandemic has exacerbated the spread of these pathogens.

Abstract:
Introduction: Foodborne pathogens are a major cause of disease and death among the global population. illnesses related to contaminated food may vary from person to person from temporary to long-term complications. Nevertheless, therapid rise of multidrug-resistant bacteria worldwide with a declination in antibiotics developments and production make bacteriophages an attractive tool to overcome bacterial resistance. Bacteriophages have become widely recognized for several potential applications in food industry. Theyrepresent an ideal tool for a rapid and sufficient diagnostic assays with great potentials in controlling the spread of harmful pathogens. Their abundance in nature of and high specificity against a specific host bacterium allow them to eradicate, prevent foodborne illness and recalls and provide safe food to consumers.

Purpose: The purpose of this topic is to shed a light on the existing phage based application such as immobilization of phages and using them as a biosensor for foodborne pathogen detection in food.

Methods: In our research, a phage capture-amplification assay based on the phage immobilization on bioactive paper were used. Experiments started with as isolation of very specific phage against one or more of Non O157:H7 E. coliin food. Isolated phages are screened and made to undergo a variety of phenotypic and genotypic characterisationto make sure that they meet the desirable requirement For example host range experiment , efficiency of plating, phage adsorption, growth curve of phage, whole genome sequencing, immobilization phage into colorlok paper (dipstick approach) using electrostatic properties of phages and the surface. The data were collected from three independents trials where the averages and standard deviations were determined. Detection limits were calculated and compared using an Independent-Samples T-Test using IBM SPSS.

Results: The result have statistically significant differences (p<0.05) in the detection of E. coli O45:H2. The cycle threshold (Ct) values were averaged for each concentration and compared to the average Ct values for the incubated control paper without phage. Using phages as biosensor enabled the detection of as few as 10 CFU/mL of the Big Six Shiga Toxin producing E. coli strains in both TSB media and ground beef using both a plaque assay and real-time PCR to detect phage progeny.

Significance: These data suggest that the immobilization of bacteriophages onto paper offers a promising approach for the detection of foodborne pathogens. The bacteriophage dipstick assay had the ability to detect as few as 10 cells of E. coliO45:H2 in only 8 hours in both TSB and ground beef.

Candida tropicalis is a common member of human and animal microbiota, with invasive candidiasis having an overall mortality ranging from 55% to 60% in adults, and 26% to 40% in pediatric patients and is included as an emergent pathogen in the “high group” of the World Health Organization Antimicrobial Resistance Division fungal priority pathogens list.

We studied the antifungal activity of gold nanoparticles (AuNPs) stabilized with cetyltrimethylammonium bromide (CTAB-AuNPs) and those conjugated with cysteine, in combination with Amphotericin B (AmB), on sessile and persister cells (PCs) of Candida tropicalis biofilms.

The PC model was used and synergistic activity was tested by the checkerboard assay. Biofilms were studied by crystal violet and scanning electron microscopy. The fractional inhibitory concentration (FIC) is a widely accepted means of measuring interactions by calculating the fractional inhibitory concentration index (FICi) using the following equations: FICi = (MBIC in combination/MBIC AuNPs alone) + (MBIC in combination/MBIC AmB alone). The Bliss independence model was chosen as a theoretical approach to compare the effects of AuNPs in combination with AmB. The data interpretation was made by response surface analysis, which served to calculate a theoretical response surface of an indifferent interaction.

Mature biofilms decreased in biomass after the combination of both AuNPs and AmB. A biofilm reduction of 97.29% was obtained with CTAB-AuNPs plus AmB, corresponding to 0.05 FICi points (synergism). Meanwhile, by conjugating cys-AuNPs with AmB, a 91.66% biofilm reduction was obtained, resulting in a FICi value of 0.75 (additive). Analysis of the checkerboard data by the response surface approach led to similar results. For the combination of CTAB-AuNPs with AmB, the SUM-SYN-ANT metric ranged from 5.69 to 36.06, with a mean of 30.91.

Our improved understanding of how these AuNPs aid in the fight against biofilm-associated candidiasis and the development of new approaches to eradicate PC biofilms have great clinical relevance in the treatment of mycoses, which has seen an increasing number of at-risk patients.